Aminomethylene-Phosphonate Analogue as a Cu(II) Chelator: Characterization and Application as an Inhibitor of Oxidation Induced by the Cu(II)-Prion Peptide Complex

Article abstract of DOI:10.1021/acs.inorgchem.9b00287

Recently, we reported on a series of aminomethylene-phosphonate (AMP) analogues, bearing one or two heterocyclic groups on the aminomethylene moiety, as promising Zn(II) chelators. Given the strong Zn(II) binding properties of these compounds, they may fi

Full text of DOI:10.1021/acs.inorgchem.9b00287

Article
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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Aminomethylene-Phosphonate Analogue as a Cu(II) Chelator:
Characterization and Application as an Inhibitor of Oxidation
Induced by the Cu(II)−Prion Peptide Complex
Natalie Pariente Cohen,† Eliana Lo Presti,‡ Simone Dell’Acqua,‡ Thomas Jantz,†
Linda J. W. Shimon,§ Naomi Levy,† Molhm Nassir,† Lior Elbaz,† Luigi Casella,*,‡
and Bilha Fischer*,†
†Department of Chemistry, Bar-Ilan University, Ramat Gan 5290002, Israel
‡Department of Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy
§Faculty of Chemistry, Crystallography Unit, Weizmann Institute, Rehovot 76100, Israel
S
* Supporting Information
ABSTRACT: Recently, we reported on a series of aminomethylene-phosphonate (AMP) analogues, bearing one or two
heterocyclic groups on the aminomethylene moiety, as promising Zn(II) chelators. Given the strong Zn(II) binding properties
of these compounds, they may find useful applications in metal chelation therapy. With a goal of inhibiting the devastating
oxidative damage caused by prion protein in prion diseases, we explored the most promising ligand, {bis[(1H-imidazol-4-
yl)methyl]amino}methylphosphonic acid, AMP-(Im)2, 4, as an inhibitor of the oxidative reactivity associated with the Cu(II)
complex of prion peptide fragment 84−114. Specifically, we first characterized the Cu(II) complex with AMP-(Im)2 by
ultraviolet−visible spectroscopy and electrochemical measurements that indicated the high chemical and electrochemical
stability of the complex. Potentiometric pH titration provided evidence of the formation of a stable 1:1 [Cu(II)-AMP-(Im)2]+
complex (ML), with successive binding of a second AMP-(Im)2 molecule yielding ML2 complex [Cu(II)-(AMP-(Im)2)2]+ (log
K′ = 15.55), and log β′ = 19.84 for ML2 complex. The CuN3O1 ML complex was demonstrated by X-ray crystallography,
indicating the thermodynamically stable square pyramidal complex. Chelation of Cu(II) by 4 significantly reduced the oxidation
potential of the former. CuCl2 and the 1:2 Cu:AMP-(Im)2 complex showed one-electron redox of Cu(II)/Cu(I) at 0.13 and
−0.35 V, respectively. Indeed, 4 was found to be a potent antioxidant that at a 1:1:1 AMP-(Im)2:Cu(II)-PrP84−114 molar ratio
almost totally inhibited the oxidation reaction of 4-methylcatechol. Circular dichroism data suggest that this antioxidant activity
is due to formation of a ternary, redox inactive Cu(II)-Prp84−114-[AMP-(Im)2] complex. Future studies in prion disease animal
models are warranted to assess the potential of 4 to inhibit the devastating oxidative damage caused by PrP.
Recently, we reported on a series of aminomethylene-
phosphonate (AMP) analogues, 3−9, bearing one or two
heterocyclic moieties (imidazolyl, pyridyl, and thiazolyl) on the
aminomethylene group, as potential Zn(II) chelators (Figure
1).1 Analogues 3−9 formed stable water-soluble 2:1 L:Zn(II)
complexes. ML2-type Zn(II) complexes of AMP, bearing either
an imidazolyl or pyridyl moiety, 3, 5, and 6, exhibited high log
INTRODUCTION
■
Aminomethylenephosphonic acid (AMPA), 1, is similar to
glycine, 2, the simplest amino acid, where the carboxylic acid
group is replaced by phosphonic acid, yet AMPA is dibasic
compared to the monobasic glycine, thus allowing better
binding for metal ions and increased water solubility.
Therefore, for the synthesis of the new Zn(II)/Cu(II)
chelators targeted for medicinal applications, we previously
selected the aminomethylene-phosphonate scaffold.1
Received: February 6, 2019
© XXXX American Chemical Society
A
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values (22.25, 21.00, and 18.28, respectively) higher than those
of analogues bearing one heterocyclic moiety.
Given the strong Zn(II) binding properties of these
compounds, they may find useful applications in metal
chelation therapy. As a preliminary assessment of this
potential, we investigated the most promising of these ligands,
AMP-(Im)2 (4), as an inhibitor of the oxidative reactivity
associated with the Cu(II) complex of prion peptide fragment
84−114 (Prp84−114, Ac-PHGGGWGQGGGTHSQWNKPSK-
PKTNMKHMAG-NH2), which includes the high-affinity
Cu(II) binding site.2 Prion diseases make up a family of
transmissible neurodegenerative disorders affecting humans
and animals associated with the conformational conversion of
the cellular prion protein (Prp, ∼210 residues) into an
oligomeric, β-sheet rich form.3 A peculiar aspect of these
proteins is their ability to bind several copper(II) ions,4
although the physio-pathological implications of this inter-
action are not known.
Figure 1. Structures of previously reported aminomethylene-
phosphonate derivatives 3−9.1
With a goal of inhibiting the devastating oxidative damage
caused by PrP in prion diseases, we explored AMP-(Im)2, 4, as
an inhibitor of the oxidative reactivity associated with the
Cu(II) complex of the Prp84−114 fragment. Specifically, we
report here on the characterization of a complex of Cu(II) with
AMP-(Im)2, including the stoichiometry, geometry, and
β values (17.68, 16.92, and 16.65, respectively), while for the
AMP-thiazolyl-Zn(II) complex, 9, log β = 12.53. Ligands 4, 7,
and 8, bearing two heterocyclic moieties, presented log β
Figure 2. (A) Titration of a 4 × 10−5 M solution of copper(II) perchlorate in 5 mM Hepes buffer (pH 7.4) with AMP-(Im)2, from 0 to 1.0 equiv
(cell path of 10 cm, room temperature). (B) Variation of absorbance data at 294 nm, corrected for background and dilution vs the AMP-
(Im)2:Cu(II) molar ratio from 0 to 2.5 equiv. (C) Titration of a 2 × 10−4 M solution of copper(II) perchlorate in 5 mM Hepes buffer (pH 7.4)
with AMP-(Im)2 from 0 to 2.5 equiv (cell path of 10 cm, room temperature). (D) Variation of absorbance data at 850 nm, corrected for
background and dilution vs the AMP-(Im)2:Cu(II) molar ratio.
B
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coordination sphere. In addition, we describe the chemical and
electrochemical stability of the complex and the effect of the
chelator, AMP-(Im)2, on the redox potential of copper ion.
Furthermore, we explored the ability of AMP-(Im)2 to inhibit
the oxidation of a catecholamine model compound (4-
methylcatechol) by Cu(II)-PrP84−114 and to inhibit oxidation
and chemical modifications of PrP84−114 itself under these
conditions. Finally, we studied the mode of action of AMP-
(Im)2 by circular dichroism (CD) and cyclic voltammetry.
formation of a high-affinity [Cu(II)-AMP-(Im)2]+ complex
(Figure 2D).
Determination of Stability Constants of Cu(II)-AMP-
(Im)2 Complexes. Analyzing the stability of Cu(II) complexes
with ligands often encounters problems due to the strong
affinity of this divalent ion compared to those of other first row
transition metals and in particular Zn(II). Moreover, ligands
with soft binding sites (e.g., N-heterocycles, thiols, or those
with π-bonds) usually form stronger complexes with Cu(II)
than with Zn(II).9 Although AMPA analogues 3−9 formed
strong complexes with Zn(II) with log K values from 6.69 to
12.68, AMP-Th formed the least stable Zn(II) complex, with
the lowest log K value, 6.69.1 In this study, we determined the
stability constants for Cu(II)-AMP-Th and Cu(II)-AMP-(Im)2
complexes by potentiometric pH titration. All stability
constants were calculated using Hyperquad software,10 and
the data are listed in Table 1.
RESULTS AND DISCUSSION
■
Characterization of the Cu(II) Complexes Formed
with AMP-(Im)2. Replacement of the hydrogen atoms of the
amino group in AMPA, 1, with nitrogen-containing hetero-
cyclic moieties, e.g., AMP-(Im)2, 4, makes the latter a potential
copper and zinc ion chelator. Heterocyclic moieties such as the
imidazolyl group mimic histidine, which is known as the best
amino acid ligand for Cu(II) and Zn(II) ions in proteins and
peptides.5 Indeed, we recently reported that AMP-(Im)2 forms
a most stable complex with Zn(II) (log β = 22).1 AMP-(Im)2
was synthesized by formation of an intermediate Schiff base
from imidazolyl aldehyde and AMPA followed by Schiff base
reduction by 2-picoline-borane. Various aminomethylene-
phosphonate derivatives bearing heterocyclic substituents
(i.e., imidazolyl, pyridyl, or thiazolyl) at the aminomethylene
carbon atom were also reported as Cu(II) and Ni(II) ion
chelators.6
Table 1. Stability Constants for AMPA-Th and AMP-(Im)2
with Zn(II) and Cu(II) for the 1:1 and 2:1 Complexes and
Overall Log β Values
M, ligand
log KMM(L)
log KLM(L )
log βLM(L )
2
2
a
Zn, AMP-Th
Cu, AMP-Th
6.69 0.14
10.20 0.14
12.02 0.16
15.55 0.14
5.84 0.11
6.24 0.14
10.24 0.12
4.29 0.16
12.53 0.24
16.44 0.28
22.25 0.23
19.84 0.30
b
a
b
Zn, AMP-(Im)2
Cu, AMP-(Im)2
a
b
It should be noted that under neutral conditions AMP-(Im)2
is expected to coordinate to Cu(II) as a monoanionic ligand,
due to the capability of copper(II) to reduce the acidity of the
secondary amino group, and therefore, both a 1:1 complex
[Cu(II)-AMP-(Im)2]+ and a 1:2 complex [Cu(II)-(AMP-
(Im)2)2] can be formed depending on the ligand concen-
tration. To investigate the AMP-(Im)2 binding properties
toward Cu(II), we performed a spectrophotometric study
monitoring the charge transfer band developing in the range
between 250 and 400 nm, with an apparent maximum near
294 nm, upon addition of the ligand to a 4 × 10−5 M solution
of copper(II) perchlorate in 5 mM Hepes buffer (pH 7.4). The
near-ultraviolet band contains contributions from π(imidazole)
→ Cu(II)7 and σ(amine) → Cu(II) LMCT8 and is more easily
followed than the spectral changes occurring at higher energies,
where an overlap between intraligand (imidazole) π → π* and
σ (imidazole) → Cu(II) LMCT transitions occur (Figure 2A).
An isosbestic point at 278 nm can be observed in the initial
part of the titration, up to an ∼1:1 ligand:Cu(II) molar ratio.
Upon further addition of the ligand, the increase in absorbance
is smaller, indicating that further coordination of AMP-(Im)2
molecules occurs with reduced affinity (see Figure S1). The
plot in Figure 2B shows the stepwise formation of a high-
affinity 1:1 Cu(II)-AMP-(Im)2 complex, [Cu(II)-AMP-
(Im)2]+, and the successive binding of a second AMP-(Im)2
molecule yielding [Cu(II)-(AMP-(Im)2)2]. This result is
confirmed by performing the titration experiment in the low-
energy spectral range, where the d−d transitions of the Cu(II)
chromophore can be followed. As shown in Figure 2C, the
addition of AMP-(Im)2 to a 2 × 10−4 M solution of Cu(II)
under the same conditions produces a broad increase in
absorption above 650 nm, extending to the near-infrared range.
As described above, an isosbestic point can be noted in the
initial part of the titration [up to the intermediate point
outlined in Figure 2C (see selected spectra in Figure S2)]. The
plot of the absorbance change taken at 850 nm confirms the
Data from ref 1. In this study, stability constants determined by pH
potentiometric titration.
The potentiometric pH titration (Figures S3 and S4) gives a
high stability constant for the 1:1 complex of AMP-(Im)2 with
Cu(II) [log K′(Cu/AMP‑(Im) ) = 15.55 0.14], whereas the log β
2
value [log β′(Cu/(AMP‑(Im) ) ) = 19.84
0.30] indicates that
2
2
binding of the second ligand to form the ML2 species is much
weaker for Cu(II) than for Zn(II). The much higher stability
of the ML complex with Cu(II) and the huge difference
between the stability constants of the ML species for Cu(II)
with respect to Zn(II) might be both explained by the different
geometry of the complexes. While Zn(II) usually prefers an
octahedral coordination sphere, Cu(II) forms stable complexes
with four donor ligands in a square planar arrangement and
weaker bonding in the axial position, due to strong Jahn−
Teller effect.11
AMP-Th, 9, has three coordination sites (N-thiazolyl, N-
amine, and O-phosphonate), so that half of the coordination
sites of Zn(II) are filled. When a second ligand chelates, too,
the symmetry of the whole complex is quite high and the
stability constants show that the contribution of the two
ligands to the complex is almost equal [log K′(Zn/AMP‑Th) = 6.69
0.14, and log β′(Zn/(AMP‑Th) ) = 5.84 0.11].
2
In the 1:1 Cu(II)-(AMP-Th) complex, the ligand can bind
with three donors (N2O set) in the Cu(II) equatorial
coordination plane. In the Cu(II)-(AMP-Th)2 complex, it is
likely that both ligands will act as bidentate ligands or, less
likely, one AMP-Th will bind as a tridentate ligand and the
second as a monodentate ligand. Therefore, the stability
constant for binding of the second ligand in the 1:2 complex is
much lower (∼4 log units) as compared to that in the 1:1
complex.
AMP-(Im)2, 4, has four coordination sites (two N-
imidazolyl, N-amine, and O-phosphonate), so the formation
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of the 1:1 complex, [Cu(II)-AMP-(Im)2]+, is particularly
favored because the ligand binds with three strong N donors in
a system of two five-membered chelate rings. The stability
constant for 1:1 complex formation is 3.5 log units higher for
copper(II) than for zinc(II). This is confirmed by the crystal
structure of Cu(II)-AMP(Im)2 described below. On the other
hand, the stability constant for binding the second ligand to
form the 1:2 complex [Cu(II)-(AMP(Im)2)2] is much lower
than that of the 1:1 complex, because only one equatorial
position is available and binding of further donor groups could
occur only opposite to, or displacing, the axial phosphonate
ligand in a weak coordination position.
Table 2. Short List of Important Bonds and Angles in the
Cu(II)-AMP-(Im)2Cl Complex and Known Related
Complexes
[Cu(II)-AMP-
a
a
(Im)2Cl]
PIXPAN
2.018
2.050
2.144
2.078
PIXPER
2.043
2.003
2.124
2.094
Cu−N1 (Å)
Cu−N3 (Å)
Cu−N5 (Å)
1.943
1.921
2.124
2.442
Cu−O (vertical
position) (Å)
(Cu−N)
(Cu−N)
Cu−Cl (Å)
2.241
2.245
2.226
N1−Cu−N3 (deg)
Cl−Cu−N5 (deg)
Cl−Cu−O1 (deg)
154.05
176.56
92.30
123.66
179.67
101.23
124.69
177.56
101.70
In contrast, zinc(II) can form complexes with higher
coordination numbers, and for this reason, the formation of
the ML2 complex is particularly strong, with stability constants
for the two successive binding equilibria that occur with almost
a
From ref 12.
similar values [log K′(Zn/AMP‑(Im) ) = 12.02
0.16, and log
coordinated by three nitrogen atoms, one oxygen atom, and
one chloride ion, resulting in a square pyramidal structure
(equatorial positions are occupied by three nitrogen atoms and
a chloride ion, while the apical position is occupied by the O
atom of the phosphonate group).
Chelation by AMP-(Im)2 Significantly Reduces the
Oxidation Potential of Cu(II). Because free Cu(II) ion acts
as a moderate oxidizing agent, we explored the effect of ligand
4 on the reduction potential of the copper ion. Cyclic
voltammograms (CVs) were measured for Cu(ClO4)2- and
AMP-(Im)2-chelated copper ions.
2
K″(Zn/(AMP‑(Im) ) ) = 10.24 0.12].
2
2
Crystal Structure of [Cu(II)-AMP-(Im)2Cl]. A single
crystal of the Cu-AMP(Im)2 complex, crystallized in water
under an acetone atmosphere, using a 1:2 Cu(II):ligand 4
ratio, was analyzed by X-ray diffraction. The crystal data are
listed in Table S1. The Cu(II) ion was found to be coordinated
by five atoms giving rise to a square pyramidal geometry, with
the tridentate 3N moiety in the equatorial plane, bound in two
fused five-membered chelate rings. This coordination mode
represents a strong binding site and the best possible
arrangement for a Cu(II) complex (Figure 3). The basal
Comparison between the voltammograms of the electrolyte
solution [50 mM Hepes buffer (pH 7.4)] devoid of the metal
ion and the ligand (black line in Figure 4) and the same
Figure 3. Crystal structure of the [Cu(II)-AMP-(Im)2Cl] complex.
coordination plane is completed by the chloride ion, while the
apical position is occupied by the O atom of the phosphonate
group. The Cu−O bond distance of the apical position is larger
than the other copper bond distances of the basal plane,
indicating the Jahn−Teller distortion in this complex.
We attempted to obtain a single crystal of the ML2 complex
using 2.5 equiv of ligand 4. No crystals were obtained, but a
precipitate was obtained. The latter was analyzed by HRMS as
a mixture of ML2 and ML complexes (see the Experimental
Section).
Figure 4. Cyclic voltammograms of 50 mM Hepes buffer (pH 7.4)
without the ligand and copper ions (black), with 20 mM AMP-(Im)2
(red), with 0.25 mM Cu(ClO4)2 in Hepes buffer (blue), and with a
mixture of AMP-(Im)2 and Cu(ClO4)2 in different ratios (green,
purple, brown, and turquoise). All of the measurements were
conducted in deaerated solutions at a scan rate of 50 mV/s.
The crystal structures of related copper(II) complexes of
tripodal triimidazolyl methylamine ligands were previously
reported [PIXPAN and PIXPER (Table 2)].12 The cupric ion
in these complexes is coordinated by four nitrogen atoms and a
chloride ion in distorted trigonal bipyramidal structures, where
three imidazole nitrogen atoms occupy equatorial positions of
the trigonal bipyramid, while the amine nitrogen atom and
chlorine atom occupy apical sites. Unlike these complexes, in
the [Cu(II)-AMP-(Im)2Cl] complex the cupric ion is
solution after the addition of AMP-(Im)2 (red line in Figure 4)
clearly shows that the ligand is not electrochemically active in
the voltage range from 0.6 to −0.1 V versus Ag|AgCl. The CV
of the electrolyte solution containing 0.25 mM Cu(ClO4)2
without the ligand (blue line in Figure 4) disclosed a clear
redox reaction at 0.06 V versus Ag|AgCl, which corresponds to
the one-electron redox of Cu(II)/Cu(I) (note that the
D
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voltammogram shape suggests a high resistance of the system,
due to the relatively low concentration of the electrolyte
solution, specifically chosen to keep the experimental
conditions of the different measurements, e.g., potentiometric
titration, constant). The AMP-(Im)2 ligand was gradually
added to the solution until the characteristic copper peak
completely disappeared at a 1:1 ligand:Cu(II) molar ratio.
Upon further addition of the ligand, there was still no
electrochemical response, indicating that the chelated copper
ions are electrochemically inactive within the measured
window. This confirms the formation of a 1:1 Cu(II) complex
under these conditions.
oxidation at a Cu(II):Prp84−114 molar ratio of 1:2. Indeed, it
was previously reported that under these conditions almost all
copper(II) is bound to the peptide even in a dilute solution.14
When catechol oxidation by Cu(II)-Prp84−114 was studied in
the presence of AMP-(Im)2, a rapid decrease in the rate was
observed as a function of the added ligand (Figure 6). Actually,
AMP-(Im)2 Is Chemically Stable. We have also analyzed
the pH-dependent chemical stability of AMP-(Im)2, 4, by
ultraviolet (UV) measurements at room temperature.
UV spectra (Figure S5) showed that the absorption maxima
at pH 1, 7, and 13 remained at 225 and 275 nm. The band at
225 nm at pH 13 has a lower intensity, which can be explained
by the deprotonation of the ammonium group.
The band at 275 nm has the highest intensity at pH 1. This
band can be related to the AMP-(Im)2 species that is
protonated at all nitrogen donor groups. The UV spectra
show that AMP-(Im)2 did not decompose under alkaline (pH
13) or acidic (pH 1) conditions at room temperature.
The Effect of AMP-(Im)2 on Cu(II)/Prp84−114 Induced
Oxidative Activity. Encouraged by the promising properties
of AMP-(Im)2 described above, we next explored its
application to the inhibition of the devastating oxidative
activity of the Cu(II)-PrP complex.13 Specifically, AMP-(Im)2
by acting as copper ion chelator may remove the metal ion
from the copper-peptide complex or form a ternary complex
that prevents reduction of the cupric ion. For these
experiments, we used the Prp84−114 fragment, which includes
the high-affinity Cu(II) binding site of the protein, comprising
His85 and two adjacent deprotonated amide groups within the
octarepeat sequence (PHGGGWGQ) and His111 (or His96)
as copper(II) ligands (Figure 5).2 The copper(II) complexes
Figure 6. Kinetic profiles of oxidation of 4-methylcatechol (3 mM) in
50 mM Hepes buffer (pH 7.4) at 25 °C in the presence of (a) Cu(II)
(25 μM) (red); (b) Cu(II) (25 μM) and Prp84−114 (50 μM) (dark
blue); (c) Cu(II) (25 μM), Prp84−114 (50 μM), and AMP-(Im)2 (10
μM) (light blue); (d) Cu(II) (25 μM), Prp84−114 (50 μM), and AMP-
(Im)2 (25 μM) (orange); and (e) Cu(II) (25 μM), Prp84−114 (50
μM), and AMP-(Im)2 (50 μM) (green ).
at a 1:1 AMP-(Im)2:Prp84−114 molar ratio, the reaction was
almost totally quenched. This suggests that AMP-(Im)2 either
acts as strong chelator of Cu(II), abstracting it from the
Prp84−114 complex, or is able to form a ternary, redox inactive
Cu(II)-Prp84−114-[AMP-(Im)2] complex. Notably, the Cu(II)-
AMP-(Im)2 complex used as a control displayed no significant
reactivity toward MC oxidation (Figure S6).
Analysis of Prp84−114 Oxidative Modifications. As
shown by our recent study, the copper redox activation
promoted by coordination of PrP peptides not only affects
MC, the external substrate, but also produces several types of
modifications at the endogenous PrP peptide.14 It is therefore
important to establish whether the presence of the ligand
AMP-(Im)2 also inhibits the redox reactivity of copper toward
the peptide. To this end, samples of the reaction mixtures of
Cu(II)-Prp84−114, MC, and AMP-(Im)2 as used in the kinetic
studies were analyzed at various times by liquid chromatog-
raphy−mass spectrometry (LC−MS). The data collected
(Table 3) show that indeed the presence of AMP-(Im)2
protects the peptide from oxidation and modification, even
though complete protection would require larger amounts of
the ligand.
Figure 5. Proposed structure of the Cu(II) complex with prion
2
fragment Prp84−114
.
The types of modifications identified by LC−MS analysis
include (a) insertion of an O atom into Met109, Met112, or
His96 (which can be hardly differentiated) or His85 (+16 mass
increment), (b) insertion of two atoms at the two Met residues
or a Met and a His residue (+32), (c) addition of methyl
quinone (MQ) to His96 or His111 (+120), and (d) a double
modification consisting of addition of MQ to one His residue
(+120) and insertion of an O atom into Met or His (+16),
yielding a total mass increment of +136 (see Figure 7).
CD Investigation of the Interaction of AMP-(Im)2 with
the Cu(II)/Prp84−114 Complex. The far-UV CD spectrum in
neutral buffer of Prp84−114 is characterized by a negative
minimum near 198 nm, indicative of its unstructured nature in
solution (Figure 8). The CD spectrum of the corresponding
with this and related PrP peptides have been recently used to
show the amplification of toxic effects associated with Cu redox
cycling in the presence of catecholamines and related
catechols.14
The inhibition experiments were carried out by assessing the
effect of AMP-(Im)2 in the aerobic oxidation of the model
compound 4-methylcatechol (MC) promoted by the Cu(II)-
Prp84−114 complex. The reaction produces the corresponding 4-
methylquinone (MQ) in the initial phase and more complex
oligomeric products at longer times.14 Given the low
concentration range of the Cu-peptide complex and the
competing binding properties of MC (used in large excess)
toward Cu(II), we chose to investigate the kinetics of MC
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Table 3. Modifications of Prp84−114 Detected by LC−ESI-
MS Analysis upon Oxidation of MC (3 mM) Promoted by
Cu(II) (25 μM) and Prp84−114 (50 μM), in the Absence and
Presence of AMP-(Im)2 (50 μM)
time (min) Prp84−114 (%) +16 (%) +32 (%) +120 (%) +136 (%)
Cu(II)-Prp84−114 + MC
20
110
200
86.2
45.2
37.8
4.0
16.0
20.4
1.5
11.5
14.8
8.3
11.0
10.5
0.0
16.3
16.4
Cu(II)-Prp84−114 + MC + AMP-(Im)2
20
110
200
93.6
85.9
85.5
4.6
2.6
6.9
0.6
0.9
1.0
1.2
10.5
6.6
0.0
0.0
0.0
Figure 8. Far-UV CD spectra of Prp84−114 (50 μM) in 5 mM
phosphate buffer (pH 7.3) (black line) and after addition of 1 equiv of
Cu(II) (red line) and after further addition of 1 equiv of AMP-(Im)2
(blue line).
copper(II) complex can be almost superimposed with that of
the free peptide, with only a slight decrease in the intensity of
the 198 nm peak. Also, the addition of AMP-(Im)2 has a
negligible effect on the CD spectrum, indicating that the
presence of this ligand does not affect the structure of
Prp84−114
.
The visible portion of the CD spectrum of Cu(II)-Prp84−114
is shown in Figure 9. The main features are the weak positive
band around 525 nm and a stronger negative CD activity at a
higher energy, between 300 and 350 nm.15 Addition of 1 equiv
of AMP-(Im)2 to the Cu(II)-Prp84−114 complex yields a red
shift of the 525 nm band to ∼600 nm and an inversion of the
near-UV CD activity in the range of 300−350 nm. These
changes suggest the formation of a Cu(II)/Prp84−114/AMP-
(Im)2 ternary complex rather than copper abstraction by the
ligand from the Cu(II)-Prp84−114 complex, as the Cu(II)-AMP-
(Im)2 complex would give a flat CD curve because it is not
optically active.
Figure 9. Visible CD trace of Prp84−114 (740 μM) in 5 mM phosphate
buffer (pH 7.3) (black line) and spectra after the addition of 1 equiv
of Cu(II) (red line) and after further addition of 1 equiv of AMP-
(Im)2 (blue line).
CONCLUSION
Indeed, AMP-(Im)2, 4, was shown in this study to form a
highly chemically and electrochemically stable complex with
Cu(II). This complex was proven to exist predominantly as a
ML species, N3O1(Cl). X-ray crystallography indicated that
the thermodynamically stable complex is square pyramidal.
■
The design of AMP-(Im)2, 4, as a Cu(II)/Zn(II) chelator, was
inspired by both EDTA and L-histidine, which are known to be
excellent chelators of soft and borderline M(II) ions such as
Cu(II) and Zn(II).
Figure 7. Schematic illustration of the type of chemical modifications undergone by the Prp84−114 fragment upon catechol-induced Cu redox
cycling.
F
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Article
titration. The absorbance readings were corrected for background, at
510 nm, and for dilution. The titration experiment with AMP-(Im)2
(0.03 M) was repeated on a 2 × 10−4 M Cu(II) solution in the same
buffer following the absorbance changes in the visible and near-
infrared range, with optical readings recorded at 850 nm. Also in this
case, correction for background at 510 nm and for dilution was made.
Potentiometric pH Titration of Cu-AMP-(Im)2 Complexes.
Potentiometric titrations were performed in an aqueous solution with
0.1 M NaNO3 as the supporting electrolyte in a thermostated cell at
25 °C under a nitrogen atmosphere. Protonation constants of AMP-
(Im)2 were determined at a constant ionic strength, carrying out the
experiments on 10 mL of a 5 × 10−4 M solution of the ligand
(containing NaNO3 salt) to which an excess of HNO3 was added.
Titrations were performed by adding small aliquots of standard
NaOH (0.1 M), from pH 2.5 to 12. Copper binding constants were
determined under the same conditions, starting from a 3.5 × 10−4 M
solution of [Cu-(AMP-(Im2))2]. The standard electrochemical
potential of the glass electrode was previously determined with a
titration in accordance with the method of Gans.16 The experimental
data were processed with Hyperquad to determine equilibrium
constants.10
Crystallization of [Cu(II)-AMP-(Im)2Cl]. Compound 4 (20 mg)
was dissolved in HPLC grade water (1000 μL), and 0.11 M CuCl2
(680 μL in water) was added. The mixture was mixed for 1 h and then
freeze-dried for 2 days. The residue was dissolved in HPLC grade
water (300 μL), and the vial was immersed in an acetone
environment. After 2 days (at room temperature), blue single crystals
were obtained. HRMS ESI (positive m/z): [C9H13CuN5O3P]+• calcd
333.0047, found 333.0049; [C9H14CuN5O3P]+ calcd 334.0125, found
334.0101.
Electrochemical Measurements. All electrochemical measure-
ments were conducted in a three-electrode Teflon cell, where the
working electrode was a glassy carbon disk electrode (0.196 cm2), the
counter electrode was a glassy carbon rod, and the reference electrode
was a Ag/AgCl electrode. Throughout all measurements, Ar gas was
purged into the cell to keep the system inert from oxygen.
Peptide Synthesis. The Prp84−114 peptide (Ac-PHGGGWGQG-
GGTHSQWNKPSKPKTNMKHMAG-NH2, molecular weight of
3297.65) was synthesized using the standard fluorenyl methoxy
carbonyl (Fmoc) solid phase method in dimethylformamide,
following a literature procedure.14 Purification was performed by
HPLC, using a linear gradient of water, containing 0.1% trifluoro-
acetic acid (TFA), and acetonitrile, also containing 0.1% TFA, from 0
to 100%. The column was a Jupiter 4u proteo 90A 250 mm × 10 mm,
4 μm column; elution was carried out at flux rate of 5 mL/min. The
retention time of the peptide was 12 min. The product was
lyophilized, yielding a white solid, which was characterized by ESI-
MS (direct injection, MeOH, positive ion mode, capillary temperature
of 200 °C, m/z): +472.14 (Prp84−114H77+), +550.65 (Prp84−114H66+),
+660.52 (Prp84−114H55+), +825.3 (Prp84−114H44+), +1099.99
(Prp84−114H33+).
Furthermore, potentiometric pH titrations indicated a high
stability constant for the 1:1 complex with Cu(II) [log
K′(Cu/AMP‑(Im) ) = 15.55
0.14, and log β′(Cu/(AMP‑(Im) ) ) =
2
2 2
19.84 0.30]. While CuCl2 showed redox reaction at 0.13 V
versus Ag|AgCl, one-electron redox of Cu(II)/Cu(I), the 1:2
CuCl2-AMP-(Im)2 complex, showed an irreversible reduction
process peaking at −0.35 V versus Ag|AgCl. The observation of
the high stability of Cu(II) complexes of 4, together with the
antioxidant nature of 4, encouraged us to explore its potential
as an inhibitor of oxidation induced by the Cu(II)−prion
peptide complex.
Indeed, ligand 4 was found to be a potent antioxidant that at
a 1:1 AMP-(Im)2:Cu(II)-Prp84−114 molar ratio almost totally
inhibited the oxidation reaction of methylcatechol. CD data
suggest that this antioxidant activity is due to the formation of
a ternary, redox inactive Cu(II)-Prp84−114-[AMP-(Im)2]
complex. Future studies in prion disease animal models are
warranted to assess the potential of 4 for delaying the
progression of signs of neurodegeneration by inhibiting the
devastating oxidative damage caused by PrP.
EXPERIMENTAL SECTION
■
General. CD spectra were recorded with a Jasco J-710
spectropolarimeter. Mass spectra and LC-MS/MS data were obtained
with a LCQ ADV MAX ion-trap mass spectrometer, with an ESI ion
source. The system was run in automated LC-MS/MS mode and
using a surveyor high-performance liquid chromatography (HPLC)
system (Thermo Finnigan, San Jose, CA) equipped with a
Phenomenex Jupiter 4u Proteo column (4 μm, 150 mm × 2.0
mm). For the analysis of peptide fragments, Bioworks 3.1 and
Xcalibur 2.0.7 SP1 software were used (Thermo Finnigan). UV−
visible (UV−vis) spectra and kinetic data were recorded on an Agilent
8453 diode array spectrophotometer, equipped with a thermostated,
magnetically stirred optical cell. Nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker AVANCE 400 spectrometer.
Absorption data were measured with a Shimadzu UV−vis 2401PC
spectrophotometer. Compounds were characterized by NMR spec-
troscopy with Bruker spectrometers. H, 31P, and 13C NMR spectra
1
were measured with a Bruker DPX-300 spectrometer (300 and 75
MHz for 1H and 13C, respectively) and an AV-400 spectrometer (400,
1
162, and 100.6 MHz for H and 13C). All spectra were measured at
room temperature with D2O samples. High-resolution mass spectra
were recorded with an AutoSpec-E FISION VG (Waters) mass
spectrometer by chemical ionization. Primary purification of ligand 4
was achieved on an LC (Isco UA-6) system using a column of
Sephadex DEAE-A25, swollen in 1 M NaHCO3 or 1 M TEAB at 4 °C
for 24 h. The resin was washed with deionized water before use. LC
separation was monitored by UV detection at 220 and 190 nm.
Procedure for the Synthesis of AMP-(Im)2, Compound 4.
AMP-(Im)2, 4, was synthesized as described previously.1 Purification
of compound 4 was performed by subjecting the reaction’s residue to
ion-exchange chromatography (on DEAE-Sephadex A-25 chloride
form, swollen overnight in a 1 M NaHCO3 solution at 4 °C). The
product was eluted with a gradient from 0 to 0.5 M TEAB
(triethylammonium bicarbonate) at pH 7.6 (adjusting the pH with a
5% HCl solution). The product was obtained at 0.05 M TEAB.
Freeze-drying provided the product as a white solid (325 mg, 27%
yield).
Titration of Cu(II) with Compound 4 Monitored by UV−Vis
Spectroscopy. Titrations of Cu(II) with compound 4 were
performed in a quartz optical cell with a path length of 10 cm. To
monitor the spectral changes in the near-UV range (collecting data at
294 nm), a 4 × 10−5 M solution of copper(II) perchlorate in 5 mM
Hepes buffer at pH 7.4 (22 mL) was titrated with small portions of a
slightly acidic aqueous solution of AMP-(Im)2 (0.03 M) at room
temperature, with ligand additions of 0−2.5 equiv. The final pH of the
titrated solution was measured to exclude any pH variation during the
Kinetics of 4-Methylcatechol (MC) Oxidation by Cu(II)-
Prp84−114 in the Presence of AMP-(Im)2. The dependence of the
rate of MC oxidation promoted by Cu(II)-Prp on AMP-(Im)2
concentration was studied in 50 mM Hepes buffer (pH 7.4) and at
25 °C. The experiments were carried out spectrophotometrically,
following the increase in the intensity of the band at 401 nm due to
formation of 4-methyl quinone (ε = 1550 M−1 cm−1). Initially, MC (3
mM) autoxidation, MC (3 mM) oxidation promoted by Cu(II) salt
[25 μM copper(II) nitrate], and MC (3 mM) oxidation by a 2:1
Prp84−114-Cu(II) complex [50 μM Prp84−114 and 25 μM Cu(II)] were
evaluated. Then, the dependence of the rate on AMP-(Im)2
concentration was investigated by comparison of kinetic profiles at
increasing AMP-(Im)2 concentrations (from 10 to 50 μM),
maintaining all other reagents unchanged.
Characterization of Modified Prp84−114 by HPLC−ESI-MS
Analysis. The competitive modification of Prp84−114 during MC
oxidation was investigated by HPLC−ESI-MS analysis, preparing
samples of 25 μM copper(II) nitrate, 50 μM Prp84−114, and 3 mM MC
in the absence and presence of AMP-(Im)2 (50 μM), in 50 mM
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Article
Hepes buffer (pH 7.4). LC−ESI-MS analyses were performed at
different reaction times. The elution of reaction mixtures was carried
out with a linear gradient of H2O (with 0.1% HCOOH) and
acetonitrile (with 0.1% HCOOH), at a flow rate of 0.2 mL/min. The
identification of specific modifications was performed with a Bioworks
database.
CD Measurements. The far-UV CD spectrum of a 50 μM
solution of Prp84−114 in 5 mM phosphate buffer (pH 7.3) was
recorded in a cell with a path length of 0.1 cm. The spectral changes
upon addition of 1 equiv of Cu(NO3)2 to the Prp84−114 solution and
the effect of further addition of 1 equiv of AMP-(Im)2 were
subsequently assessed. Spectra were recorded with a scanning rate of
100 nm/min with 10 accumulations.
The visible CD spectrum of a solution of Prp84−114 (740 μM) in the
same buffer was recorded in a cell with a path length of 1 cm. Spectra
were then recorded after addition of 1 equiv of Cu(NO3)2 to the
peptide solution and upon further addition of 1 equiv of AMP-(Im)2
to the Cu(II)-Prp84−114 solution. Spectra were recorded with a
scanning rate of 100 nm/min with six accumulations.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: bilha.fischer@biu.ac.il. Fax: 972-3-6354907. Phone:
972-3-5318303.
*E-mail: luigi.casella@unipv.it. Fax: +39-0382-528544. Phone:
+39-0382-987331.
■
ORCID
Simone Dell’Acqua: 0000-0002-1231-4045
Linda J. W. Shimon: 0000-0002-7861-9247
Lior Elbaz: 0000-0003-4989-5135
Luigi Casella: 0000-0002-7671-0506
Bilha Fischer: 0000-0001-8837-0978
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the Italian Ministry of Education,
University, and Research (MIUR)-Research Projects of
National Interest (PRIN) 2015, prot. 2015T778JW, for
funding. Prof. Valeria Amendola and Dr. Ana Miljkovic are
gratefully acknowledged for their help in performing and
analyzing potentiometric titration.
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