Copper-Aβ Peptides and Oxidation of Catecholic Substrates: Reactivity and Endogenous Peptide Damage

Article abstract of DOI:10.1002/chem.201603824

The oxidative reactivity of copper complexes with Aβ peptides 1–16 and 1–28 (Aβ16 and Aβ28) against dopamine and related catechols under physiological conditions has been investigated in parallel with the competitive oxidative modification undergone by the peptides. It was found that both Aβ16 and Aβ28 markedly increase the oxidative reactivity of copper(II) towards the catechol compounds, up to a molar ratio of about 4:1 of peptide/copper(II). Copper redox cycling during the catalytic activity induces the competitive modification of the peptide at selected amino acid residues. The main modifications consist of oxidation of His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine, and the formation of a covalent His6-catechol adduct. Competition by the endogenous peptide is rather efficient, as approximately one peptide molecule is oxidized every 10 molecules of 4-methylcatechol.

Full text of DOI:10.1002/chem.201603824

DOI: 10.1002/chem.201603824
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Medicinal Chemistry |Hot Paper|
Copper-Ab Peptides and Oxidation of Catecholic Substrates:
Reactivity and Endogenous Peptide Damage
Valentina Pirota, Simone Dell’Acqua, Enrico Monzani, Stefania Nicolis, and Luigi Casella*[a]
Abstract: The oxidative reactivity of copper complexes with
Ab peptides 1–16 and 1–28 (Ab16 and Ab28) against dopa-
mine and related catechols under physiological conditions
has been investigated in parallel with the competitive oxida-
tive modification undergone by the peptides. It was found
that both Ab16 and Ab28 markedly increase the oxidative
reactivity of copper(II) towards the catechol compounds, up
to a molar ratio of about 4:1 of peptide/copper(II). Copper
redox cycling during the catalytic activity induces the com-
petitive modification of the peptide at selected amino acid
residues. The main modifications consist of oxidation of
His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine,
and the formation of a covalent His6-catechol adduct. Com-
petition by the endogenous peptide is rather efficient, as ap-
proximately one peptide molecule is oxidized every 10 mole-
cules of 4-methylcatechol.
aggregation of Ab peptides,[10] and treatment of AD brain ex-
tracts with metal chelators can dissolve these deposits.[11]
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative
condition that results in synaptic failure and neuronal death.
Characteristic pathological hallmarks in the brains of those suf-
fering from the disease include the presence of extracellular
senile plaques, intracellular neurofibrillary tangles, and altered
levels of neurotransmitters.[1] Amyloid plaques are composed
of aggregated amyloid-b (Ab) peptides, of 39 to 43 residues in
length, that derive from a membrane-bound amyloid-b precur-
sor protein (APP).[2] According to the amyloid cascade hypothe-
sis,[3,4] an increase in Ab production and its accumulation leads
to the formation of oligomers, protofibrils and, in a sequence
of steps, fibrils, the main constituent of amyloid plaques.[5]
However, a number of studies suggest that small soluble, oli-
gomeric forms of Ab are the most toxic species, rather than
more extensively aggregated fibrils or protofibrils.[3,5]
Ab binds Cu2+ with high affinity (Kd =10ꢀ10–10ꢀ11 m)[12] in the
hydrophilic N-terminal portion (residues 1–16, DAEFRHDS-
GYEVHHNK), in which His residues (His6, His13, and His14) are
important for Cu2+ coordination and Cu2+-mediated Ab aggre-
gation.[13] Ab is a disordered peptide that can adopt a multitude
of different coordination sites with similar energies and, indeed,
two different Ab coordination models for Cu2+ coexist around
physiological pH.[14–16] In the first coordination mode, Cu2+ bind-
ing involves the ꢀNH2 terminal group, the adjacent carbonyl
group function from the Asp1-Ala2 peptide bond, the imidazole
ring of His6, and the imidazole ring of His13 or His14.[15] In the
second coordination mode, copper is bound to the deprotonat-
ed Asp1-Ala2 amide bond, the N-terminal amine, the carbonyl
group from the Ala2-Glu3 peptide bond, and an imidazole ring
of either His6, His13, or His14.[15] Also, Cu+ plays an important
role in the interaction with Ab and, in fact, the brain is a rather
reducing environment both in the intracellular and the extracel-
lular media. Furthermore, cultured neurons are sensitive to Cu–
Ab complex-catalyzed production of ROS, which involves redox
Metal ions have been shown to play a critical role in Ab neu-
rotoxicity, thus prompting an intense investigation into the for-
mation of metal–Ab complexes.[6] In fact, remarkably high con-
centrations of several transition-metal ions, mainly Cu2+, Zn2+
,
and Fe3+, were found in amyloid deposits of AD-affected
brains. In particular, the finding of high copper concentration
(400 mm),[7] compared to the normal brain extracellular concen-
tration of 0.2–1.7 mm,[8] suggests that its complexation with Ab
is linked to ROS-related neurotoxicity, Ab aggregation, and for-
mation of amyloid plaques.[9] In addition, both in vitro and in
vivo studies revealed that copper (and zinc) may mediate the
cycling between Cu+ and Cu2+ [14]
. The electron-donation re-
quired for Ab-mediated Cu2+ reduction can involve either inter-
nal Ab amino acid side chains or an external reductant.[8] In the
reduced form, copper binds only to His13 and His14 in a linear,
two-coordinate structure.[17]
The exact role of copper binding to Ab peptides is far from
being completely elucidated and a controversy still exists re-
garding the pro-oxidant[18–20] versus antioxidant[21–24] effects of
this complexation.
In a previous study,[21] we found that the formation of the
Cu–Ab complexes, both [Cu2+ꢀAb16] and [Cu2+ꢀAb28], re-
duced the ability of free copper to catalyze the oxidation of
phenols and catechols. However, the experiments were per-
formed in the presence of the chromophoric reagent 3-
[a] Dr. V. Pirota, Dr. S. Dell’Acqua, Prof. E. Monzani, Dr. S. Nicolis,
Prof. L. Casella
Dipartimento di Chimica, Universitꢀ di Pavia
Via Taramelli 12, 27100 Pavia (Italy)
E-mail: luigi.casella@unipv.it
Supporting information and ORCIDs from the authors for this article are
available on the WWW under http://dx.doi.org/10.1002/chem.201603824.
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methyl-2-benzothiazolinone hydrazone (MBTH), as a trap for
the quinone products,[19,25] but it was found that MBTH is not
redox innocent and it also participates in the reaction as a re-
ducing agent for copper(II).[26] The radical scavenging effect of
Ab in this system is in line with the previously reported hy-
droxyl radical scavenging property of the peptide.[22,23]
This evidence shows that the choice of experimental condi-
tions is crucial in studies that aim at reproducing in vitro the
physiopathological conditions of the cellular environment. In
this context, among the potential external substrates the most
important, in terms of redox reactivity, are neurotransmitter
catecholamines, such as dopamine (DA), and their metabolites,
due to their presence in many brain areas, and in particular in
substantia nigra neurons.[27] These molecules bear a rich redox
chemistry, potentially acting as one- and two-electron reduc-
tants, and their oxidation products are very electrophilic.[28]
However, oxidation of catecholamines is a complex process be-
cause of the high reactivity of their primary quinone prod-
uct,[28] which leads to rapid formation of insoluble melanic ag-
gregates, thus making difficult the conduction and interpreta-
tion of kinetic experiments.[29] Therefore, in this paper, besides
DA, we chose to investigate the oxidation of two catechol
compounds lacking functional groups in the aromatic ring side
chain, that is, 4-methylcatechol (MC) and 3,5-di-tert-butylcate-
chol (DTBC), as substrates for catecholase activity in biomimet-
ic studies.[21,26] It is also important to take into account that an-
other possible target of ROS generated by copper redox cy-
cling is Ab itself, since there is ample evidence of endogenous
oxidative damage of the peptide by metal-catalyzed oxidation
(MCO).[30–32] In general, the presence of oxidative damage on
neuronal proteins is evidence of a link between oxidative
stress and AD.[30] Herein, we report on the reactivity of the
copper complexes with the N-terminal peptides Ab16
Figure 1. Kinetic profiles of DA (3 mm) oxidation with time, in 50 mm Hepes
buffer solution at pH 7.4 and 258C in the presence of Cu2+ (25 mm) (blue)
and with addition of A) Ab16 (1 equiv: yellow; 2 equiv: red; 4 equiv: brown);
B) Ab28 (1 equiv: yellow; 2 equiv: red; 4 equiv: brown). In both cases, the
absorption profiles were corrected for DA autoxidation under the same con-
ditions.
(1DAEFRHDSGYEVHHNK16-NH2)
and
Ab28
(1DAEFRHDSGYEVHHNKLVFFAEDVGSNK28-NH2) towards cate-
cholic substrates and the competitive endogenous oxidative
modification undergone by the peptide backbone.
either Ab16 or Ab28. In both cases, the DA oxidation rate in-
creases up to a 4:1 Ab/Cu2+ ratio, with a slightly stronger
effect in the presence of Ab28. The reaction rates increase by
saturating the solution with dioxygen instead of air (Figure S2,
Supporting Information), indicating that the rate-determining
step involves O2. The low [O2] in Hepes buffer (240 mm) may be
partly responsible for the slow reaction with oxygen. Notwith-
standing the low concentration of O2, which is substoichiomet-
ric with respect to DA, the peculiar kinetic profile is not associ-
ated to O2 consumption, since only a very low fraction of DA is
converted into DAC during the observed reaction times (Fig-
ure S3, Supporting Information).
Results and Discussion
The oxidation of DA by Cu2+ in 4-(2-hydroxyethyl)-1-piperazine
ethanesulfonic acid (Hepes) buffer at pH 7.4 was studied in the
presence of increasing amounts of either Ab16 or Ab28, in par-
allel experiments. Dopamine oxidation was monitored by UV/
vis spectroscopy through the development of the absorption
band of dopaminochrome (DAC) at 475 nm. This product accu-
mulates in the initial stages of the reaction.[29,33] After approxi-
mately 20 min of reaction time, other absorption bands at
about 405 and 620 nm appear (Figure S1C, Supporting Infor-
mation) and a general increase of absorption over the whole
wavelength range, due to the formation of melanin, is ob-
served. Within about 1 h, formation of insoluble melanic prod-
ucts can be noted; therefore, we focused our attention on the
first 30 min of reaction. Under these conditions, DA autoxida-
tion is not negligible and needs to be subtracted from the ki-
netic profile. Figure 1 shows that DA (3 mm) oxidation cata-
lyzed by Cu2+ (25 mm) is strongly promoted by the addition of
Since DA oxidation is rather slow and occurs with formation
of a mixture of products and precipitate, this substrate is not
suitable to perform a more detailed kinetic study. Therefore,
the oxidation of the more reactive MC (with a lower semiqui-
none/catechol redox potential)[26] can be conveniently studied
as a model substrate of DA. Oxidation of MC (3 mm) promoted
by Cu2+ (25 mm) and Ab peptides at pH 7.4 (50 mm Hepes
buffer) proceeds with a biphasic behavior where an initial step,
concluded in about 100 s, is followed by a second linear phase
(Figure 2). As for DA, also with MC the two-step kinetic profile
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Cu2þ þ Ab Ð Cu2þ ꢀ Ab
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
þ
Cu2þ ꢀ Ab þ MC ! Cu ꢀ Ab þ MC
Cþ
Cuþ ꢀ Ab þ MC Ð Cuþ ꢀ Ab ꢀ MC
þ
*
Cu ꢀ Ab-MC þ O2 ! Cu ꢀ Ab ꢀ MC ꢀ O2
2þ
ꢀ
Cþ
*
Cu ꢀ Ab ꢀ MC ꢀ O2 !! Cu ꢀ Ab þ MC þ O2
According to the mechanism proposed, the biphasic behav-
ior is not associated with a change in the rate-determining
step of the reaction but in the product accumulated. The 4-
methylcatechol semiquinone, MC +, disproportionates to MC
C
and MQ. In the presence of excess MC, an addition product to
MQ is formed:
Cþ
2 MC ! MC þ MQ
ð6Þ
ð7Þ
MC þ MQ ! addition products
Therefore, in the initial phase of the reaction, MQ is formed
and it accumulates in solution, but after about 100 s it is con-
sumed by the formation of the addition products, according to
Equation (7). These addition products absorb at higher wave-
lengths, giving rise to a reduction in the slope shown in
Figure 2. This reaction path describes the biphasic kinetic trace
observed. It is interesting to note that the reaction of Equa-
tion (2) proceeds in spite of the slightly larger one-electron
standard reduction potential of the MC +/MC couple[26] with re-
C
Figure 2. Kinetic profiles of MC (3 mm) oxidation with time, in 50 mm Hepes
buffer solution at pH 7.4 and 258C in the presence of Cu2+ (25 mm) (blue)
and with addition of A) Ab16 (1 equiv: yellow; 2 equiv: red; 4 equiv: brown);
B) Ab28 (1 equiv: yellow; 2 equiv: red; 4 equiv: brown). In both cases, the
absorption profiles were corrected for MC autoxidation under the same con-
ditions.
spect to those of Cu2+–Ab/Cu+–Ab peptides.[35] This happens
because the semiquinone species does not accumulate in solu-
tion but is consumed by the reaction in Equation (6). It is likely
that also a Fenton-type mechanism, generating ROS, contrib-
utes to the catalytic oxidation of MC promoted by the copper
complex of Ab peptides.
DTBC is a widely used substrate in biomimetic studies of cat-
echolase activity. Due to the low solubility of DTBC in water,
the oxidation of this substrate is usually studied in organic sol-
vents.[36] When the Cu2+-mediated DTBC oxidation was carried
out in a mixed methanol/aqueous Hepes buffer 50 mm (80:20
v/v) at pH 7.4, the reaction was completely quenched by the
addition of two equivalents of Ab16 (see Figure 3) or Ab28
peptides (data not shown). This behavior was previously
noted,[21] and suggests that solvent also plays an important
role in the reaction. For this reason, we performed the DA and
MC oxidation experiments in the same methanol/Hepes buffer
mixture. In the case of the Cu2+-promoted DA oxidation, the
addition of Ab16 increases the oxidation rate but to a minor
extent with respect to aqueous buffer (Figure S1A, Supporting
Information). It is also worth noting that the DA oligomeriza-
tion process observed in aqueous buffer (Figure S1C, Support-
ing Information) is suppressed in the mixed organic medium,
in which only the absorption band of dopaminochrome at
475 nm can be observed (Figure S1B, Supporting Information).
The Cu2+-promoted MC oxidation in the methanol/Hepes
buffer mixture exhibits similar behavior, as the reaction rate is
progressively reduced when increasing amounts of Ab16 are
is not associated to low [O2] in solution. The biphasic behavior
can be noted also for DA oxidation but the presence of several
absorbing species in solution makes the observation less clear.
During the time course of MC oxidation, a shift of the develop-
ing absorption bands occurs from the initial value of 401 nm,
due to the quinone (MQ, e=1550mꢀ1 cmꢀ1), to higher wave-
lengths, which is related to the formation of the quinone addi-
tion product with catechol.[34] The rate of both steps increases
by saturating the solution with dioxygen instead of air (data
not shown), indicating that the slow step of the reaction in
both phases involves the formation of a Cu/O2 intermediate
[Eq. (4)]. Moreover, by increasing the peptide concentration,
the rate of both steps is accelerated reaching a saturation
value at Ab/Cu2+ ratio of 2:1 or slightly above (Figure 2).
Again, a slightly stronger effect is observed in the presence of
Ab28.
We can therefore propose the following mechanism, in
which the rate-limiting step is the formation of some reactive
copper–dioxygen intermediate [Eq. (4)]. This species is generi-
cally indicated as Cu–Ab–substrate–O2* because its nature has
not been characterized yet.
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a linear increase of the absorbance with time. The first step is
stoichiometric with respect to copper concentration and its
rate did not change by saturating the solution with pure
oxygen. On the other hand, the second step was oxygen-con-
centration dependent and decreased by the addition of pep-
tide. These results suggest that copper(II) is reduced by the
catechol, according to the reaction in Equation (2), giving rise
to the formation of MQ and thus to the MBTH–MQ adduct in
the first, oxygen-independent phase. Then, it is also possible
that MBTH competes with MC for coordination to [Cu+ꢀAb28]
or [Cu+ꢀAb16] [Eq. (3)], decreasing the accumulation of the
Cu+–Ab–MC adduct in the pre-equilibrium step and, conse-
quently, the rate of the oxygen-dependent determining step of
the reaction [Eq. (4)].
The reactivity described so far could have important conse-
quences not only for oxidation of specific targets, such as DA
and related catecholic substrates, but also for nonspecific oxi-
dation reactions promoted by CuI–Ab species generated
during turnover. Unlike copper enzymes that activate oxygen
for specific reactions,[38] Cu–Ab complexes generate Cu/O2 spe-
cies capable of nonselective oxidations. This behavior is due to
Fenton’s reaction yielding harmful ROS that give rise to oxida-
tive protein damage, through oxidation of amino acid residues,
structural alteration, and loss of function.[8,30,31,39] These reac-
tions contribute to the oxidative damage of several biomole-
cules observed in AD, including Ab itself, which has been
found to be oxidized in amyloid plaques in vivo.[40]
Figure 3. Kinetic traces of absorbance at 407 nm versus time for the oxida-
tion of DTBC (3 mm) in MeOH-50 mm Hepes buffer (80:20 v/v) at pH 7.4 and
258C in the presence of free Cu2+ (25 mm) (blue trace), and Cu2+ (25 mm)
and Ab16 (50 mm) (red trace).
added (Figure S4, Supporting Information). Also in this case,
the spectral profiles during the reactions are quite different in
Hepes buffer versus methanol/Hepes buffer mixture. The pres-
ence of alcohol, in fact, favors the accumulation of MQ (ab-
sorption band at 401 nm, Figure S4B, Supporting Information),
preventing the subsequent reactions observed in aqueous
buffer (Figure S4C, Supporting Information). These results sug-
gest that methanol may stabilize the copper(I) redox state in
the presence of Ab16. At the same time, a less polar solvent
disfavors deprotonation of the catechol hydroxyl group and
change of the redox potential of the species involved. The
former effect prevents coordination of DTBC or MC to the [Cu+
ꢀAb16] intermediate, shifting to the left the pre-equilibrium re-
action in Equation (3) above. In addition, an increase of the
substrate redox potential will make substrate oxidation more
difficult. The spectra recorded over reaction time in a metha-
nol/buffer mixture (Figures S1B and S4B, Supporting Informa-
tion) compared to the same situation in the absence of metha-
nol (Figures S1C and S4C, Supporting Information) seem to
further confirm this hypothesis, because the reduced deproto-
nation of catechol in MeOH inhibits the nucleophilic attack to
the quinone formed and thus the formation of condensation
products is easily detectable in aqueous solvent.
To identify the metal-catalyzed oxidation of Cu-Ab in the re-
ductive environment generated during catechol oxidation, we
used LCMS analysis. Although DA is the most relevant among
the catecholic substrates in this study, the LCMS analysis of the
fragmentation pattern of modified peptides is very complicat-
ed due to the number of products generated by DA oxidation
and the resulting adducts with Ab. We therefore performed
the analysis of Ab modifications produced upon MC oxidation,
following a protocol developed for the analysis of copper–a-
synuclein peptide complexes.[26]
In these experiments, a solution of copper and Ab peptides
was incubated in the presence of MC under the same condi-
tions as in the catalytic oxidations ([Ab16]=75 mm; [Cu2+]=
25 mm; MC=3 mm; in 50 mm Hepes buffer pH 7.4). After
90 min of incubation, only about half of the peptide remained
unmodified (peak with tR =21 min), whereas the remaining
part underwent a series of modifications to products with
higher retention times (Figure 4).
In the case of DA, the catechol deprotonation is favored by
the positive charge of the protonated amine group in the side
chain at physiological pH (pKa ꢁ9).[37] Therefore, coordination
of DA to [Cu+ꢀAb16] is favored. In addition, the oxidation of
DA in MeOH is faster than in aqueous buffer, also because the
concentration of dioxygen in the MeOH containing solvent in-
creases (from 240 mm in aqueous buffer to 320 mm in the
mixed solvent). In conclusion, the peptide stabilizes mostly the
copper(I) form and the oxidation of the substrate greatly de-
pends upon the conditions of the reaction.
In order to identify the oxidative modifications of Ab16, we
have to consider that aromatic amino acids are among the pre-
ferred targets for ROS attack.[41] In previous studies, it was re-
ported that [Cu2+ꢀAb]/ascorbate/O2 or [Cu2+ꢀAb]/H2O2 sys-
tems can induce histidine oxidation.[20,42–44] In our experiments,
the most abundant modification (about 32%, see Table 1) cor-
responds to the peak with tr =23.5 min. The a, b (N-terminal
fragments obtained upon peptide fragmentation at CHꢀCO
and COꢀNH bonds, respectively), and y (C-terminal fragments
obtained upon peptide fragmentation at COꢀNH bonds) ion
series observed in the MS/MS spectra indicate that either one
His residue or Phe4 undergo oxygen insertion (Ab16+16), and
The absorbance changes with time at 500 nm observed in
the previous study using MBTH[21] show a biphasic behavior,
with an initial fast phase followed by a slower phase, with
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completely exclude oxidation of Tyr10. The formal addition of
one oxygen atom deduced from MS data most likely corre-
sponds to conversion of His to 2-oxohistidine (Scheme S1, Sup-
porting Information)[45] or Phe to ortho-tyrosine,[40] respectively.
It is not possible to discern which His residue (6, 13, or 14) is
involved in the oxidation, but other studies report that oxida-
tion at the His6 residue was not detected in the fragments of
Ab peptides resulting from digestion with trypsin.[20] A mass in-
crement of 32 Da was detected for the peak at 26.3 min
(Ab16+32) that corresponds to a modification of about 7% of
the total peptide after 90 min of reaction time. MS/MS spectra
indicate that this peak likely corresponds to simultaneous oxi-
dation of Phe4 and one histidine residue (His13 or His14).
The peak at 24.4 min corresponds to the addition of MQ to
one His residue (mass increment of 122 Da; Ab16-MC); the
fragmentation spectra indicate that the involved residue is
probably His6.
This result is in agreement with the previous evidence that
His6 is less prone to oxidation, whereas it is the main target of
the reaction with quinones giving rise to Ab16-MC adduct. The
resulting peptide–catechol adduct is further oxidized by Cu2+
,
thus explaining the peak at 29.6 min (Ab16-MQ) with a 120 Da
mass increment with respect to the starting peptide
(Scheme S2, Supporting Information), the fragmentation spec-
tra of which corroborate the selective derivatization at His6.
Table 1 reports the variation over time of the percent modifica-
tion of the native peptide.
As expected, the overall amount of oxidized peptide increas-
es with time. The single oxidized derivative (Ab16+16) reaches
its maximum amount after 90 min, then this species is further
oxidized forming Ab16+32 species. In addition, the Ab16–MC
species is appreciably detected after 90 min, and then it is
partly converted to Ab16–MQ. As a blank experiment, Ab16
was incubated in the presence of copper(II) but in the absence
of MC; under these conditions, the peptide is not oxidized
even after 4 h. However, a further control experiment interest-
ingly shows that the incubation of Ab16 with MC alone (Fig-
ure 5A) results in the development of the peaks corresponding
to Ab16-MC and Ab16-MQ species (peaks at 25 and 28.3 min,
respectively), albeit to lower extent with respect to the experi-
ments with copper(II). Again, the formation of a covalent
adduct between Ab and MC or MQ is more favorable at His6.
At longer incubation time a peak at 25.7 min clearly appears,
corresponding to an Ab16–MC species, in which addition of
MQ occurs on His6 residue, and a further double oxidation
occurs at Phe4 and either His13 or His14 (Ab16-MC+32;
154 Da mass increment) (Figure 5B). It is also possible to
detect a minor peak at 23 min, assignable to a singly oxidized
Ab16 species (Ab16+16), the low intensity and proximity of
which to the main Ab16 peak preclude its quantification.
These results indicate that catechol autoxidation in the pres-
ence of atmospheric oxygen at physiological pH can induce
a slow Ab16 oxidation even with the small impurities of redox
active metals present in reagents and solvents. The depend-
ence over time of these modifications is summarized in
Table 2.
Figure 4. HPLC-MS elution profiles of Ab16 (75 mm) in Hepes buffer (50 mm)
pH 7.4 at 258C, in the presence of copper(II) (25 mm) and MC (3 mm) at the
beginning (A), and after 90 min incubation time (B). The assignment of the
peaks is shown.
Table 1. Modification of Ab16 peptide (75 mm) detected by LCMS analy-
sis, in the presence of 25 mm copper(II) nitrate and 3 mm MC in Hepes
buffer (50 mm) pH 7.4 at 258C.
Incubation t
Ab16
Ab16+16 Ab16–MC Ab16+32 Ab16–MQ
[min]
[%]
[%]
[%]
[%]
[%]
0
90
180
270
360
100
45
35
24
16
0
32
27
25
24
0
11
7
11
4
0
5
17
25
31
0
7
14
15
25
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It is worth noting that, when the same process is studied in
a MeOH/Hepes buffer mixture, the modifications on Ab16
occur to a lower extent. LCMS analysis of the reaction mixture
containing Cu2+, Ab16, and MC showed the presence of four
main peaks in different proportions, corresponding to modifi-
cation of 20% of Ab16 after 90 min, which is half of that ob-
tained in aqueous buffer (Figure S5, Supporting Information).
In particular, three principal peptide modifications were ob-
served, which are characterized by mass changes of +16, ꢀ45,
and +120 Da. MS/MS fragmentation spectra allowed the oxi-
dation of one His or Phe4 residue (Ab16+16), the decarboxyla-
tion/deamination of Asp1 (Ab16-45), and the addition of MQ
to one His residue (probably His6) with subsequent oxidation
(Ab16–MQ), respectively, to be identified. In particular, the
peak at 25.6 min, corresponding to the decarboxylation/deami-
nation of Asp1 to pyruvate, is a modification that was previ-
ously detected also on Ab fragments of human and mouse b-
amyloid.[24,44] The proposed mechanism for Asp1 oxidative
cleavage, as reported in Scheme S3, Supporting Information,
proceeds through an alkoxyl-radical pathway,[41] making the
carbon atom at the a-position of the amino acid residue sensi-
tive to attack by ROS. Table S1, Supporting Information, reports
the percentages of peptide modification in MeOH/Hepes mix-
ture over time. Under these conditions, peptide modifications
due to the simple MC autoxidation in the absence of Cu2+ are
negligible for incubation times lower than 8 h.
The modification of Ab28 in Hepes buffer was also analyzed
in order to compare its behavior with that of Ab16 under the
same reaction conditions. After 90 min incubation of Ab28
with MC alone (Figure 6A), both Ab28 and the 1DAEFRHD7
fragment were detected with a mass increment of 120 Da
(Ab28–MQ, tR =40.2 min; 6%, and 1DAEFRHD7-MQ, tR =
33.5 min; 3%, respectively) corresponding to the covalent link-
ing of MQ to one His residue. Interestingly, the latter result
confirms beyond any doubt that the addition of MQ occurs se-
lectively at the His6 residue, as previously assumed also for
Ab16. In the same analysis other peptide fragments were iden-
tified, resulting from the spontaneous hydrolysis of the native
peptide at physiological pH: 19FFAEDVGSNK28 (tR =30.7 min;
Figure 5. HPLC-MS elution profiles of Ab16 (75 mm) in Hepes buffer (50 mm)
pH 7.4 at 258C in the presence of MC after A) 90 min and B) 450 min reac-
tion time. The assignment of the peaks is shown.
1%),
15QKLVFFAEDVGSNK28
(tR =38.3 min;
3%),
and
17LVFFAEDVGSNK28 (tR =41.4 min; 4%).
When the peptide was incubated for 90 min in the presence
of copper(II) and MC (Figure 6B), more products of peptide hy-
drolysis were observed (Table 3). In this case, it was possible to
identify a peak at 42.9 min linked to the oxidation of the
native peptide at Phe residues 19 or 20 that also reports the
covalent Ab28-MQ adduct. The ion fragmentation analysis is
compatible with MQ insertion into His6, a result also support-
ed by the most abundant modification detected in the peak at
Table 2. Modification of Ab16 peptide (75 mm) detected by LCMS analy-
sis, in the presence of 3 mm MC in Hepes buffer (50 mm) pH 7.4 at 258C.
Incubation t
Ab16
Ab16–MC
Ab16–MC +32
Ab16–MQ
[min]
[%]
[%]
[%]
[%]
1
33.5 min (16%), which corresponds to the DAEFRHD7-MQ spe-
0
90
100
83
76
73
74
71
67
64
0
4
6
7
8
10
11
12
0
0
3
5
6
8
11
14
0
13
15
15
12
11
11
10
cies, as mentioned before.
Additionally, it was possible to identify a peptide fragment
at 47.6 min with a mass increase of +64 Da, in which His13,
His14, Phe19, and Phe20 residues were oxidized at the same
time to 2-oxohistidine and to ortho-tyrosine, respectively, in
agreement with the results obtained by Cassagnes et al.[20]
180
270
360
450
540
630
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Regarding the possible oxidation of other amino acid resi-
dues, Berlett et al. reported that oxidation of the Tyr residue
can form 3,4-dihydroxyphenylalanine,[41] with a 16 Da mass in-
crement with respect to the peptide, but under our conditions
we can exclude this amino acid oxidation in Ab28 (as above re-
ported for Ab16). Furthermore, although there is evidence sup-
porting metal-catalyzed damage to proteins with insertion of
an oxygen atom at arginine and lysine residues,[46] MS/MS
spectra analysis for the Ab16 and Ab28 fragments exclude
with high probability the occurrence of such modifications.
It is apparent, therefore, that the principal targets of oxida-
tive modifications are phenylalanine and histidine residues. In
particular, His6, which is involved in Cu2+ coordination but not
in Cu+ binding, is the main target for quinone addition. This
process does not appear to be strictly copper-dependent,
since it is also observed in the modification process derived
from catechol autoxidation. On the other hand, ROS generated
by Fenton’s reactivity of Cu+–Ab complexes preferentially
react with the imidazole rings that are directly bound to the
metal site, that is, His13 and His14. Also Asp1, Phe4, Phe19,
and Phe20 are targets of MCO, probably for their proximity to
the metal site (Asp1) or chemical propensity to undergo oxida-
tive reactions. In addition, under the experimental conditions
described here, no evidence for the formation of Tyr–Tyr cross-
linking has been found. This product was previously observed
when Ab peptide was incubated in the presence of H2O2 and
copper,[47] or heme.[48] Furthermore, LCMS analysis reveals that
Ab28 peptide is more prone to hydrolysis when compared to
Ab16. A more detailed analysis regarding hydrolysis of Ab28
and the role of metal ions in controlling this type of modifica-
tion will be reported separately.
It is important to understand to which extent the oxidation
of catechol competes or affects endogenous peptide modifica-
tion processes. To this end, the reaction mixtures resulting
from MC oxidation under the same conditions described
above were analyzed by HPLC. In particular, in parallel experi-
ments, solutions of MC (3 mm) in 5 mm Hepes buffer pH 7.4
were incubated for 90 min under the following conditions:
a) MC alone (autoxidation); b) MC and copper(II) (25 mm);
c) MC, copper(II) (25 mm), and Ab16 (75 mm); d) MC, copper(II)
(25 mm), and Ab28 (75 mm). The analytical separation by HPLC
of the reaction mixtures was performed after mixing and after
90 min incubation, using p-chlorophenol (3 mm retention time
9.3 min) as an internal standard (added just before injection in
the HPLC).
Figure 6. HPLC-MS elution profiles of Ab28 (75 mm) incubated in the pres-
ence of A) MC, B) copper(II) and MC, after 90 min in Hepes buffer (50 mm)
pH 7.4 at 258C. The assignment of the peaks for A is shown, whereas for B
they are reported in Table 3.
Table 3. Modification of Ab28 peptide (75 mm) detected by LCMS analy-
sis, in the presence of 3 mm MC and 25 mm Cu2+ after 90 min in Hepes
buffer (50 mm) pH 7.4 at 258C.
The HPLC profiles are shown in Figures S6, S7, and S8, Sup-
porting Information. The progress of MC oxidation with time is
shown by the decrease in intensity of the peak at 5.9 min. The
amount of MC oxidized by copper(II) or copper(II)/peptides
could be obtained after subtraction of the contribution of MC
autoxidation. After 90 min, a decrease in [MC] of 0.36, 0.47,
and 0.55 mm was observed with free copper(II), CuAb16, and
CuAb28, respectively. These data are in agreement with the re-
sults reported in Figure 2, showing that Ab peptides increase
the reactivity of copper(II) in the oxidation of MC and that the
effect of Ab28 is slightly higher with respect to that exerted by
Ab16. Moreover, the HPLC profiles shown in Figures S6 to S8,
tR [min]
Species
Amount [%]
28.7
30.9
33.5
34.4
35.2
36.9
38.3
40.6
41.6
42.9
47.6
1DAEFRHDSGYEV12
19FFAEDVGSNK28
1DAEFRHD7-MQ
11EVHHQKLVFFAEDVGSNK28
18VFFAEDVGSNK28
Ab28
2
2
16
3
4
42
11
2
10
3
15QKLVFFAEDVGSNK28
Ab28-MQ
17LVFFAEDVGSNK28
Ab28-MQ+16
8SGYEVHHQKLVFF20 +64
6
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Supporting Information, are in agreement with the formation
of MQ and of addition products of MC (peaks at 7.2 and
7.6 min retention time, respectively), as indicated in Equa-
tion (7).
physiologic conditions, also considering the larger size of Ab
isoforms present in vivo (39 to 43 residues).
Moreover, we noticed that formation of catechol–Ab cova-
lent adducts is not negligible in the absence of copper. This
behavior is due to catechol autoxidation in solution that gen-
erates reactive quinone species yielding addition products
with Ab nucleophilic residues. This behavior could be observed
clearly only for MC, but it is probably present also for DA, that
is a more relevant substrate for this kind of reaction. The situa-
tion in the presence of copper is different, since more oxida-
tive reaction pathways are promoted. In this case, the forma-
tion of covalent Ab–catechol adducts occurs to a lower extent,
while peptide oxidation with insertion of oxygen atoms at His
and Phe residues becomes more relevant.
The data obtained in these experiments can be compared
with those obtained in the HPLC-MS/MS analysis (Figures 4B
and 5B and Tables 1 and 3). After 90 min incubation time in
the presence of copper(II) and MC, 55% of Ab16 (75 mm) un-
dergoes modification with concomitant consumption of
0.47 mm MC, indicating approximately a ratio of one molecule
peptide modified every 10 molecules MC reacted. It should be
noted that, according to the mechanism proposed, the reac-
tion proceeds with the formation of MC·+ and subsequent dis-
proportionation to MC and MQ in solution. Then MQ under-
goes addition by the nucleophiles in solution. Considering that
a relevant fraction of the peptide is modified by the addition
of MC/MQ, even if the MC is in excess, the data indicate that
part of the reactive species formed during catalysis has the en-
dogenous peptide as the target. Of particular relevance is the
observation of oxidative modification of Ab16 at His and Phe
residues, as this reaction requires strongly reactive species.
Thus, the easily oxidized MC substrate does not act as an effi-
cient scavenger of the reactive species formed at the copper
center and responsible for oxidative modification of the bound
peptide.
The comparison of the efficiency in catechol oxidation
versus Ab modification indicates that the oxidative reactivity
towards the peptide is relevant, showing that Cu–Ab com-
plexes may have an important role in vivo in radical scaveng-
ing. We can therefore deduce a dual role for Ab peptides: on
one hand the formation of Cu–Ab complex exacerbates the Cu
ability to promote oxidation of external substrates, and on the
other hand, in this oxidative environment, Ab peptides bear
a strong tendency to undergo oxidation by Cu redox cycling.
The latter aspect is in agreement with the hypothesis that Ab
peptides act as radical scavengers.[23]
A similar conclusion can be drawn for Ab28. In this case,
after 90 min incubation time in the presence of copper(II)
(25 mm) and MC (3 mm), 58% of Ab28 (75 mm) undergoes
modification with the concomitant consumption of 0.55 mm
MC. These data confirm that CuAb28 is slightly more efficient
than CuAb16 in MC oxidation.
Experimental Section
Materials and instrumentation
Protected amino acids, rink amide resin, and other reagents for
peptide synthesis, were purchased from Novabiochem. All other
chemicals were reagent grade from Sigma–Aldrich. Peptide purifi-
cations were performed on a Jasco HPLC instrument equipped
with two PU-1580 pumps and a MD-1510 diode array detector
(working range: 195–659 nm), using a Phenomenex Jupiter 4U
Proteo semipreparative column (4 mm, 250ꢁ10 mm). Mass spectra
and LCMS/MS data were obtained with a LCQ ADV MAX ion-trap
mass spectrometer, with an ESI ion source. The system was run in
automated LCMS/MS mode and using a surveyor HPLC system
(Thermo Finnigan, San Jose, CA, USA) equipped with a Phenomen-
ex Jupiter 4u Proteo column (4 mm, 150ꢁ2.0 mm). MS/MS experi-
ments by collision-induced dissociation (CID) were performed with
an isolation width of 2Th (m/z); the activation amplitude was
around 35% of the ejection radiofrequency (RF) amplitude of the
instrument. ESI-MS direct injection experiments were performed
using a positive or negative ion mode, capillary temperature at
2008C and MeOH as the eluent. For the analysis of peptide frag-
ments, Bioworks 3.1 and Xcalibur 2.0.7 SP1 software were used
(Thermo Finnigan, San Jose, CA (USA)). UV/Vis spectra and kinetic
experiments were recorded on an Agilent 8453 diode array spec-
trophotometer, equipped with a thermostated, magnetically stirred
optical cell. In the case of MC consumption, HPLC analysis was per-
formed on a 1260 Infinity Agilent system using a XSelectꢂ HSS C18
analytical column (2,5 mm, 4,6ꢁ50 mm).
Conclusion
In this study, we have characterized two important aspects of
the oxidative reactivity of copper–Ab peptide complexes: the
oxidation of exogenous catecholic substrates and the endoge-
nous modifications induced by this reactivity on bound Ab. Al-
though this type of reactivity was considered in previous stud-
ies, this is the first time where the two effects have been evalu-
ated simultaneously and quantitatively.
Both copper complexes with Ab16 and Ab28 peptides in-
crease the rate of the catalytic oxidation of catecholic sub-
strates, such as the neurotransmitter DA and its biomimetic an-
alogues MC and DTBC. This work integrates previous data
from our group[21] in which the catecholase reactivity was in-
vestigated in the presence of MBTH, which is indeed redox
noninnocent and efficiently competes with the substrate in
copper(II) reduction. From these data, it is evident that the ex-
perimental conditions and the choice of the substrate are cru-
cial to reproduce more likely physiologic conditions.
A different situation was revealed by LCMS analysis of the
modifications undergone by the two Ab peptides. Compared
to Ab16, which principally undergoes oxidative modification,
Ab28 is also prone to hydrolysis processes. These data suggest
that hydrolysis of Ab peptides may play an important role in
Peptide synthesis
Ab16
(1DAEFRHDSGYEVHHNK16-NH2)
and
Ab28
(1DAEFRHDSGYEVHHNKLVFFAEDVGSNK28-NH2) peptides were syn-
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thesized using the standard fluorenyl methoxycarbonyl (Fmoc)
solid-phase synthesis in DMF. Rink-amide resin was used as a solid
support, which yielded the peptides amidated at the C-terminus.
For Ab28 peptide, a low loading Rink-amide resin (substitution
0.36 mmolgꢀ1) was used. After deprotection of the resin with
20 mL of 20% (v/v) piperidine in DMF, the first amino acid (3 mol e-
quiv vs. resin sites), was added in the presence of 3 equiv of N-hy-
droxybenzotriazole, 3 equiv of benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate, and 6 equiv of N,N-diisopro-
pylethylamine. After 45 min, the same coupling procedure was re-
peated. After recoupling of each amino acid, a capping step was
performed by using 20 mL of 4.7% acetic anhydride and 4% of
pyridine in DMF. Deprotection of the Fmoc group was performed
by treating the resin twice, for 3 and 7 min, respectively, with
15 mL of 20% piperidine in DMF. At the end of the synthesis, the
protections of the side chains of the amino acids were removed
with a solution of 95% trifluoroacetic acid (TFA, 25 mL for 1 g of
resin), triisopropyl silane (2.5%), and water (2.5%), which serves
also to release the peptide from the resin. After stirring for 3 h, the
solution was concentrated under vacuum and cold diethyl ether
was added to precipitate the peptide. The mixture was filtered and
the precipitate washed with cold diethyl ether; then, it was dis-
solved in water and purified by HPLC, using a 0–100% linear gradi-
ent of 0.1% TFA in water to 0.1% TFA in CH3CN over 40 min (flow
rate of 3 mLminꢀ1, loop 2 mL), as the eluent. The product was
then lyophilized, yielding a white solid, which was characterized by
ESI-MS. ESI-MS data (direct injection, MeOH, positive-ion mode, ca-
pillary temperature 2008C): m/z: 1955 (Ab16H+), 978 (Ab16H22+),
652 (Ab16H33+), 489.5 a.m.u (Ab16H44+); 1631 (Ab28H22+), 1088
(Ab28H33+), 816 (Ab28H44+), 653 a.m.u (Ab28H55+).
c) 3,5-Di-tert-butylcatechol oxidation: The catalytic oxidation of
DTBC by Cu2+ was studied at room temperature (258C)), in
a mixed solvent of 80% methanol and 20% 50 mm aqueous Hepes
buffer at pH 7.4 (v/v), saturated with atmospheric oxygen. The re-
action was monitored by UV/Vis spectroscopy through the absorp-
tion band of 3,5-di-tert-butylquinone at 407 nm (e=
1500mꢀ1 cmꢀ1).[26] The experiment was performed by adding cop-
per(II) nitrate (25 mm) to the solution containing DTBC (3 mm).
DTBC autoxidation is negligible under these conditions. In the ex-
periment with copper(II)–Ab complex, Ab16 (50 mm) was added to
a solution of DTBC (3 mm), followed by copper(II) nitrate (25 mm).
The measurements were performed in triplicate.
Determination of the [O2] in the reaction media
The oxygen concentration in 50 mm aqueous Hepes buffer at
pH 7.4 and in the mixed solvent of 80% methanol and 20%
50 mm aqueous Hepes buffer at pH 7.4 (v/v), saturated with atmos-
pheric oxygen at 258C were determined with the classical Winkler
method.[49]
Identification and characterization of oxidized peptides by
HPLC-ESI-MS
Peptide modification was analyzed by HPLC-ESI-MS, performing ex-
periments under the same conditions used for MC oxidation stud-
ies. Samples were prepared with copper(II) nitrate (25 mm), Ab16
(75 mm) and MC (3 mm) in Hepes buffer (50 mm) pH 7.4 at different
reaction times: 0, 90, 180, 270, 360 min. The experiment was re-
peated with MC 3 mm and Ab16 (75 mm) in Hepes buffer (50 mm)
pH 7.4, but in absence of copper, at the following reaction times:
0, 90, 180, 270, 360, 450, 540, 630 min. The effect of methanol as
a co-solvent was evaluated by performing the analysis in the pres-
ence of copper(II) nitrate (25 mm), Ab16 (75 mm) and MC (3 mm) in
80:20 (v/v) MeOH/HEPES buffer (50 mm) at different reaction times:
0, 90, 180, 270, 360 min. In the case of Ab28, samples were pre-
pared in the presence of copper(II) nitrate (25 mm), Ab28 (75 mm),
and MC (3 mm) in Hepes buffer (50 mm) pH 7.4 and analyzed after
90 min reaction time. LCMS and LCMS/MS data were obtained by
using the LCQ ADV MAX ion-trap mass spectrometer, as described
in the Materials and instrumentation Section. The elution was per-
formed by using 0.1% HCOOH in distilled water (solvent A) and
0.1% HCOOH in acetonitrile (solvent B), with a flow rate of
0.2 mLminꢀ1; elution started with 98% solvent A for 5 min fol-
lowed by a linear gradient from 98 to 55% A in 65 min.
Kinetics of oxidation of catecholic substrates
a) Dopamine oxidation: The catalytic oxidation of DA by Cu2+ was
studied at room temperature (258C) in 50 mm Hepes buffer at
pH 7.4, saturated with atmospheric oxygen. The reaction was
monitored by UV/Vis spectroscopy through the absorption band of
dopaminochrome at 475 nm.[33] Firstly, a DA (3 mm) autoxidation
experiment was evaluated; the kinetic trace obtained in the autoxi-
dation experiment was subsequently subtracted from the kinetic
profile of the reactions catalyzed by copper(II) and copper(II)–pep-
tide complexes. In the former case, the experiments were carried
out by adding copper(II) nitrate (25 mm) to the solution containing
DA (3 mm). In the experiments with copper–peptide complexes,
Ab16 or Ab28 were added at 25–100 mm concentrations, to a solu-
tion of DA (3 mm), followed by copper(II) nitrate (25 mm) as the last
reagent. All measurements were performed at least in triplicate. In
order to observe the dioxygen concentration effect on DA oxida-
tion, the above-mentioned experiments were repeated saturating
the solutions with pure molecular oxygen at 1 atm., instead of at-
mospheric oxygen.
HPLC quantification of oxidized catechol
b) 4-Methylcatechol oxidation: The catalytic oxidation of MC by
Cu2+ was studied at room temperature (258C) in 50 mm Hepes
buffer at pH 7.4, saturated with atmospheric oxygen. The reaction
was monitored by UV/vis spectroscopy through the absorption
band of 4-methylquinone at 401 nm (e=1550mꢀ1 cmꢀ1),[26] which
subsequently undergoes a shift to 480 nm due to the addition re-
action to MQ by excess MC. Firstly, MC (3 mm) autoxidation was
evaluated, and the corresponding kinetic trace was subsequently
subtracted from the kinetic profile of the catalytic reactions. The
latter reactions with free copper(II) and with copper(II)–Ab com-
plexes were performed under the same conditions used for DA oxi-
dation. All kinetic experiments were performed at least in triplicate.
The consumption of MC was monitored by recording HPLC chro-
matograms (95–50% linear gradient of 0.1% TFA in water using
0.1% TFA in CH3CN as an organic solvent over 12 min; flow rate of
1.4 mLminꢀ1; loop 10 mL; lobs =256 nm) carrying out experiments
under the same conditions used for MC oxidation studies and
HPLC-ESI/MS analysis. A stock solution of MC in milliQ water was
diluted in 5 mm Hepes buffer at pH 7.4 to 3 mm concentration and
analyzed at 0 time and after 90 min of incubation. p-Chlorophenol
(3 mm) was used as an internal standard, and it was added just
before the HPLC injection. The same profiles were recorded with
MC (3 mm) and Ab16 or Ab28 (75 mm) in the absence and presence
of copper nitrate (25 mm) in 5 mm Hepes buffer at pH 7.4.
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[24] R. C. Nadal, S. E. Rigby, J. H. Viles, Biochemistry 2008, 47, 11653–11664.
[25] J. N. Rodrꢅguez-Lꢆpez, J. Escribano, F. Garcꢅa-Cꢇnovas, Anal. Biochem.
1994, 216, 205–212.
[26] S. Dell’Acqua, V. Pirota, C. Anzani, M. M. Rocco, S. Nicolis, D. Valensin, E.
Monzani, L. Casella, Metallomics 2015, 7, 1091–1102.
[27] L. Zecca, C. Bellei, P. Costi, A. Albertini, E. Monzani, L. Casella, M. Gallori-
ni, L. Bergamaschi, A. Moscatelli, N. J. Turro, M. Eisner, P. R. Crippa, S. Ito,
K. Wakamatsu, W. D. Bush, W. C. Ward, J. D. Simon, F. A. Zucca, Proc.
Natl. Acad. Sci. USA 2008, 105, 17567–17572.
Acknowledgements
This work was supported by the Italian MIUR, through the
PRIN (Programmi di Ricerca di Rilevante Interesse Nazionale)
project 2015T778JW. The COST CM1003 Action and CIRCMSB
are also gratefully acknowledged.
[28] A. Napolitano, P. Manini, M. d’Ischia, Curr. Med. Chem. 2011, 18, 1832–
1845.
[29] A. N. Pham, T. D. Waite, J. Inorg. Biochem. 2014, 137, 74–84.
[30] D. A. Butterfield, T. Reed, S. F. Newman, R. Sultana, Free Radical Biol.
Med. 2007, 43, 658–677.
Keywords: Alzheimer’s disease
complexes · dopamine · oxidation
· Ab peptides · copper
[1] a) C. Haass, D. J. Selkoe, Nat. Rev. Mol. Cell. Biol. 2007, 8, 101–112; b) M.
Jucker, L. C. Walker, Nature 2013, 501, 45–51.
[2] D. J. Selkoe, Nature 1999, 399, A23–31.
[31] S. Chassaing, F. Collin, P. Dorlet, J. Gout, C. Hureau, P. Faller, Curr. Top.
Med. Chem. 2012, 12, 2573–2595.
[3] J. Hardy, D. J. Selkoe, Science 2002, 297, 353–356.
[4] D. J. Selkoe, Physiol. Rev. 2001, 81, 741–766.
[5] W. L. Klein, W. B. Stine Jr., D. B. Teplow, Neurobiol. Aging 2004, 25, 569–
580.
[32] A. M. Swomley, S. Fçrster, J. T. Keeney, J. Triplett, Z. Zhang, R. Sultana,
D. A. Butterfield, Biochim. Biophys. Acta - Molecular Basis of Disease
2014, 1842, 1248–1257.
[33] E. Herlinger, R. F. Jameson, W. Linert, J. Chem. Soc. Perkin Trans. 2 1995,
259–263.
[6] K. P. Kepp, Chem. Rev. 2012, 112, 5193–5239.
[7] M. A. Lovell, J. D. Robertson, W. J. Teesdale, J. L. Campbell, W. R. Mar-
kesbery, J. Neurol. Sci. 1998, 158, 47–52.
[34] S. Dell’Acqua, V. Pirota, E. Monzani, F. Camponeschi, R. De Ricco, D. Va-
lensin, L. Casella, Inorg. Chem. 2016, 55, 6100–6106.
[8] D. G. Smith, R. Cappai, K. J. Barnham, Biochim. Biophys. Acta Biomembr.
2007, 1768, 1976–1990.
[35] L. Guilloreau, S. Combalbert, A. Sournia-Saquet, H. Mazarguil, P. Faller,
ChemBioChem 2007, 8, 1317–1325.
[9] X. Huang, C. S. Atwood, R. D. Moir, M. A. Hartshorn, R. E. Tanzi, A. I.
Bush, J. Biol. Inorg. Chem. 2004, 9, 954–960.
[36] G. Battaini, A. Granata, E. Monzani, M. Gullotti, L. Casella, Adv. Inorg.
Chem. 2006, 58, 185–233.
[10] V. Tꢃugu, A. Tiiman, P. Palumaa, Metallomics 2011, 3, 250–261.
[11] A. I. Bush, R. E. Tanzi, Neurotherapeutics 2008, 5, 421–432.
[12] C. J. Sarell, C. D. Syme, S. E. Rigby, J. H. Viles, Biochemistry 2009, 48,
4388–4402.
[13] I. V. Murray, L. Liu, H. Komatsu, K. Uryu, G. Xiao, J. A. Lawson, P. H. Axel-
sen, J. Biol. Chem. 2007, 282, 9335–9345.
[37] S. Corona-AvendaÇo, G. Alarcꢆn-Angeles, G. A. Rosquete-Pina, A. Rojas-
Hernꢇndez, A. Gutierrez, M. T. Ramꢅrez-Silva, M. Romero-Romo, M. Palo-
mar-Pardavꢄ, J. Phys. Chem. B 2007, 111, 1640–1647.
[38] E. I. Solomon, D. E. Heppner, E. M. Johnston, J. W. Ginsbach, J. Cirera, M.
Qayyum, M. T. Kieber-Emmons, C. H. Kjaergaard, R. G. Hadt, L. Tian,
Chem. Rev. 2014, 114, 3659–3853.
[14] P. Faller, C. Hureau, Dalton Trans. 2009, 1080–1094.
[15] C. Hureau, P. Dorlet, Coord. Chem. Rev. 2012, 256, 2175–2187.
[16] R. De Ricco, D. Valensin, S. Dell’Acqua, L. Casella, C. Hureau, P. Faller,
ChemBioChem 2015, 16, 2319–2328.
[17] R. A. Himes, G. Y. Park, G. S. Siluvai, N. J. Blackburn, K. D. Karlin, Angew.
Chem. Int. Ed. 2008, 47, 9084–9087; Angew. Chem. 2008, 120, 9224–
9227.
[39] C. L. Hawkins, M. J. Davies, Biochim. Biophys. Acta Bioenerg. 2001, 1504,
196–219.
[40] C. Hureau, P. Faller, Biochimie 2009, 91, 1212–1217.
[41] B. S. Berlett, E. R. Stadtman, J. Biol. Chem. 1997, 272, 20313–20316.
[42] C. Schçneich, T. D. Williams, Chem. Res. Toxicol. 2002, 15, 717–722.
[43] K. Inoue, C. Garner, B. L. Ackermann, T. Oe, I. A. Blair, Rapid Commun.
Mass Spectrom. 2006, 20, 911–918.
[18] a) G. F. da Silva, L. J. Ming, Angew. Chem. Int. Ed. 2005, 44, 5501–5504;
Angew. Chem. 2005, 117, 5637–5640; b) C. Opazo, X. Huang, R. A.
Cherny, R. D. Moir, A. E. Roher, A. R. White, R. Cappai, C. L. Masters, R. E.
Tanzi, N. C. Inestrosa, A. I. Bush, J. Biol. Chem. 2002, 277, 40302–40308;
c) K. Reybier, S. Ayala, B. Alies, J. V. Rodrigues, S. Bustos Rodriguez, G. La
Penna, F. Collin, C. M. Gomes, C. Hureau, P. Faller, Angew. Chem. Int. Ed.
2016, 55, 1085–1089; Angew. Chem. 2016, 128, 1097–1101.
[19] G. F. Z. da Silva, L. J. Ming, Angew. Chem. Int. Ed. 2007, 46, 3337–3341;
Angew. Chem. 2007, 119, 3401–3405.
´
[44] T. Kowalik-Jankowska, M. Ruta, K. Wisniewska, L. Łankiewicz, M. Dyba, J.
Inorg. Biochem. 2004, 98, 940–950.
[45] T. G. Huggins, M. C. Wells-Knecht, N. A. Detorie, J. W. Baynes, S. R.
Thorpe, J. Biol. Chem. 1993, 268, 12341–12347.
[46] J. R. Requena, C. C. Chao, R. L. Levine, E. R. Stadtman, Proc. Natl. Acad.
Sci. USA 2001, 98, 69–74.
[47] C. S. Atwood, G. Perry, H. Zeng, Y. Kato, W. D. Jones, K.-Q. Ling, X.
Huang, R. D. Moir, D. Wang, L. M. Sayre, M. A. Smith, S. G. Chen, A. I.
Bush, Biochemistry 2004, 43, 560–568.
[20] L.-E. Cassagnes, V. Hervꢄ, F. Nepveu, C. Hureau, P. Faller, F. Collin, Angew.
Chem. Int. Ed. 2013, 52, 11110 – 11113 ; Angew. Chem. 2013, 125, 11316–
11319.
[48] G. Thiabaud, S. Pizzocaro, R. Garcia-Serres, J.-M. Latour, E. Monzani, L.
Casella, Angew. Chem. Int. Ed. 2013, 52, 8041–8044; Angew. Chem.
2013, 125, 8199–8202.
[21] D. Ciregna, E. Monzani, G. Thiabaud, S. Pizzocaro, L. Casella, Chem.
Commun. 2013, 49, 4027–4029.
[49] L. W. Winkler, Ber. Dtsch. Chem. Ges. 1888, 21, 2843–2846.
[22] R. Baruch-Suchodolsky, B. Fischer, Biochemistry 2009, 48, 4354–4370.
[23] J. T. Pedersen, S. W. Chen, C. B. Borg, S. Ness, J. M. Bahl, N. H. Heegaard,
C. M. Dobson, L. Hemmingsen, N. Cremades, K. Teilum, J. Am. Chem.
Soc. 2016, 138, 3966–3969.
Received: August 10, 2016
Published online on && &&, 0000
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Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
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ÝÝ These are not the final page numbers!

Full Paper
FULL PAPER
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Medicinal Chemistry
V. Pirota, S. Dell’Acqua, E. Monzani,
S. Nicolis, L. Casella*
&& – &&
Copper-Ab Peptides and Oxidation of
Catecholic Substrates: Reactivity and
Endogenous Peptide Damage
Competitive oxidative experiments
with exogenous catechols show that
copper complexes with both Ab16 and
Ab28 peptides can significantly increase
the oxidation rate of copper(II) towards
the catechol compounds and promote
endogenous peptide oxidation (see
scheme). The main modifications consist
of oxidation of His13/14 to 2-oxohisti-
dine and Phe19/20 to ortho-tyrosine,
and the formation of a covalent His6-
catechol adduct.
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
11
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ