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EPR Spectroscopy of a Clinically Active (1:2) Copper(II)-Histidine Complex Used in the Treatment of Menkes Disease: A Fourier Transform Analysis of a Fluid CW-EPR Spectrum


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Redox active transition metal ions (e.g., iron and copper) have been implicated in the etiology of many oxidative stress-related diseases including also neurodegenerative disorders. Unbound copper can catalyze formation of reactive oxygen species (hydroxyl radicals) via Fenton reaction/Haber-Weiss chemistry and therefore, under physiological conditions, free copper is potentially toxic and very rarely exists inside cells. Copper(II) bound to the aminoacid L-histidine represents a species discovered in blood in the mid 60s and since then extensive research on this complex was carried out. Copper bound to L-histidine represents an exchangeable pool of copper(II) in equilibrium with the most abundant blood plasma protein, human serum albumin. The structure of this complex, in aqueous solution, has been a subject of many studies and reviews, however without convincing success. The significance of the (1:2) copper(II)-L-histidine complex at physiological pH documents its therapeutic applications in the treatment of Menkes disease and more recently in the treatment of infantile hypertrophic cardioencephalomyopathy. While recently the (1:2) Cu(II)-L-His complex has been successfully crystallized and the crystal structure was solved by X-ray diffraction, the structure of the complex in fluid solution at physiological pH is not satisfactorily known. The aim of this paper is to study the (1:2) Cu(II)-L-histidine complex at low temperatures by X-band and S-band EPR spectroscopy and at physiological pH at room temperature by Fourier transform CW-EPR spectroscopy.
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Molecules 2014, 19, 980-991; doi:10.3390/molecules19010980
ISSN 1420-3049
EPR Spectroscopy of a Clinically Active (1:2) Copper(II)-
Histidine Complex Used in the Treatment of Menkes Disease:
A Fourier Transform Analysis of a Fluid CW-EPR Spectrum
Lukas Gala 1,†, Michael Lawson 1,†, Klaudia Jomova 2, Lubomir Zelenicky 2,
Andrea Congradyova 2, Milan Mazur 1 and Marian Valko 1,*
1 Faculty of Chemical and Food Technology, Slovak Technical University, Bratislava SK-812 37,
Slovakia; E-Mails: (L.G.); (M.L.); (M.M.)
2 Faculty of Natural Sciences, Constantine The Philosopher University, Nitra SK-949 74, Slovakia;
E-Mails: (K.J.); (L.Z.); (A.C.)
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail:;
Tel.: +421-2-593-25-750; Fax: +421-2-524-93-198.
Received: 27 November 2013; in revised form: 23 December 2013 / Accepted: 26 December 2013 /
Published: 15 January 2014
Abstract: Redox active transition metal ions (e.g., iron and copper) have been implicated
in the etiology of many oxidative stress-related diseases including also neurodegenerative
disorders. Unbound copper can catalyze formation of reactive oxygen species (hydroxyl
radicals) via Fenton reaction/Haber–Weiss chemistry and therefore, under physiological
conditions, free copper is potentially toxic and very rarely exists inside cells. Copper(II)
bound to the aminoacid L-histidine represents a species discovered in blood in the mid 60s
and since then extensive research on this complex was carried out. Copper bound to
L-histidine represents an exchangeable pool of copper(II) in equilibrium with the most
abundant blood plasma protein, human serum albumin. The structure of this complex, in
aqueous solution, has been a subject of many studies and reviews, however without convincing
success. The significance of the (1:2) copper(II)-L-histidine complex at physiological pH
documents its therapeutic applications in the treatment of Menkes disease and more recently in
the treatment of infantile hypertrophic cardioencephalomyopathy. While recently the (1:2)
Cu(II)-L-His complex has been successfully crystallized and the crystal structure was
solved by X-ray diffraction, the structure of the complex in fluid solution at physiological
Molecules 2014, 19 981
pH is not satisfactorily known. The aim of this paper is to study the (1:2) Cu(II)-L-histidine
complex at low temperatures by X-band and S-band EPR spectroscopy and at physiological
pH at room temperature by Fourier transform CW-EPR spectroscopy.
Keywords: copper-histidine complex; copper metabolism; Menkes disease; EPR
spectroscopy; FT-EPR spectroscopy
1. Introduction
Copper is an essential metal element necessary for all living organisms [1]. It is an integral part of
many copper-containing enzymes involved in various biological processes, such as photosynthesis,
respiration, redox-metal metabolism, neurological functions and other processes.
The balance between copper needs and toxicity is tightly regulated at the cellular level and at the
tissue and organ levels [2]. Free (unbound) copper in excess of physiological requirements is toxic,
since it is known to catalyse formation of free radicals (mainly via the Fenton reaction) which in turn
may lead to oxidation of important biological molecules including DNA, proteins, as well as biological
membranes [3]. Therefore copper is present in biological systems as an integral part of enzymes or it is
transported by proteins such as serum albumins. Also, chelation of copper by biologically active small
molecular weight molecules has many physiological functions and in addition to this, copper chelation
reduces its capacity to catalyse formation of free radicals [4].
One of the very important copper chelators is the amino acid L-histidine, which plays an important
role in copper transport prior to its entry into cellular transport systems and incorporation into enzymes
and proteins (Figure 1) [5]. Copper(II) bound to L-histidine is in equilibrium with human serum
albumin and may undergo mutual exchange which modulates the bioavailability of copper to the cell.
Figure 1. A potential tridentate ligand, L-histidine (HL).
The breakdown of copper metabolism results in various disease states of an organism [6]. Menkes
disease is a genetic disease usually affecting male infants, characterized by a disorder of copper
transport which affects levels of copper in the body with clinical manifestation of copper deficiency [7].
Clinical diagnosis of Menkes Disease is often difficult due to the fact that affected newborns usually
appear healthy at birth, with almost no symptoms for the first 2–3 months. Currently there is no
effective treatment for Menkes diseases, however copper treatment in the form of copper salts (copper
chloride, copper sulfate, copper acetate) or copper histidine are available. The medicinal potential
of copper-histidine complex has resulted in the increased interest of scientists in solving its structure.
The exact role of L-histidine in copper-albumin interaction (albumin is a copper carrier) in cellular
Molecules 2014, 19 982
uptake of copper as well as in Menkes disease is not well understood. The exact characterization of
copper-L-histidine complex in aqueous environment has been attempted, however without a detailed
structural description [8–16].
The problems associated with the structural characterization of copper(II)-histidine species in
solution are caused by the variability of L-histidine in coordinating metal ions. L-Histidine has three
potential metal-binding sites and can bind to metals as mono-, bi- and tri-dentate ligands [5].
Moreover, its binding mode depends on the pH of the solution.
The only success with respect to direct structural elucidation of Cu(II)-L-histidine complex has been
achieved in solid state by means of X-ray diffraction studies. The X-ray results have shown that the
(1:2) copper(II)-L-histidine complex in the solid state is five-coordinate, possessing distorted
square-pyramidal geometry with bidentate and tridentate L-histidine ligands [17].
EPR spectroscopy is a very sensitive technique for the elucidation of the structure of paramagnetic
species in solution [18]. A unique feature of EPR is its ability to elucidate the directly bonded
magnetically active atoms to a paramagnetic center of the complex and to describe the stereochemistry
around a metal ion. This is substantiated by the resolution of superhyperfine (shf) structure in EPR
spectra due to the magnetically active nuclei of donor atoms in metal complexes which provide
valuable information about the nature of metal-ligand bonding [19].
In order to characterize the exact nature of directly bonded ligand atoms to copper as well as the
stereochemistry around copper ion in aqueous solution, we present here the results of EPR study of
(1:2) copper(II)-L-histidine complex. We believe that the detailed structural characterization of
copper-L-histidine species in a fluid environment has biological importance and significance in view of
a better understanding of the mechanism of action of the therapeutically active copper(II) complex.
2. Results and Discussion
2.1. EPR Theory
In our approach we assume that second order dynamical shifts are negligible and that the linewidth
is independent of the magnetic field. Under these assumptions the line pattern generated by copper
hyperfine interaction (I = 3/2) is given by [20,21]:
F(f ) 1
4cos(2 mA ) 1
Cu Cu
   
The second component producing a shift in the field domain can be written in Fourier space:
exp( )2
i (2)
where σ is half of the sweep width of the spectrum.
According to deconvolution theorem and assuming that the nitrogen hyperfine constants are all the
same, in agreement with the above assumptions we may write an EPR spectrum in the form:
CuCu AAAi )]2cos(21[
where n is the number of nitrogens and
() is a FT-derivative Lorentzian line shape function given by:
Molecules 2014, 19 983
() exp( )
22i (4)
where is the half-height width in Gauss.
2.2. EPR Spectroscopy of (1:2)Copper(II)-L-Histidine
The EPR spectrum of the (1:2) Cu2+-L-His complex at pH 7.3 recorded at X-band at 77 K is
presented in Figure 2A. The spectrum is resolved in parallel direction of the g-tensor and shows three
of four well-resolved low-field parallel lines with a hyperfine splitting of 554 MHz and g = 2.237.
The detailed inspection of the perpendicular band reveals a slight rhombic distortion with g values of
2.044 and 2.047. The super-hyperfine splitting due to the directly bonded atoms is not seen in the
low-temperature spectrum. Better resolution was observed in the X-band EPR spectrum measured at
room temperature, however one should bear in mind that the structure of the copper-histidine complex
at low temperature may differ from the structure equilibrated at room temperature. Nevertheless, the
X-band EPR spectrum measured at room temperature shows an isotropic quartet, with partially
resolved hyperfine structure on the high field line (see Inset I, Figure 2). However, detailed
interpretation of the partially resolved multiplet is not straightforward.
Figure 2. The EPR spectra of [Cu(his)2] measured at 77 K and at room temperature.
(A) spectrum measured at X-band; (B) spectrum measured at S-band. Inset I: High-field
band of the spectrum measured at X-band at room temperature. Inset II: 2nd derivative of
the high field band of the S-band EPR spectrum at 77 K. E-experiment, S-computer
simulation (EPR data are given in Table 1).
Molecules 2014, 19 984
In order to obtain better resolution in the superhyperfine splitting due to the directly bonded ligands,
S-band EPR spectra at both room temperature and at 77 K were measured (shown in Figure 2B).
It is generally known, that the EPR measurements at frequencies lower that 9 GHz (S-band or L-band)
can result in: (a) reduced local strain effects, the so called “g-strain” and correlated “g-A” strain;
(b) increased resolution of super-hyperfine splitting due to the directly bonded ligand atoms to metal;
(c) suppression of spectral line-width and nuclear quadrupole interaction; and finally (d) improved
resolution of ligand super-hyperfine splitting due to the enhanced admixture of electronic and nuclear
spin functions which in turn increase probability of spectral transitions [22–26].
Generally, L-histidine is one of the most strongly coordinating aminoacids to metal ions possessing
three potential metal-binding sites (Figure 1) [27]. They are imidazole imido nitrogen, amino nitrogen
and carboxylate oxygen. The dissociation of the second proton of the imidazole group does not occur
at physiological pH (pKa = 14). Nitrogen has two stable isotopes, 14N (natural abundance 99.632%,
spin S = 1) and 15N (natural abundance 0.368%, spin S = ½). In the EPR spectra only couplings to
nitrogen-14 can be detected. The EPR spectrum measured at low temperature at X-band is shown in
Figure 2A. The resolution of the hyperfine splitting structure was further improved by the use of
S-band EPR spectroscopy. While the S-band spectrum measured at low temperature (Figure 2B) shows
well resolved superhyperfine patterns in the perpendicular region of the spectrum, the EPR spectrum
taken at room temperature lacks sufficient resolution (spectrum not shown). The second derivative
high-field band of the spectrum (Figure 2B, Inset II), shows nine resolved lines. The best fit to
experimental spectrum was achieved assuming the 4N:3NO ratio of 0.8:0.2 with superhyperfine
splitting constants summarized in Table 1. The spectral assignment would correspond to a major
abundance of metal species formed with both histidines in a histamine-like (4-N) coordination
(see below) [28,29], however presence of minor species containing 3-NO coordination mode cannot be
excluded (approximately 4:1 ratio in favour of 4-N coordination).
Table 1. Parameters used to simulate EPR spectra at S-band for [Cu(his)2] a,b.
Complex g1g2 g3A1(Cu) A2(Cu) A3(Cu) A1(N) A2(N) A3(N)
[Cu(his)2] 2.044 2.047 2.237 27 27 555 38/33
c 38/33
c 38/33 c
a Hyperfine and superhyperfine coupling constants are given in MHz; b Fluid solution EPR data (X-band and
S-band): giso = 2.117, Aiso
Cu = 199 MHz. Nitrogen shf structure not satisfactorily resolved; c Simulation of low
temperature EPR spectrum was performed using a mixture of four nitrogens (4N, splitting constant = 38 MHz)
and three nitrogens (3NO, splitting constant = 33 MHz) assuming the ratio 4N:3NO = 0.8:0.2.
Spin Hamiltonian parameters g|| and A|| reflect the donor atoms from the ligands. Plots of g|| versus
A|| are called Peisach – Blumberg diagrams and suggest the nature of the directly bonded donor atoms
to copper ion for cupric complexes [30]. The g|| value increases and A|| value decreases as oxygen
replaces either nitrogen or sulphur atom. A typical g|| value is 2.21 and a typical A|| value is 570 MHz
for 4 nitrogen donor set, while g|| of 2.42 and A|| of about 390 MHz can be expected for 4 oxygen
donor atoms. The spin Hamiltonian parameters for Cu-His (1:2) complex obtained by computer
simulation (Table 1) show the g|| value of 2.237 and A|| value of about 555 MHz. The data are in
agreement with either a homogenous 4-N donor set or a mixed 4-N/3-NO donor set with minor
abundance of 3-NO structures.
Molecules 2014, 19 985
2.3. Structural Characterization of Cu(L-His)2 Complex
L-Histidine can bind as mono-, bi- and tri-dentate ligand and its binding mode to a metal depends
on the pH of the solution. At physiological pH the major structure of L-histidine is shown in Figure 1.
Upon increase of pH the proton from the amino group is deprotonated to give a monoanionic
L ligand. Conversely, upon decrease of pH imidazole nitrogen and carboxylate oxygen are protonated.
Copper(II)-L-histidine species characterized upon increase of pH are: [Cu(HisH)]2+ (MHL),
[Cu(His)]+ (ML), [Cu(HisH)2]2+ (MH2L2), [Cu(His)(HisH)]+ (MHL2), [Cu(His)2] (ML2) and
[Cu(His)2(OH)] (MH1L2) [5]. The most abundant structures are MHL, MHL2 and ML2.
At physiological pH, the predominant structures (more than 99%) in solution are Cu(L-His)2 (ML2).
Copper(II) salt mixed with two equiv of L-histidine in aqueous solution results in the pH of
approximately 3.7 [5]. The solid state monocrystal X-ray structural analysis of this complex shows
four coordinate square-planar arrangement around copper ion with nitrogen and oxygen atoms in trans
coordination (Figure 3) [31].
Figure 3. The structure of the Cu(L-His)2 complex at acidic pH.
Upon increase of pH to a physiological value (7.2), the structural rearrangement of histidine ligands
takes place leading to formation of a square pyramidal copper(II) complex. The electronic spectrum of
(1:2) copper-histidine complex in fluid solution is shown in Figure 4. The absorption maximum was
observed at relatively long wavelength of 645 nm, in agreement with weak apical chelation by a donor
atom of greater ligand field strength than water, as proposed on the basis of ESEEM analysis [32].
Thus, in this complex, copper is coordinated by carboxylate oxygen and amino nitrogen of one
histidine molecule and imidazole and amino nitrogens and carboxylate oxygen in apical position of
another histidine molecule via semi-coordination (see Figure 5). The imidazole of L-histidine plays an
important role in copper chelation, not only in various metalloenzymes but also in thermodynamic
stabilization of various copper-amino acid complexes [33]. The tridentate coordination of L-histidine
to copper ion provides enhanced stability over binary copper(II) amino acid complexes.
Molecules 2014, 19 986
Figure 4. Absorption spectrum of (1:2) copper-histidine complex in PBS at room temperature.
Figure 5. The structure of the Cu(L-His)2 complex at physiological pH.
2.4. Fourier Transform of the CW-EPR Spectrum of Cu(L-His)2
In addition to detailed interpretation of CW-EPR spectra, Fourier transformation of the CW-EPR
spectrum measured at room temperature can be of help to discriminate between even and odd number
of directly bonded nitrogens to copper (see Figure 6). It has been observed that fourier transforms
consist of a few significant peaks separated by long regions where all the hyperfine components are
approaching an extreme value, while the flat regions correspond to those points where one or more of
the hyperfine components are small [20,21]. As already observed by other authors, the central region
of the fourier transform is very sensitive to the parity of directly bonded magnetically active atoms
(nitrogens) [20,21]. The phase of the enlarged region is inverted by varying number of the nitrogens.
Fourier transform of the fluid EPR spectrum of Cu(L-His)2 measured at room temperature is shown in
Figure 6. Inset B shows the phase-sensitive enlarged pattern of the Fourier transform of the fluid EPR
spectrum of Cu(L-His)2 at pH 7.3. Insets C and D are calculated FT-EPR spectra according to eqn. (3)
assuming four and three nitrogens, respectively.
Molecules 2014, 19 987
Figure 6. Fourier transform of the X-band CW-EPR spectrum of the complex [Cu(L-His)2]
([Cu] = 2 mM) in the phosphate buffer at pH = 7.3. (A) Fourier transform of the X-band
CW EPR spectrum measured at room temperature. Insets: High-field band of the EPR
spectrum measured at room temperature. (A) Real part of the FT-EPR spectrum;
(B) Enlargement of the circled region; (C) Part of the simulated FT-EPR spectrum assuming
four nitrogens; (D) Part of the simulated FT-EPR spectrum assuming three nitrogens.
FT simulation
pattern of A
3290 3340 3390
Fourier Transform
Looking at the match between Fourier transform of the experimental spectrum and simulated
FT-EPR spectra using three/four coordinated nitrogens, one can see that the experimental spectrum is a
superposition of both simulated spectra with major abundance of species containing four coordinated
nitrogens to copper ion and a minor abundance of species with three coordinated nitrogens.
Thus in fluid buffer solution at pH 7.3 we can expect an equilibrium between histamine-like (4-N)
and glycine-like (3-NO) coordination of two molecules of histidine to copper atom in the [Cu(L-His)2]
complex (Figure 7).
Figure 7. Glycine-like (3-NO) and histamine-like (4-N) types of coordination of two
molecules of histidine to copper(II) ion in an aqueous solution at pH 7.3.
_ _
Glycine-like coordination Histamine-like coordination
Molecules 2014, 19 988
3. Experimental
3.1. Metal-Chelate Solutions
L-Histidine and CuCl2·2H2O were obtained from Sigma Chemical Co. (Milwaukee, WI, USA).
Chelate solutions were prepared by mixing the ligand with the metal salt in Hepes buffer
(pH 7.3). The concentration of the metal was [Cu] = 2 mM. The metal to chelator ratio was 1:2
throughout the work. The pH was measured using a glass electrode connected to Digi-Sense
pH/mV/ORP meter (Cole-Parmer, Vernon Hills, IL, USA).
3.2. EPR Measurements
The EPR spectra were recorded on a Bruker EMX spectrometer (X-band) (Bruker BioSpin,
Karlsruhe, Germany) coupled to an Aspect 2000 computer and equipped with a variable temperature
unit. For low-temperature measurements cylindrical quartz sample tubes with 3.5 mm o.d. (3.0 mm i.d.)
were used. The low-temperature EPR spectrum of copper-L-histidine was also recorded at S-band on a
Bruker ESP 300 E spectrometer at the National EPR Research Facilities at the University of
Manchester, UK. Room temperature measurements at X-band were carried out using a flat cell. In g
factor evaluations field gradients were corrected using the internal reference standard marker
(gM = 2.0052). The CW-EPR spectra were simulated on an IBM compatible computer PC using the
QPOW program [34,35] and a program developed in our laboratory [36].
4. Conclusions
In this work we have demonstrated the potential of Fourier-transform CW-EPR spectroscopy in
the elucidation of magnetically active atoms directly bonded to metal ions. The results based on the
analysis of Peisach-Blumberg plots for spin Hamiltonian parameters and Fourier transform-CW
EPR spectroscopy of [Cu(L-His)2] complex in water solution at physiological pH revealed an
equilibrium between major abundance of histamine-like (4N) and minor abundance of glycine-like
(3NO) species.
We thank VEGA (#1/0856/11 and #1/0289/12) and APVV #0202-10 and APVV #0339-10 for
financial support. We thank Eric McInnes and Frank Mabbs for the access to EPR facilities at the
National EPR Centre, University of Manchester, UK. We thank one of the anonymous referees for
very constructive criticism.
Conflicts of Interest
The authors declare no conflict of interest.
Molecules 2014, 19 989
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Sample Availability: Not available.
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... ATP7A is an ATP-ase enzyme responsible for regulating copper levels in the body [152]. Menkes disease affects many systems in the body, clinical manifestations of which include neurological degeneration, "steely" hair, degeneration of connective tissues, and hypopigmentation of skin and hair [153,154]. ...
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In total, twenty elements appear to be essential for the correct functioning of the human body, half of which are metals and half are non-metals. Among those metals that are currently considered to be essential for normal biological functioning are four main group elements, sodium (Na), potassium (K), magnesium (Mg), and calcium (Ca), and six d-block transition metal elements, manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn) and molybdenum (Mo). Cells have developed various metallo-regulatory mechanisms for maintaining a necessary homeostasis of metal-ions for diverse cellular processes, most importantly in the central nervous system. Since redox active transition metals (for example Fe and Cu) may participate in electron transfer reactions, their homeostasis must be carefully controlled. The catalytic behaviour of redox metals which have escaped control, e.g. via the Fenton reaction, results in the formation of reactive hydroxyl radicals, which may cause damage to DNA, proteins and membranes. Transition metals are integral parts of the active centres of numerous enzymes (e.Dg. Cu,Zn-SOD, Mn-SOD, Catalase) which catalyze chemical reactions at physiologically compatible rates. Either a deficiency, or an excess of essential metals may result in various disease states arising in an organism. Some typical ailments that are characterized by a disturbed homeostasis of redox active metals include neurological disorders (Alzheimer's, Parkinson's and Huntington's disorders), mental health problems, cardiovascular diseases, cancer, and diabetes. To comprehend more deeply the mechanisms by which essential metals, acting either alone or in combination, and/or through their interaction with non-essential metals (e.g. chromium) function in biological systems will require the application of a broader, more interdisciplinary approach than has mainly been used so far. It is clear that a stronger cooperation between bioinorganic chemists and biophysicists - who have already achieved great success in understanding the structure and role of metalloenzymes in living systems - with biologists, will access new avenues of research in the systems biology of metal ions. With this in mind, the present paper reviews selected chemical and biological aspects of metal ions and their possible interactions in living systems under normal and pathological conditions.
... The conclusions about the planar-square structure of the obtained Cu(II) complexes based on the results of the IR spectra were additionally confirmed by EPR spectroscopy data [44][45][46][47]. In the EPR spectra of the studied compounds ( Figure 9) at room temperature, the hyperfine lines from the magnetic interaction of the unpaired electron spin with the copper atom nuclear spin were well resolved. ...
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New coordination oligomers and polymers of Sn(IV)-tetra(4-sulfonatophenyl)porphyrin have been constructed by the chelation reaction of its diaxialphenolates with Cu2+. The structure and properties of the synthesized polyporphyrin arrays were investigated by 1H Nuclear Magnetic Resonance (1H NMR), Infra Red (IR), Ultra Violet - Visible (UV-Vis) and fluorescence spectroscopy, mass spectrometry, Powder X-Rays Diffraction (PXRD), Electron Paramagnetic Resonance (EPR), thermal gravimetric, elemental analysis, and quantum chemical calculations. The results show that the diaxial coordination of bidentate organic ligands (L-tyrazine and diaminohydroquinone) leads to the quenching of the tetrapyrrole chromophore fluorescence, while the chelation of the porphyrinate diaxial complexes with Cu2+ is accompanied by an increase in the fluorescence in the organo-inorganic hybrid polymers formed. The obtained results are of particular interest to those involved in creating new ‘chemo-responsive’ (i.e., selectively interacting with other chemical species as receptors, sensors, or photocatalysts) materials, the optoelectronic properties of which can be controlled by varying the number and connection type of monomeric fragments in the polyporphyrin arrays.
... More detailed information on each of the modules is found in Dataset S1 was presented roughly as having half-Lorentzian and half-Gaussian broadenings with a g value of 2.16. The signal matched the signal observed with a standard solution of Cu(I)-cysteine at acidic pH ( Figure S5B), 33 fitting the spectrum of copper sulphate at acidic pH (3 N HCl). This signal was characterized by a copper-chloride/ water complex in fast dynamics and/or coupled by exchange and magnetic dipolar interaction with spectroscopic parameters consistent with data reported for Cu(II). ...
Background and Aims: Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, with its main pathology attributed to copper-mediated oxidative damage. The limited therapeutic effect of copper chelators and the early occurrence of mitochondrial deficits, however, undermine the prevalence of this mechanism. Methods: We characterized mitochondrial DNA copy number and mutations as well as bioenergetic deficits in blood from patients with WD and in livers of tx-j mice, a mouse model of hepatic copper accumulation. In vitro experiments with hepatocytes treated with CuSO4 were conducted to validate in vivo studies. Results: Here, for the first time, we characterized the bioenergetic deficits in WD as consistent with a mitochondrial DNA depletion-like syndrome. This is evidenced by enriched DNA synthesis/replication pathways in serum metabolomics and decreased mitochondrial DNA copy number in blood of WD patients as well as decreased mitochondrial DNA copy number, increased citrate synthase activity, and selective Complex IV deficit in livers of the tx-j mouse model of WD. Tx-j mice treated with the copper chelator penicillamine, methyl donor choline or both ameliorated mitochondrial DNA damage but further decreased mitochondrial DNA copy number. Experiments with copper-loaded HepG2 cells validated the concept of a direct copper-mitochondrial DNA interaction. Conclusions: This study underlines the relevance of targeting the copper-mitochondrial DNA pool in the treatment of WD separate from the established copper-induced oxidative stress-mediated damage.
... The immersion of discs of F(3)-Cu-D in an aqueous/methanol solution buffered at physiological pH gave rise to the discolouration of the initially blue films and the colouration initially colourless solution. The interaction of the amino acids with the F(3)-Cu-D lead to the initial material F(3), with the concomitant displacement of the dye and the formed new colourless complex Cu-(amino acid) [46][47][48][49][50][51][52][53][54][55][56][57], initially to the swelled film and finally to the solution. ...
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We anchored a colourimetric probe, comprising a complex containing copper (Cu(II)) and a dye, to a polymer matrix obtaining film-shaped chemosensors with induced selectivity toward glycine. This sensory material is exploited in the selectivity detection of glycine in complex mixtures of amino acids mimicking elastin, collagen and epidermis, and also in following the protease activity in a beefsteak and chronic human wounds. We use the term inducing because the probe in solution is not selective toward any amino acid and we get selectivity toward glycine using the solid-state. Overall, we found that the chemical behaviour of a chemical probe can be entirely changed by changing its chemical environment. Regarding its behaviour in solution, this change has been achieved by isolating the probe by anchoring the motifs in a polymer matrix, in an amorphous state, avoiding the interaction of one sensory motif with another. Moreover, this selectivity change can be further tuned because of the effectiveness of the transport of targets both by the physical nature of the interface of the polymer matrix/solution, where the target chemicals are dissolved, for instance, and inside the matrix where the recognition takes place. The interest in chronic human wounds is related to the fact that our methods are rapid and inexpensive, and also considering that the protease activity can correlate with the evolution of chronic wounds.
... EPR spectroscopy is widely used in the study of copper complex formation. However, room temperature EPR measurements allowing a direct comparison with mole-ratio and kinetics data are quite limited by comparison to frozen solution studies [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. In the present study a series of solutions containing [CuCl 2 (en)] or [CuCl 2 (terpy)] complexes and ligand molecules (1,2,4-triazole, imidazole, or pyrazine, respectively) in the molar ratios 1:1 and 1:2 have been analyzed using room-temperature EPR. ...
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Comprehensive mole-ratio, kinetic, EPR and X-ray studies were performed to investigate substitution reactions between square-planar [CuCl2(en)] (en = ethylenediamine) and square-pyramidal [CuCl2(terpy)] (terpy = 2,2’:6’,2”-terpyridine) complexes with bio-relevant nucleophiles at 295 K in 0.010 M Tris-HCl buffer (pH = 7.4) in the presence of 0.010 M NaCl. According to mole-ratio and EPR studies, Tris buffer reacts with [CuCl2(en)], and likely the complex geometry remains square-planar. Kinetic results have shown that the order of reactivity of nucleophiles towards [CuCl2(en)] in solution for both reaction steps is 1,2,4-triazole > imidazole > pyrazine. The order of reactivity of the azole and diazine ligands toward [CuCl2(terpy)] for the first reaction step is imidazole > 1,2,4-triazole > pyrazine, while for the second one is pyrazine > 1,2,4-triazole > imidazole. X-ray studies indicate great affinity of ethylenediamine toward Cu(II), which results in crystal structures of [Cu(en)2]Cl2·H2O and [Cu(en)2][ZnCl4], instead of dinuclear or heteronuclear complexes with a bridging pyrazine ligand. The recorded EPR spectra demonstrate that all ligands interact with Cu(II) complexes, except in the case of the [CuCl2(en)] complex, where the competition between ligand and Tris molecules might be observed.
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A biosensing scheme requiring only one-step sample incubation before signal collection, and using a compact “three-in-one” probe of target-binding, signal conversion, and amplification, may greatly simplify the design of biosensors. Therefore, sparing the multi-step addition of enzymes, protein, and nanomaterial, as well as the associated complexity and non-specific interactions. In this work, a peptide probe aimed at such compact features has been designed, based on protein-triggered, conformation-driven, and Cu (II) facilitated side-chain di-tyrosine cyclization. This design can use target-probe recognition to induce discriminated cross-linking and self-cleavage of the probe, resulting in retention or dissociation of a signal amplification motif from the search and consequently quantitative detection performance. The method has also been tested preliminarily in fractioned osteosarcoma clinical samples, showing an acceptable coherence between signal readout and clinical diagnosis. On the basis of these early findings, it is reasonable to assume that the proposed probe will be beneficial for the next development of tumor screening and prognosis sensors.
A series of thirty six complexes obtained from acetonitrile solutions of Cu(NO3)2, Ln(NO3)3 and 2,2‐biyridine or 5,5‐dimethyl‐2,2‐bipyridine (Ln=La‐Yb excluding Pm) have been characterized. The crystals may be grouped into six types: Type‐I, [Cu(bpy)3][La(NO3)5(OH2)]; Type‐II, [Cu(bpy)3][Ln(NO3)5(OH2)] ⋅ 2CH3CN (Ln=Ce, Pr); Type‐IIa, [Cu(5,5‐dmbpy)3][Ln(NO3)5(OH2)] ⋅ 2CH3CN (Ln=La, Ce); Type‐III, [Cu(bpy)3][Ln(NO3)5] (Ln=Nd, Sm–Lu); Type‐IIIa [Cu(5,5‐dmbpy)3][Ln(NO3)5] (Ln=Pr, Nd, Sm‐Lu); Type‐IV, [Cu(5,5‐dmbpy)2(NO3)]2[Ln(NO3)5(OH2)]⋅CH3CN [Ln=La‐Nd, Sm‐Tb). The coordination numbers of the nitrato and aquonitrato lanthanide anions are 10 and 11, respectively. The tris‐chelates in Types I, II and IIIa undergo static Jahn‐Teller distortion while Types IIa and III support fluxional tris‐chelate ions. Type‐IV contains distortion isomers of [Cu(5,5‐dmbpy)2NO3]+. The structural trends are analysed in terms of lanthanide contraction and non‐covalent interactions in the crystals. EPR and electronic spectral characteristics are discussed for the six types of compounds. Cu(II) nitrate and lanthanide(III) nitrates combine with bipyridyl type ligands in acetonitrile to form a series of tris‐chelate and bis‐chelate copper complexes having 10 and 11 cooordinate lanthanide nitrate and aquonitrate complexes as counterions. The tris‐chelates have a range of tetragonalities influencing their EPR spectra. Acetonitrile solvent exerts a structure directing role, especially for aquonitrate complexes leading to H‐bonded anion dimers and chains.
Extracellular matrix (ECM) enzymes such as lysyl oxidase (LOX) provide a new possibility to contain the invasive progress of cancer. Unlike conventional enzymes, the activity of ECM enzymes is not simply the conversion of the substrate to the product; the amount of enzymes such as matrix metalloproteinases in the ECM changes the structural integrity and morphology of the ECM. These are all important aspects that must be monitored in a spatiotemporally coupled fashion to fully understand their procancerous effect. To achieve this goal, a new molecular probe is developed, which, unlike antibodies or aptamers, can interact with the target enzyme in a more interactive way: the probe can withdraw the metal ion cofactor of the enzyme and modulate its catalytic ability. This can lead to self-propagated cross-linking of the probes to form a network not dissimilar to the collagen and elastin network of the ECM, formed through LOX activity. Thus, the biosensing process itself is a biomimetic of what may occur in vivo in the ECM, and three distinct types of signal readouts can be simultaneously recorded on the sensing surface to provide a fuller picture of ECM enzyme activity, not achievable with traditional designs. Using this method, a parallel between the detected signal and the progress of colorectal cancer can be observed. These results may point to prospective application of this method in evaluating ECM-related tumor invasiveness in the future.
Several types of biological samples, including hair strands, are found at crime scenes. Apart from the identification of the value and the contributor of the probative evidence, it is important to prove that the time of shedding of hair belonging to a suspect or victim matches the crime window. To this end, to estimate the ex vivo aging of hair, we evaluated time-dependent changes in melanin-derived free radicals in blond, brown, and black hairs by using electron paramagnetic resonance spectroscopy (EPR). Hair strands aged under controlled conditions (humidity 40%, temperature 20-22°C, indirect light, with 12/12 hour of light/darkness cycles) showed a time-dependent decay of melanin-derived radicals. The half-life of eumelanin-derived radicals in hair under our experimental settings was estimated at 22 ± 2 days whereas that of pheomelanin was about 2 days suggesting better stabilization of unpaired electrons by eumelanin. Taken together, this study provides a reference for future forensic studies on determination of degradation of shed hair in a crime scene by following eumelanin radicals by utilizing the non-invasive, non-destructive, and highly specific EPR technique.
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Copper(II) complex systems containing 3,5-di, 4-, or 5-chlorosalicylic acids (X-ClsalH) and different copper(II) salts (copper acetate (Cu(ac)2) or copper sulphate (CuSO4)), with varying 2,6-pyridinedimethanol (pydime) concentration, [Cu(ac)2(aq) or CuSO4(aq) + 2 (X-ClsalH(solv)) + x pydime(solv)], where X = 3,5-di, 4-, or 5- and x = 0, 1, 2, 4, or 8, were prepared. The effects of two copper(II) salts (containing anions of different basicity) and N-donor ligand (pydime) with varying ligand-to-metal ratio (x) on the formation of resulting complexes were studied by electron paramagnetic resonance (EPR) spectroscopy in frozen water/methanol (1:3 v/v) solutions. For x ≥ 2, unusual Cu(II) EPR spectra with "inverse" axial g values of (g ⊥ > g ∥ > 2.0023) were observed, which can indicate the compressed octahedral geometry of the central copper atom with the unpaired electron/hole localized on the $ d_{{z^{2} }} $ orbital. However, for x = 1, composite Cu(II) EPR spectra with both "usual" and "inverse" axial g values were detected. Finally, for x = 0 (ligand not present) Cu(II) EPR spectra only with the ‘usual’ axial g values of g ∥ > g ⊥ > 2.0023 were collected, which can indicate the elongated octahedral geometry of the central copper atom with the unpaired electron/hole localized on $ d_{{x^{2} - y^{2} }} $ orbital. The above described observations are independent of the usage of different copper(II) salts and X-chlorosalicylic acids.
A simple method based on Fourier analysis of EPR spectra is proposed for a straightforward discrimination between an even or odd number of nitrogen nuclei bound to copper in solution. The method is tested on a ternary complex of copper with phenantroline and oxidized glutathione (Cu-Phen-GSSG), verifying the structural arrangement at room temperature previously defined by other techniques.
A method of reducing EPR spectra of free radicals in solution is presented in detail. This method is based on the use of the fast Fourier transform algorithm and curve fitting in the Fourier space by weighted least-squares minimization. Comparison with previous work is shown for EPR spectra of methyl viologen.
Co-ordination modes for the various copper(II) complexes of glycine(Gly)-containing di- and tri-peptides (HL) with non-co-ordinating side-chains have been investigated. The e.s.r. spectra of predominant species at 1 : 1, 2 : 1, and 50 : 1 ligand : metal concentration ratios in the region pH ≈ 6–13 have been recorded in fluid and frozen aqueous solutions, and evaluated by computer simulation. The energies of the d–d electronic transitions have been determined by Gaussian analysis of the visible absorption spectra. Molecular-orbital coefficients characteristic of metal–ligand bonds for the various 1 : 1 and 1 : 2 complexes have been calculated assuming effective D4h symmetry. At ligand excess in alkaline solution, the temperature strongly affects the chemical equilibria: low temperature promotes the formation of 1 : 2 complexes: [Cu(LH–1)L]– at pH ≈ 9, and [Cu(LH–1)2]2– at pH ≈ 13 in the case of X-Gly type dipeptides. In the predominant isomers of these complexes one of the dipeptide molecules is co-ordinated equatorially through its amino nitrogen, deprotonated peptide nitrogen, and carboxylate oxygen atoms. The amino group of the other dipeptide occupies an axial position, while the fourth equatorial donor atom is either the peptide oxygen (pH ≈ 9) or the deprotonated peptide nitrogen (pH ≈ 13) of the second ligand. In the latter case, axial co-ordination of the second carboxylate group is also likely. Competition can be observed between the σ and π bonds in the equatorial plane on the one hand, and between the σ bonds of different symmetries on the other hand. The influence of the co-ordination modes, the type of ligand, and the temperature on the covalent character of the metal–ligand bonds is discussed.