Rapid halogen substitution and dibenzodioxin formation during tyrosinase-catalyzed oxidation of 4-halocatechols

Article abstract of DOI:10.1021/tx100315n

4-Fluoro-1,2-benzoquinone, generated by tyrosinase oxidation of 4-fluorocatechol in aqueous buffer, rapidly undergoes substitution by O-nucleophiles (water or catechols) with release of fluoride. 4-Chloro- and 4-bromocatechol behave similarly. The reactions, which have toxicological implications, have been monitored by spectrophotometry and HPLC/MS, and intermediate and final products, including dibenzodioxins, identified.

Full text of DOI:10.1021/tx100315n

ARTICLE
pubs.acs.org/crt
Rapid Halogen Substitution and Dibenzodioxin Formation during
Tyrosinase-Catalyzed Oxidation of 4-Halocatechols
†
‡
,§
Michael R. L. Stratford, Patrick A. Riley, and Christopher A. Ramsden*
†
Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive,
Oxford OX3 7DQ, U.K.
‡
Totteridge Institute for Advanced Studies, The Grange, Grange Avenue, London N20 8AB, U.K.
§
Lennard-Jones Laboratories, School of Physical and Geographical Sciences, Keele University, Staffordshire ST5 5BG, U.K.
S Supporting Information
b
ABSTRACT: 4-Fluoro-1,2-benzoquinone, generated by tyrosinase oxida-
tion of 4-fluorocatechol in aqueous buffer, rapidly undergoes substitution
by O-nucleophiles (water or catechols) with release of fluoride. 4-Chloro-
and 4-bromocatechol behave similarly. The reactions, which have toxico-
logical implications, have been monitored by spectrophotometry and
HPLC/MS, and intermediate and final products, including dibenzodioxins,
identified.
’
INTRODUCTION
intervals shows a steady decay of the initially formed ortho-
quinone 2 (λmax 400 nm) with well-defined isosbestic points
corresponding to the formation of the para-quinone isomer 3.
When4-fluorocatechol 4a was used as substrate, similar behavior
was observed, with an ortho-quinone chromophore (λmax 410 nm)
decaying over 10 min. Since 4-fluoro-ortho-quinone 5a cannot
rearrange to a quinomethane, another transformation must account
for the relatively fast decay of the ortho-quinone 5a.
Quinones represent a class of toxicological intermediates that
can cause a variety of hazardous effects in vivo, including acute
1
cytotoxicity, immunotoxicity, and carcinogenesis. Quinones
possess several reactivities, and the mechanisms by which they
cause toxic effects may involve complex pathways. We have had a
long-standing interest in the toxicity of ortho-quinones generated
by tyrosinase [E.C.1.14.18.1] activity, in particular because of the
possibility of utilizing the action of quinones in selective toxicity
to pigment cells in targeted chemotherapy for disseminated
malignant melanoma. It was shown that the major cytotoxic
product of the noncyclizing tyrosinase substrate 4-hydroxyani-
Scheme 1
2
,3
sole is the corresponding ortho-quinone, and the use of
melanogenesis as a targeting strategy in melanoma was pro-
4
posed. A series of investigations showed that the acute cyto-
toxicity is due to rapid reaction of the tyrosinase-generated
5-8
ortho-quinones with cellular thiols,
and, because the cellular
thiol concentration is high, the efficiency of various noncyclizing
tyrosine analogues as antimelanoma agents is limited. Alternative
cytotoxic strategies based on the pigment cell specificity of
tyrosinase have been studied, including utilizing the spontaneous
cyclization of ortho-quinones with appropriate side chain struc-
9
-11
12
ture
to release cytotoxic agents from prodrugs, and a variety
13,14
of other targeting mechanisms.
In this article, as part of our
continuing interest in ortho-quinone toxicity, we describe the results
of an investigation of the tyrosinase-catalyzed oxidation of 4-fluoro-,
4-chloro-, and 4-bromocatechols and the potentially toxic conse-
quences of 4-halo-ortho-quinone formation.
In a previous study, related to the suicide inhibition of tyrosinase by
15-17
catechols,
we demonstrated the clean rearrangement of 4-n-
alkyl-ortho-quinones 2to para-quinomethanes 3over a period of 10-
18
2
0 min in aqueous buffer. Fresh tyrosinase rapidly oxidizes 4-n-
alkylcatechols 1 to the corresponding ortho-quinones 2 (Scheme 1).
Monitoring the UV-vis spectrum of the reaction solution at 30 s
Received: September 14, 2010
Published: February 09, 2011
r 2011 American Chemical Society
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|Chem. Res. Toxicol. 2011, 24, 350–356

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Tyrosinaseis unique in that itoxidizesboth phenols (oxygenase
linear gradient to 75% over 5 min. Bromide was separated on a Dionex
activity) and catechols (oxidase activity) to ortho-quinones, via
AS4ASC column (250 ꢀ 4 mm) and AS4ASC guard column (50 ꢀ 4
19-24
subtly different mechanisms.
The natural substrate for the
2 4 7
mm) using eluents A, water, and B, 70 mM Na B O . Starting conditions
enzyme is tyrosine 6g. The resulting ortho-quinone 5g rapidly
cyclizes leading in several steps to melanin pigments. Since
tyrosinase, and related enzymes, can oxidize a wide variety of
phenol and catechol substrates, the formation of reactive ortho-
quinones, which cannot rapidly cyclize, from xenobiotic substrates
has toxicological implications. 4-Benzyloxyphenol 6d is a potent
skin-depigmenting agent which induces permanent loss of mela-
nocytes and onset of occupational vitiligo. This condition can also
be induced by other industrial chemicals such as 4-hydroxyanisole
were 7% B, followed by a linear gradient to 40% B over 8 min. The flow
-
1
rate was 1.5 mL min
.
HPLC/MS. Separations were carried out using a Waters 2695
separations module (Waters, Elstree, UK) with an Atlantis dC18, 150
ꢀ
3.0 mm HPLC column (Waters) maintained at 35 °C. The HPLC
eluents were as follows: A, 10 mM formic acid; B, acetonitrile, with gradients
of 10-65% B (F), 10-70% B (Cl), and 20-70% B (Br) over 5 min. The
-1
flow rate was 0.5 mL min , which was split after diode array detection to
-1
give a flow rate of ∼200 μLmin to the mass spectrometer. The eluent was
monitored using a Waters 2996 diode array detector (215-450 nm, 2.4 nm
resolution) and a Waters Micromass ZQ mass detector (mass range 115-
6
e and 4-tert-butylphenol 6f. These phenols are all oxidized by
tyrosinase, and a possible mechanism leading to vitiligo is covalent
500 Da) in negative ion mode using electrospray ionization, and using
bonding of the ortho-quinones 5d-f to a protein, possibly
Waters Empower 2 software. The mass detector employed the following
25
tyrosinase, resulting in the formation of a neo-antigen.
conditions: capillary voltage, 2.1 kV; cone voltage, 20 V; source temperature,
Since haloquinones are bifunctional species that could simi-
larly lead to antigen formation or take part in other potentially
deleterious reactions, we have undertaken an investigation of the
products of halocatechol decay. The results, which we now
describe, demonstrate extremely fast displacement of halide from
the ortho-quinones 5a-c by oxygen nucleophiles, including
water, followed by nucleophilic addition to the resulting ortho-
quinones 7 to give products that include potentially toxic
dibenzodioxins (Scheme 2).
-1
1
20 °C; desolvation temperature, 425 °C; desolvation gas flow, 450 L h ;
-1
cone gas flow, 100 L h
.
The major product 16 (X = Cl) from the oxidation of 4-chlorocatechol by
tyrosinase was isolated as follows: 4-chlorocatechol (43 mg) was dissolved in
50 mM sodium phosphate buffer, pH 6.8 (500 mL), and tyrosinase (1 mg/
mL) (1 mL) was added with stirring. This mixture was incubated at room
temperature (24 h) and the product isolated in 35% acetonitrile and 10 mM
formic acid by semipreparative HPLC. After the removal of acetonitrile in a
centrifugal evaporator, the aqueous sample was applied to a C18 solid phase
extraction cartridge and eluted with acetonitrile which was removed in a
centrifugal evaporator to give an amorphous solid (25 mg).
’
EXPERIMENTAL PROCEDURES
General. 4-Methylcatechol was supplied by TCI Europe N.V.,
’
RESULTS
Belgium. 3-Fluorocatechol, 4-fluorocatechol, and 4-chlorocatechol were
obtained from Sigma-Aldrich, Poole, Dorset, U.K. 4-Bromocatechol was
Combined Oximetry and Spectrophotometry. Each of the
26
synthesized by the method of Ding and co-workers. Tyrosinase (ex
halocatechols investigated, 4-fluoro-, 4-chloro-, and 4-bromoca-
techol, was oxidized by tyrosinase with utilization of oxygen and
giving rise to ortho-quinone species absorbing in the region of
Agaricus bisporus) was obtained from Sigma-Aldrich. This material
27
migrates as a single electrophoretic band and was used without further
purification. A standard solution of 300 units/mL was made up in 0.1 M
phosphate buffer (pH 6.3) and stored at 4 °C prior to use.
4
00 nm. The rates of oxygen utilization varied according to the
halogen substituent in the order: F > Cl > Br. Halogen release,
investigated by ion chromatography, was exhibited in each case.
Catechols are known to slowly inactivate tyrosinase by a
mechanism that is believed to be associated with the reduc-
tion of copper in the active site; a process known a suicide
inactivation.
Combined Oximetry and Spectrophotometry. Experiments
were conducted at room temperature (24 °C) using the apparatus
28
previously described. The test catechol was dissolved in deionized
water and added to 0.1 M phosphate buffer (pH 6.3) in a silica cuvette
positioned in a Hewlett-Packard diode-array spectrophotometer (Model
15-17,29
Tyrosinase-catalyzed oxidation of the halo-
8
452A) and fitted with a Clark-type polarimetric electrode attached to a
catechols exhibited the characteristic suicide-inactivation ki-
netics, and, in the case of 4-fluorocatechol, it was shown that
this inactivation was not due to halide release since the activity of
the enzyme against the standard substrate (4-methylcatechol)
was unaffected by fluoride addition up to 10 times the concen-
tration present in the substrate. Because of the suicide inactiva-
tion concomitant with catechol oxidation by tyrosinase, the total
extent of oxidation can be determined by the initial amount of
enzyme added; in the case of 4-fluorocatechol, the total oxygen
Yellow Springs Instruments oxygen meter (Model 5300). Tyrosinase
was added to the mixture through a narrow orifice in a stopper and the
oxygen utilization and spectral changes recorded. Unless otherwise
stated, the catechol concentration was 600 μM. For kinetic investiga-
tions of halogen release, sample aliquots of 100 μL were withdrawn from
the spectrophotometer cuvette at intervals and the reaction stopped by
1:1 dilution in acetonitrile. Experiments to measure total halogen release
were conducted in 2 mL ampules using 1 mL of buffer containing the
halocatechol substrate at a concentration of 740 μM. Tyrosinase in
varying amounts (1 to 20 units, see details below) was added to the vials
and samples analyzed after 5 min of incubation.
1
utilized is approximately 20 nmol/unit. Using different amounts
of tyrosinase (between 1.5 and 9 units/mL), it was shown that
halogen release was linearly dependent on oxygen uptake (see
Figure 1).
Investigation of the initial reactions in the case of 4-fluoroca-
techol showed that the kinetics of fluoride release closely follows
the oxygen uptake (see Figure 2). However, the absorbance at
Halogen Release Assay. The quantification of the halogen
released was performed by ion chromatography using a Waters 616
gradient pump, a Waters 717 autosampler (Waters, Elstree, UK), and a
Dionex ED40 detector and ASRS suppressor run in external water mode.
Fluoride was separated on a Dionex AS12A column (250 ꢀ 4 mm) and
an AG12A guard column (50 ꢀ 4 mm) using eluents A, water, and B, 50
mM Na B O and 37.5 mM NaOH. Starting conditions were 15% B
4
10 nm showed different kinetics (Figure 3), and in all cases, the
observed quinone spectrum was relatively stable and decayed
2
4 7
held for 1 min, followed by a linear gradient to 75% B over 7 min. The
flow rate was 1.5 mL min-1. Chloride was separated using the same
column and eluents, but with a 40% start and 1 min hold followed by a
slowly over a period of tens of minutes. The first order decay of
-2
-1
the 410 nm peak was estimated as 8.0 ꢀ 10 AU min in
-2
-1
the case of 4-fluorocatechol and 1.9 ꢀ 10 AU min in the case
3
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Chemical Research in Toxicology
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Figure 1. Relationship between fluoride release and oxygen utilization.
The chart shows the data for fluoride released from the oxidation of
Figure 3. Time pattern of generation and decay of the quinone
products of 4-fluorocatechol oxidation by tyrosinase (10 units). The
inset shows the absorbance spectra recorded at 30 s. intervals and the
time trace charts the change in absorbance at 410 nm.
4
-fluorocatechol (600 μM) as a function of the oxygen utilized in the
presence of 1.5, 3, 6, and 9 units/mL tyrosinase. The calculated slope is
.91 with a coefficient of correlation of 0.9936.
1
Scheme 2
is much slower. These proposed initial reactions are consistent
with the mass spectrometric data.
We attribute halogen release from 4-haloquinones to rapid
nucleophilic substitution either by water or by a catecholic OH
(
5 f 7, Scheme 2). A number of products are theoretically
possible including a hydroxylated monomer 8 and several dimers
-17. Mechanistic considerations lead us to propose four path-
9
ways leading to the major products, and these are summarized in
Scheme 3. The products indicated in square brackets (9, 11, 12
and 14) are not observed, and we deduce that further reaction of
these species occurs rapidly.
Figure 2. Comparative kinetics of oxygen utilization (0) and fluoride
release (2). The initial 4-fluorocatechol concentration was 660 μM, and
Mass Spectrometry. The 4-halocatechol oxidation pro-
ducts were examined using LC/MS. The incubations were
made in 1 mL of 0.1 M phosphate buffer (pH 6.3). Sufficient
tyrosinase was added to limit the oxidation to the total available
dissolved oxygen in the buffer (approximately 240 μM).
Because of the kinetic differences both in the rate of oxidation
and the subsequent reactions, a range of conditions was
examined with the three halocatechols, and the optimal con-
ditions for comparison were those in which the substrate was in
slight excess of the oxidation limit, i.e., 600 μM. Under these
conditions, it was possible, by scanning specifically for the
molecular ion corresponding to quinone 8 (m/z 123), to
demonstrate small quantities of this dehalogenated product 8
of 4-fluorocatechol (RT 2.7 min). However, the main products
were identified as the peaks a-g in the chromatograms shown
in Figures 4 and 5.
10 units of enzyme were added at the time point indicated. Suicide
inactivation of the enzyme limited the total extent of oxygen utilization
to about 40 μmol, i.e., equivalent to the generation of about 80 μmol of
quinone. The fluoride assays were made on 100 μL aliquots of the
reaction mixture withdrawn at the time points indicated and the reaction
stopped by dilution 1:1 in acetonitrile.
of 4-chlorocatechol. As the absorbance observed is due to the initial
4-halo-ortho-quinone product of oxidation and the subsequent
quinone product of the halogen substitution reaction, the relatively
slow oxidation of the 4-bromocatechol prevented the accurate
assessment of the quinone decay constant for this substrate.
These results indicate that the release of halide is consistent with
rapid halogen substitution of the initial ortho-quinone product, while
the decay of the resulting ortho-quinone chromophore (Scheme 2)
3
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Chemical Research in Toxicology
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Table 1. Characteristics of Elution Products of Tyrosinase-
a
Catalyzed Oxidation of 4-Halocatechols 4
4
-F-catechol
4-Cl-catechol
m/z abs RT
4-Br-catechol
m/z abs structure
peak RT m/z abs RT
a
b
c
4.78 143/145 386 4.65 187/189 384
5.23 127 280 5.94 143/145 285 5.65 187/189 285
5.51 251 290 5.86 267/269 292 5.51 311/313 295
5
4
17
5.67
d
e
f
6.32 249 278 6.62 265/267 278 6.23 309/311 278
6.40 249 280 6.77 265/267 280 6.59 309/311 278
6.86 265 394 7.24 281/283 398 6.97 325/327 396
7.54 233 304 7.79 249/251 304 7.47 293/295 306
16
15
13
10
g
a
The principal products, identified as the peaks a-g in the absorbance
chromatograms, are characterized by their retention times in minutes
(
RT), their mass values (m/z) in negative electrospray mode (M -
H), and their absorbance in nm (abs). The putative structures are
indicated in the final column (see Scheme 3).
ortho-quinone 5, which was also seen, to a lesser extent, in the
chromatograms of the 4-chlorocatechol incubation products but
was not observed in the products of the 4-fluorocatechol
incubation. Note that, while the absorbance data clearly identi-
fied these products as quinones, the mass detected was identical
to the substrate. We have observed this in several instances, and it
is attributable to the effect of negative electrospray, which, under
conditions employed here, is able to reduce quinones with
relatively low reduction potentials. The failure to observe the
ortho-quinone product in the case of the 4-fluorocatechol oxida-
tion is attributed to the greater ease of fluoride substitution.
The relative stability of 4-bromoquinone is attributed to the
comparatively slow substitution rate of the bromine, and this
relatively slower rate of reaction was associated with the genera-
tion of a number of additional minor products not seen with the
other halogen-substituted compounds. For example, a product
with m/z 201/203 coeluted with peak a at later incubation times,
and peak e also included coeluting products with m/z 413/415
and 495/497/499; the latter product contains two bromine
atoms, deduced from the isotopic pattern, and is probably a
trimer. Also, in the bromine series, a minor product with m/z
Figure 4. Chromatograms of tyrosinase oxidation products. The panels
show the chromatograms of (A) 4-fluorocatechol after 1 min of
incubation, (B) 4-chlorocatechol after 10 min, and (C) 4-bromocatechol
after 21 min. Equivalent elution fractions in the chromatograms are
designated peaks a-g corresponding respectively to the putative
structures 5, 4, 17, 16, 15, 13, and 10 shown in Scheme 3. Peak a
was not evident in the 4-fluorocatechol products, but the existence of
the corresponding ortho-quinone 5a was inferred from the other
products. The properties of the products are summarized in Table 1.
4
13/415 was present in peak f.
In the case of the oxidation products of 4-fluorocatechol, a
double peak (indicated by c in the chromatogram obtained at
1
40 min of incubation; see Figure 5) indicated the accumulation
of isomeric products which we ascribe to the diarylethers 17
formed by the alternative minor reaction 8B þ 4 (see Scheme 3).
For the chloro- and bromocatechols, the retention time of c is
very close to that of the parent catechol b (Table 1), and only one
peak could be seen in the absorbance or total ion chromatograms.
However, extracting similarly timed chromatograms at m/z
2
69 (4-chlorocatechol) or m/z 313 (4-bromocatechol) revealed
two closely eluting peaks in each case corresponding to the
isomers of 17 (data not shown).
The relative proportions of the products varied with time and
the nature of the halogen, but in all cases, the ultimate major
product is peak d (see Figure 5) to which we assign the
dibenzodioxin structure 16 for which two isomeric forms are
possible. Peak e we attribute to a relatively stable precursor of the
final product 16, and, on the basis of its absorption spectrum, this
is believed to be the para-quinone intermediate 15. Peak f is
attributed to ethers 13, which cannot cyclize, and in all cases
Figure 5. Chromatogram of tyrosinase oxidation products of 4-fluo-
rocatechol at 140 min of incubation.
Peak b was identified as the parent substrate 4 and the earlier-
eluting peak a, which was clearly observed in the case of
4
-bromocatechol oxidation, identified as the corresponding
3
53
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Chemical Research in Toxicology
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a
Scheme 3
∼4 ppm. The sample was contaminated by a small amount of
4
-chlorocatechol, which is probably formed by oxidative decom-
position of the product 16. The mass spectrum of product 16
(
[
X = Cl) at a cone voltage of 20 V showed only the molecular ion
M - H ](m/z 265/7). At 60 V, in addition to the molecular
-
ion, a strong fragment ion at m/z 143/5 is observed, correspond-
ing to a retro-Diels-Alder fragmentation of the molecular ion.
’
DISCUSSION
We conclude that in aqueous buffer, 4-halo-ortho-quinones
5
a-c, generated by tyrosinase oxidation of the corresponding
catechols 4a-c, undergo very rapid substitution by O-nucleo-
-
philes with halide release (X ). Fluoride release is particularly
fast suggesting that addition of the nucleophile to the ortho-
quinone is the rate determining step rather than subsequent
cleavage of the strong C-F bond. Both of these steps are very
fast, and the resulting 4-O-substituted-ortho-quinones 7 react
further over a period of 10-30 min (Scheme 2).
The formation of dibenzodioxins by halide substitution of
4
-chloro-ortho-quinone was reported by Frejka and co-
31
workers, and fluoride release from phenols, including fluoro-
tyrosine, by the action of tyrosinase has been previously
32,33
32
observed.
Phillips and co-workers proposed that fluoride
release from 6-fluoro-dopaquinone was the result of intramolec-
ular nucleophilic substitution during cyclization to form cyclo-
dopa: no fluoride release occurred from 3-fluoro-dopa when
oxidized by tyrosinase. In the intermolecular reactions that we
report here, the nucleophile is a catecholic hydroxyl group or
water. The relative amounts of the products identified shows
that, despite the great concentration of water, the predominant
reactions are with the residual catechol in the incubation mixture.
Our data are consistent with the results of Battaini and co-
3
3
workers who studied tyrosinase oxidation of halophenols. We
also observed fluoride release from 3-fluorocatechol with the
formation of a product with m/z 233, consistent with the
generation of a dimer by nucleophilic substitution.
a
X = F, Cl, Br.
exhibited a clear absorbance spectrum, peaking near 400 nm,
characteristic of ortho-quinones. In the bromocatechol incuba-
tion, a significant amount of a product with m/z 413/415 was
also detected. Peak g in all cases corresponds to a compound
with the structure 10, formed by cyclization of the ethers 9
The possible therapeutic utilization of halide release by
31
tyrosinase-catalyzed oxidation has been noted but, to our
knowledge, has not been applied to targeted antimelanoma
treatment. From the toxicological viewpoint, it is possible that
the susceptibility of halogen substituted ortho-quinones to nu-
cleophilic substitution alters the pattern of their metabolism by
the action of quinone reductases and excretion through conjuga-
tion, and these pharmacokinetic implications may be of some
interest. In particular, the formation of quinone derivatives gives
(
Scheme 3).
Since the dibenzodioxin derivatives 16 are the major tyrosi-
nase oxidation products from all three halocatechols, we at-
tempted the isolation of the product 16 (X = Cl). Since there are
alternative modes of nucleophilic attack of the catechol on
quinone 8, and the proposed intermediate 15 may undergo a
Smiles rearrangement prior to cyclization, the final product 16 is
almost certainly a mixture of constitutional isomers. Semipre-
parative HPLC on a batch of 4-chlorocatechol (43 mg) incubated
with tyrosinase for 24 h gave, after centrifugal evaporation, an
25
rise to neoantigens capable of eliciting immune responses as
1,34,35
well as direct quinone toxicity.
The demonstration of
fluoride release is potentially a tool for implicating ortho-quinone
3
6
intermediates in synthetic or metabolic pathways, and the
ortho-quinone activation of halogenated aromatic carbons might
possibly be utilized in synthesis as a technique for site-directed
nucleophilic attack.
amorphous solid (∼25 mg) that corresponded to band d
1
(
Figure 5). Examination of the H NMR spectrum of a d -
6
DMSO solution showed a multiplet at ∼6.7 ppm corresponding
to three coupled aromatic protons and two sharp singlets at ∼5
ppm together accounting for one aromatic proton. We attribute
these singlets to the protons in the 3 positions of the 1,2,4-
trihydroxy ring of structure 16. The chemical shift is attributed to
the electron-rich nature of the penta-oxy ring together with the
antiaromatic character of the adjacent 1,4-dioxin ring. The
almost equal intensities (4:5) of these singlets suggests that the
constitutional isomers 16 (X = Cl) occur in almost equal
amounts. The hydroxy protons are seen as a broad signal at
Some benzodioxins are carcinogenic: 2,3,7,8-tetrachloroben-
zodioxin (TCDD) is a class 1 carcinogen. There appear to be
several mechanisms whereby ortho-quinones may be involved in
carcinogenesis. The facile generation of semiquinones and the
formation of reactive oxygen species (ROS) may cause oxidative
stress with the production of not only oxidized lipids and
proteins but also oxidized nucleic acid bases, such as 8-oxodeox-
yguanosine, which have been associated with mutagenesis in
aging and carcinogenesis. Moreover, ROS are able to activate
30
1
several signaling pathways, including protein kinase C and Ras.
3
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Chemical Research in Toxicology
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Other mechanisms of cytotoxicity implicate quinone arylation
which involves activation of signaling pathways such as the
pancreatic endoplasmic reticulum kinase (PERK) C/EBP homo-
(11) Land, E. J., Perona, A., Ramsden, C. A., and Riley, P. A. (2009)
Dopamine quinone chemistry: a study of the influence of amide, amidine
and guanidine substituents [-NH-CX-Y] on the mode of reaction.
Org. Biomol. Chem. 7, 944–950.
37
logous protein (CHOP) induction. Interestingly, catechol es-
trogen 3,4-quinones have been implicated as initiators of breast,
prostate, and other cancers through the formation of depurinat-
(
12) Jordan, A. M., Khan, T. H., Malkin, H., Osborn, H. M., Photiou,
A., and Riley, P. A. (2001) Melanocyte-directed enzyme prodrug therapy
MDEPT): development of second generation prodrugs for targeted
treatment of malignant melanoma. Bioorg. Med. Chem. 9, 1549–
558.
13) Land, E. J., Ramsden, C. A., and Riley, P. A. (2006) Toxicolo-
(
3
8,39
ing adducts.
The relative ease with which the products of
halogen-substituted catechols form adducts suggests that the
generation of dibenzodioxin derivatives from polyhalocatechols
may have toxicological and occupational health implications.
1
(
gical Aspects of Melanin and Melanogenesis, in The Pigmentary System:
Physiology and Pathophysiology (Nordlund, J. J., Boissy, R. E., Hearing,
V. J., King, R. A., Oetting, W. S., and Ortonne, J.-P.) 2nd ed., pp 354-
’
ASSOCIATED CONTENT
Supporting Information. H NMR spectrum of isomers
394, Blackwell, Oxford, U.K.
1
S
(14) Riley, P. A. (2004) Melanoma and the problem of malignancy.
b
1
6 (X = Cl). This material is available free of charge via the
Tohoku J. Exp. Med. 204, 1–9.
15) Land, E. J., Ramsden, C. A., and Riley, P. A. (2007) The
(
Internet at http://pubs.acs.org.
mechanism of suicide-inactivation of tyrosinase: a substrate structure
investigation. Tohoku J. Exp.Med. 212, 341–348.
’
AUTHOR INFORMATION
(
16) Land, E. J., Ramsden, C. A., Riley, P. A., and Stratford, M. R. L.
2008) Evidence consistent with the requirement of cresolase activity for
suicide inactivation of tyrosinase. Tohoku J. Exp.Med. 216, 231–238.
17) Ramsden, C. A., Stratford, M. R. L., and Riley, P. A. (2009) The
(
Corresponding Author
*E-mail: c.a.ramsden@chem.keele.ac.uk.
(
influence of catechol structure on the suicide-inactivation of tyrosinase.
Org. Biomol. Chem. 7, 3388–3390.
’
ACKNOWLEDGMENT
We thank John Clews (Keele) for the synthesis of 4-bromo-
(18) Land, E. J., Ramsden, C. A., Riley, P. A., and Stratford, M. R. L.
(
2008) Studies of para-quinomethane formation during the tyrosinase-
catalyzed oxidation of 4-alkylcatechols. ARKIVOC (ii) 258–267.
19) Mason, H. S., Fowlks, W. L., and Peterson, E. (1955) Oxygen
catechol and Martin Christlieb, Stuart Conway, and Jess Healy
Department of Chemistry, University of Oxford) for running
the NMR spectra.
(
(
transfer and electron transport by the phenolase complex. J. Am. Chem.
Soc. 77, 2914–2915.
’
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