# Complexes of Cu(II) Ions and Noncovalent Interactions in Systems with L-Aspartic Acid and Cytidine-5'-Monophosphate

Authors: Romualda Bregier-Jarzebowska; Anna  Gasowska; Lechosław Lomozik
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Interactions between aspartic acid (Asp) and cytidine-5-monophosphate (CMP) in metal-free systems as well as the coordination of Cu(II) ions with the above ligands were  studied. The composition and overall stability constants of the species formed in those systems were determined by the potentiometric method, and the interaction centres in the ligands were identified by the spectral methods UV-Vis, EPR, NMR, and IR. In metal-free systems, the formation of adducts, in which each ligand has both positive and negative reaction centres, was established. The main reaction centres in Asp are the oxygen atoms of carboxyl groups and the nitrogen atom of the amine group, while the main reaction centre in CMP at low pH is the N(3) atom. With increasing pH, the efficiency of the phosphate group of the nucleotide in the interactions significantly increases, and the efficiency of carboxyl groups in Asp decreases. The noncovalent reaction centres in the ligands are simultaneously the potential sites of metal-ion coordination. The mode of coordination in the complexes formed in the ternary systems was established. The sites of coordination depend clearly on the solution pH. In the molecular complexesML⋯L, metallation involves the oxygen atoms of the carboxyl groups of the amino acid, while the protonated nucleotide is in the outer coordination sphere and interacts noncovalently with the anchoring CuHx(Asp) species. The influence of the metal ions on the weak interactions between the biomolecules was  established.


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## Body

## 1. Introduction

Interactions between
metal ions and nucleic acids or their fragments affect the character of many
biological processes including that of genetic information transfer [1–6]. The
effective centres of coordination with metal ions are the donor nitrogen atoms
N(3) from pyrimidine bases and the oxygen atoms from the phosphate groups of
the nucleotide. These centres are also the sites of noncovalent interactions
with the other bioligands present in living organisms, such as small organic
polycations, polyamines, or amino acids [7, 8].Aspartic acid (Asp) is a
naturally occurring amino acid. Along with glutamic acid, it acts as a
neurotransmitter in the central nervous system [9–14]. Aspartic acid takes part
in thermogenic processes induced by prostaglandin E1 (PGE1)
[15] and is a component of the active centres of some enzymes. It influences
the solubility and ionic character of proteins, protects the liver against the
toxic effect of drugs, participates in the generation of ribonucleotides thereby
enhancing the effectiveness of the immunological system of the organism, and prevents
the destruction of neurons and the brain. The presence of metal ions in living
organisms modifies the character of bioprocesses. The reactions between the
amino acid and the metal ions are considered as models of the processes which
take place at the molecular level in the metal/protein system.Although studies of the
systems of metals with dicarboxylic amino acids have been carried out since the
1970s, [16–19], no definite conclusions as to the mode of coordination have
been obtained, in particular in the ternary systems, which is related to the
fact that aspartic acid has three functional groups (one amine group and two
carboxyl ones). To our best knowledge, no information has been reported on
interactions in the metal-free systems of aspartic acid/nucleotide or on the
character of interactions in ternary systems including metal ions.This paper presents the results
of a study on the coordination of Cu(II) ions with aspartic acid  and cytidine-5′-monophosphate (CMP) and the
interactions of these bioligands in metal-free systems.


## 2. Experimental

Cytidine 5′-monophosphate, C9H14N3O8P,
and L-aspartic acid, C4H7NO4, were purchased
from Sigma-Aldrich and were used without further purification. Cu(NO3)2 bought in POCH Gliwice (Poland)
was twice recrystallized from H2O before use. The method of
determination of Cu(II) concentration in a parent solution of a concentration
of about 2.4×10−2M was described earlier [20, 21]. Potentiometric
studies were performed on a Methrom 702 SM Titrino with a glass electrode
Methrom 6.0233.100 calibrated in terms of hydrogen ion concentration [22] with
a preliminary use of borax (pH=9.225) and phthalate (pH=4.002) standard buffers. The concentrations of the CMP and Asp
were 5⋅10−3 in the metal-free systems and from 1⋅10−3 to 2.5⋅10−3M in the systems with Cu(II). The ratio of
ligand1:ligand2 in the metal-free systems was 1:1, metal:ligand1 was 1:2.5, and
metal:ligand1:ligand2 were 1:1:1 or 1:2.5:2.5 in the ternary systems (ligand1-Asp,ligand2-CMP). Potentiometric titrations were performed at the ionic
strength μ=0.1M (KNO3),
at 20±1C° under helium, using
as a titrant CO2-free NaOH solution (about 0.2M). For each
system a series of 10 titrations was made; the initial volume of the sample was
30cm3. No precipitate formation was, observed in the entire pH
range studied. Calculations were performed using 100–350 points for each job. The
selection of the models and the determination of the stability constants of the
complexes were made using the SUPERQUAD program [23], whereas the distribution
of particular forms was determined by the HALTAFALL program [24]. The computer
procedures used for the purpose, choice of the models. and the criteria of
verification of results are described in [25–29]. The samples for C13 NMR
and P31 NMR investigation were prepared by dissolving
appropriate amounts of ligands and Cu(NO3)2 in D2O and adjusting pH by the addition of NaOD (or (C2H5)4NOH)
and DNO3, correcting pH-readings (a pH-meter C5-501 made by
Elmetron) according to the formula: pD=pHreadings+0.40 [30]. The concentration of the ligands in the samples was 0.05 M, and the
concentration ratio of Cu(II) to CMP and Asp was 1:200:200. C13 NMR
spectra were recorded on an NMR Gemini 300 VT Varian spectrometer using dioxane
as an internal standard. The positions of C13 NMR signals were
converted to the TMS scale. P31 NMR spectra were taken on an
NMR Varian Unity 300 spectrometer with H3PO4 as a standard.
UV-Vis spectra were taken on a UV 160 Shimadzu
spectrometer for the ligand and metal concentrations of the same value as in
the samples for potentiometric titrations. Electron spin resonance (EPR)
spectra were taken at 77 K in a water-glycol solution (3:1, v/v) on a
Radiopan SE/X 2547 spectrometer (CCu2+=0.002M) at the ratio of
metal:amino acid ratio 1:4 and metal:nucleotide:amino acid ratio
1:2.5:2.5. IR measurements were carried out using a Bruker ISS 66vS
spectrophotometer.The hydrolysis constants
for the metal ion Cu(II) were taken from [31] and were fully employed in the
calculations.The ligands studied are
presented in Scheme1.Scheme 1
Chemical formulae of the bioligands studied.


## 3. Results and Discussion

In
the systems of polyamine/nucleotide studied earlier at our laboratory, noncovalent
interactions were observed in the pH ranges in which one ligand was
deprotonated (nu-cleotide) and the other was protonated (polyamine), and the
molecular complex formation was a result of an ion-ion or ion-dipole reaction
[26, 32–35]. In the systems amino acid/nucleotide, the pH ranges of protonation
of both ligands overlap and each ligand has both positive and negative reaction
centres. The formation of a molecular complex can be described by the equation: HxAsp+Hy(CMP)⇆(Asp)H(x+y−n)(CMP)+nH+. The release of a proton in this
reaction permits the use of thepotentiometric method for determination of the
composition and stability constants of the adducts. Analogous procedures were
used for the investigation of the coordination compounds. The modes of
interactions were deter-mined on the basis of the spectroscopic measurements in
the pH ranges in which particular complexes dominate, as established on the
basis of the equilibrium study.


### 3.1. Asp/Nucleotide Metal-Free
Systems

Table1 presents the composition, overall stability constants (logβ), and the equilibrium constants of the
formation (logKe) of
molecular complexes appearing in Asp/CMP systems, determined from the computer
analysis of the potentiometric titration data.Table 1
Overall stability constants(logβ) and equilibrium constants (logKe) of adducts formation in
Asp/CMP system.
SpecieslogβlogKeH3Asp15.42 (3)2.06H2Asp13.36 (1)3.73HAsp9.63 (1)9.63H2CMP10.90 (1) [20]4.47HCMP6.42 (2) [20]6.42(Asp)H4(CMP)27.13 (4)2.87(Asp)H3(CMP)23.37 (3)2.84(Asp)H2(CMP)18.79 (3)2.74(Asp)H(CMP)12.01 (3)2.38As it was described earlier [28, 29, 36],
the occurrence of noncovalent interactions between the ligands and the formation
of molecular complexes in the system studied is indicated by the coincidence of
the titration curves obtained experimentally and those obtained by computer
simulation (with the use of the determined β values) as given in Figure 1.Figure 1
Experimental and simulated titration curves for the Asp/CMP system:
dotted line: experimental curve; solid line: simulated curve (an adduct formation was taken into account); dashed line: simulated curve (an adduct formation was not taken into account).Deprotonation of the phosphate group
of the nucleotide begins at low pH (logK3~0.4, [37, 38]), beyond
the range of the study. Subsequent stages of deprotonation correspond to the
abstraction of a proton from the endocyclic nitrogen atom N(3) from CMP and of
another proton from the phosphate group. The deprotonation of the aspartic acid
molecule begins with the proton abstraction from the carboxyl group C(1),
followed by dissociation of the –C(4)OOH
and the –NH3+ group [39, 40].Because of the different stoichiometric
compositions of particular species, the overall stability constantslogβ cannot be directly applied in analysis
of the character of interactions. Therefore, the efficiency of bonding was
estimated on the basis of the equilibrium constants calculated, for example,
for the species (Asp)H2(CMP): logKe=logβ(Asp)H2(CMP)−logβ(HAsp)−logβH(CMP).
On the basis of the protonation constants of the ligands (Table 1) and the pH
ranges of occurrence of individual species (Figure 2), the substrates in the
adduct formation reactions were identified.Figure 2
Distribution diagram for the Asp/CMP system; percentage of the species refers to total ligands. (1)H3Asp; (2) H2Asp; (3) HAsp; (4) Asp; (5) H2CMP; (6) HCMP; (7) CMP; (8) (Asp)H4(CMP);
(9) (Asp)H3(CMP); (10) (Asp)H2(CMP); (11) (Asp)H(CMP); CAsp=6×10−3M; CCMP=6×10−3M.The complex (Asp)H4(CMP)
appears at pH below 4.0 (Figure 2) in the range in which one of the
carboxyl groups of Asp and partly the –PO42− group from CMP are deprotonated [41–44]. In the C13 NMR spectra, the
chemical shifts of the signals assigned to C(2) and C(4) from the vicinity of N(3)
of the nucleotide changed by 0.982 and 0.760 ppm (pH 3.0), respectively, (Table 2) indicate that the protonated N(3)H from CMP is a positive centre of weak
interactions.Table 2
Differences betweenC13 NMR and P31 NMR chemical shifts for the ligands in the Asp/CMP system in relation to the free ligands [ppm].
AspCMPpHC(1)C(2)C(3)C(4)C(2)C(4)C(5)C(6)C(5′)P3.00.0490.0250.0220.0020.9820.7600.0570.0480.0440.0184.20.0000.0130.0700.0540.8610.6610.0400.0470.0600.0255.50.0530.0470.0270.0400.4400.5930.0680.0540.0990.0408.00.4541.0480.2271.3220.1910.1980.0680.0640.1110.105The lack of significant changes in
theP31 NMR spectrum suggests that the partly protonated (however
negative) phosphate group is not active, which is a result of the repulsion
from the negative carboxyl group present in the neighbourhood of –NH3+ group of Asp. The change in the chemical shift of the signal assigned to C(1) by 0.049 ppm suggests that the negative centre of interaction is thedeprotonated carboxyl group from Asp. (As established earlier, the energy of
the noncovalent interactions does not correspond directly with the chemical
shift value [27, 29, 34]). The protonated carboxyl group is not involved in the
interactions as evidenced by the lack of changes in the chemical shifts of the
NMR signals assigned to the carbon C(4) atom. In the (Asp)H3(CMP)
adduct, dominant at a pH close to 4, the N(3)H group of CMP remains a positive
centre of interactions as indicated by the changes in the NMR chemical shifts,
while the group –C(4)OO− becomes a
negative centre of interaction (Table 2), which corresponds to the
deprotonation of the second carboxyl group. The phosphate group remains
inactive. The involvement of only one centre from each ligand in the
interactions is confirmed by similar values of the equilibrium constants of the
tri- and tetraprotonated complexes of logKe=2.84 and 2.87, respectively.With increasing pH, the
deprotonation of the N(3)H group from the pyrimidine ring of CMP takes place.
The (Asp)H2(CMP) adduct starts forming from a pH close to 4 and
reaches its maximum concentration at a pH of about 5.5. The changes in the
chemical shifts of the carbon atoms C(2) and C(4), neighbouring the N(3) atom
of the nucleotide at pH 5.5, in the region of domination of the (Asp)H2(CMP)
adduct, are 0.440 and 0.593 ppm. This observation indicates that N(3) is a
centre of interactions, however, since it is already deprotonated, an inversion
of interactions takes place and the N(3) atom becomes a negative centre of
interaction. The P31 NMR spectrum of (Asp)H2(CMP) at
pH 5.5 does not show any significant changes in the chemical shift of the
phosphorus atom (Table 2), which indicates a low efficiency of the phosphate
group.Above the physiological pH, the
formation of (Asp)H(CMP) begins and it dominates at a pH of about 8. In these
complexes, the phosphate group from the nucleotide is engaged in noncovalent
interactions with amino acid as proved by the changes in the position of the
signal ofP31 NMR, by 0.105 ppm at pH=8. Changes in the
chemical shifts of the C(2) and C(4) atoms from CMP by 0.191 and
0.198 ppm, respectively, indicate that the deprotonated N(3) atom is
another negative centre of interaction. On the other hand, as follows from the changes
in the chemical shift of the signal assigned to C(2) (1.048 ppm), the protonated amine group from Asp (positive, logK1=9.6) takes part in the
interaction. In the molecule of the nucleotide, there are no positive reaction
centres, thus the intermolecular interactions of –COO− are impossible, (similar to the intramolecular interactions). Because of the
lack of another active centre in the Asp molecule, no increase in the logKe value of the
monoprotonated complex, relative to that of diprotonated one, is observed. The
unexpected changes in the chemical shift of the signals assigned to C(1) and C(4) from Asp in the C13 NMR spectrum (Table 2) are a consequence of the interaction of the carboxyl
groups (hard base) with Na+ ions (hard acids) present in the systems
studied. In the additional C13 NMR spectrum taken for the
(Asp)H(CMP) adduct in the system containing (C2H5)4NOH
instead of NaOH, the changes were insignificant (at pH 8: 0.007 and 0.020 ppm for C(1) and C(4), resp.). The positions of the
signals assigned to C(1) and C(4) from Asp in the spectra
of the Na+ free system Asp/CMP were compared to those in the spectra
of Asp, without sodium ions. (This results also explains the unexpected shifts
in the signals assigned to carbon atoms from carboxylate groups in some other
studied systems.) The above conclusions are confirmed by analysis of IR spectra
recorded in the same conditions as NMR ones. A comparison of the position of
the IR band assigned to –COO− of the amino acid (1615 and 1584cm−1) and the (Asp)H(CMP)
adduct (1617 and 1586cm−1) shows that these groups are not
involved in the ligand-ligand interactions (Figure 3), because no changes in
the band positions were observed.Figure 3
IR spectra of Asp and (Asp)H(CMP) adduct pH = 8;CAsp=0.2M, CCMP=0.2M.In all adducts studied above,
noncovalent interaction occurs with the inversion of interaction sites at a pH
close to 3.5 and close to 7, as illustrated in Scheme2. The pH values of the
inversion correspond to the values of the protonation constants, and changes in
the mode of interaction are a result of the deprotonation of the second
carboxyl group of Asp, endocyclic N(3)H, and the phosphate group of CMP. No
significant changes are noted in the acid-base equilibria of the ligands as is
usually the case  when metal-ligand bonds
are formed, which confirms that the interactions are weak, noncovalent.Scheme 2
Noncovalent interaction in the Asp/CMP system.


### 3.2. Cu/Asp Binary Systems

The
stability constants of Cu(II) complexes with Asp were determined in the same
conditions in which the heteroligand complexes in the ternary systems are
formed (Table3). The results are in agreement with the earlier reported data
[39, 40, 45, 46].Table 3
Overall stability constants(logβ) and equilibrium constants (logKe) for the complexes of Cu(II)
ions with Asp or CMP and Cu(II) ions with Asp and CMP.
EquilibriumlogβlogKeCu + CMP⇆ Cu(CMP)2.71 (7) [20]2.71Cu + CMP +H2O⇆ Cu(CMP)(OH) + H+−4.26 (6) [20]Cu +H+ + Asp ⇆ CuH(Asp)12.78 (5)3.15Cu + Asp⇆ Cu(Asp)8.76 (3)8.76Cu + 2Asp⇆ Cu(Asp)215.35 (4)6.59Cu + Asp +H2O⇆ Cu(Asp)(OH) + H+0.79 (9)Cu + 4H+ + Asp + CMP ⇆ Cu(Asp)H4(CMP)32.86 (5)Cu + 3H+ + Asp + CMP ⇆ Cu(Asp)H3(CMP)29.14 (5)5.46Cu + 2H+ + Asp + CMP ⇆ Cu(Asp)H2(CMP)25.28 (4)6.08Cu +H+ + Asp + CMP ⇆ Cu(Asp)H(CMP)20.12 (4)4.94Cu +H++ 2Asp + CMP ⇆ Cu(Asp)2H(CMP)29.87 (4)Cu + Asp + CMP +H2O⇆ Cu(Asp)(CMP)(OH) + H+4.69 (4)In the pH range from 2.5 to 6, a protonated species
CuH(Asp) is formed, while at pH 5.5, the dominant complex is Cu(Asp), binding
about 80% of copper ions. The species Cu(Asp)2 dominates  at a pH from 7 to 10, while the
hydroxocomplex Cu(Asp)(OH) begins forming from a pH close to 6 reaching a
maximum concentration above pH 10.5.The UV-Vis and EPR spectral
parameters (Table4) at pH 3, at which the CuH(Asp) complex dominates: λmax=756nm,g∥=2.413 and A∥=134, indicate
that the oxygen atoms from the Asp carboxyl groups are exclusively involved in the
coordination of the copper ions [26, 32, 47, 48], while the protonated amine group –NH3+ is blocked to coordination and
does not take part in the interactions ({Ox} chromophore). Conclusions concerning
the mode of coordination were drawn on the basis of an analysis of the relation
between the spectral parameters and the number of donor atoms in the inner
sphere of Cu(II) coordination. For
tetragonal and square pyramidal species, the ground state is normally dx2−y2 or rarely dxy. As earlier established for Cu‐Nx(x=1–6) and Cu‐NxOy(x=0–4,y=0–6) in planar bonding, the value
of g∥ decreases and that of A∥ increases [47, 49]. In
general, the value of g∥ is changed in the order CuO4>CuO2N2>CuON3>CuN4. Weak bonding of the donor atoms at
the axial position is a result of the interactions between the 4s and 4p metal
orbitals and the ligand orbitals. The hyperfine coupling constant was
experimentally observed to decrease with increasing electron density of the 4s
orbital [47].Table 4
Vis and EPR spectral data for Cu(II)/Asp and Cu(II)/Asp/CMP systems.
SpeciespHλmax [nm]ε[M−1cm−1]EPRg∥=(dm3/mol⋅cm3)A∥(10−4cm−1)CuH(Asp)3756272.413134Cu(Asp)5700402.301171Cu(Asp)28630762.257187Cu(Asp)H4(CMP)3780252.409138Cu(Asp)H2(CMP)4.5710292.275179Cu(Asp)H(CMP)6700302.269182Cu(Asp)2H(CMP)8660492.261182Cu(Asp)(CMP)(OH)10.5630962.258183The carboxyl group involvement is
confirmed by the changes in theC13 NMR chemical shifts of the carbon atoms from the two carboxyl groups: C(1) and C(4) by 1.536 and 1.163 ppm, respectively (Table 5).Table 5
Differences betweenC13 NMR and P31 NMR chemical shifts
for the ligands in the Cu(II)/Asp and
Cu(II)/Asp/CMP systems in relation to metal-free systems [ppm].
SystemsAspCMPpHC(1)C(2)C(3)C(4)C(2)C(4)C(5)C(6)C(5′)PCu(II)/Asp3.01.5360.1290.1221.163——————5.50.0500.0700.0200.050——————8.00.5950.6020.1690.755——————Cu(II)/Asp/CMP3.01.3710.4600.1471.0940.4630.5280.0750.0850.0570.1664.50.0690.6430.1130.0801.0420.9320.0520.0470.0510.0806.00.7690.7820.1420.8090.4860.7720.0620.0460.0760.1908.00.8090.7490.1520.6890.8600.8580.0710.0640.0620.129The increase in the equilibrium
constant of the formation of Cu(Asp) by approximately 5.5logKe unit relative to the value
for the protonated complex (Table 3) points to the involvement of the
deprotonated amine group –NH2 (for pH above 5) in the coordination of Cu(Asp) species, which is consistent
with the model proposed by Chaberek and Martell, later confirmed in [39, 50].
Moreover, in the electronic absorption spectrum, the λmax is shifted toward higher energy—from
756 to 700 nm, which points to the engagement of the nitrogen atom from
Asp in metal coordination, (besides the oxygen atoms). The {N,Ox} type
coordination was also confirmed by the EPR results as at pH = 5, g∥=2.301
and A∥=171. The attachment of a subsequent molecule of
amino acid to the anchoring Cu(Asp), according to the equation Cu(Asp)+Asp⇆Cu(Asp)2,
leads to a decrease in the equilibrium constant by about 2 logKe, units, so by a value suggesting the same mode of
coordination in the 1:1 and 1:2 complexes, taking into consideration the
statistical relations. The spectral parameters obtained from the UV-Vis and EPR
spectra for the Cu(Asp)2 complex are λmax=630nm,g∥=2.257nm, and A∥=187 (Table 4) indicating (in  agreement
with Gampp et al. [48]) the formation of the {N2,Ox} chromophore with the participation
of the deprotonated nitrogen and oxygen atoms from the carboxyl groups of the
amino acid, which also follows from the changes in the C13 NMR
spectra. At pH 8, at which the Cu(Asp)2 species reaches a maximum
concentration, the changes in chemical shifts of the carbon atoms C(1),C(2), and C(4) are 0.595, 0.602, and 0.755 nm,
respectively.The Cu(Asp)(OH) complex
occurs in the same pH range as that of the dominantCu(Asp)2 species
and, therefore, no spectra could be taken.


### 3.3. Cu(II)/Asp/CMP System

Analysis
of the equilibria in the ternary systems wasperformed using the protonation
constants and overall stability constants(logβ) of the complexes forming in the binary systems of Cu(II)/CMP
[20]. In the system Cu(II)/Asp/CMP, the following complexes were found: the
protonated ones Cu(Asp)H4(CMP), Cu(Asp)H3(CMP), Cu(Asp)H2(CMP),
Cu(Asp)2H(CMP), and the hydroxocomplex Cu(Asp)(CMP)(OH). Table 3
presents the results of a computer analysis of titration curves in ternary
systems with Cu(II) ions, Asp, and CMP. The Cu(Asp)H4(CMP) species
was already observed to form at a pH below 2, binding over 80% of Cu(II) ions
at pH 3 (Figure 4). Taking into regard the number of hydrogen atoms in the
species studied, the position of the d-d bands, and the EPR parameters (at pH
3, λmax=780nm,g∥=2.409, and A∥=138,
Table 4), one can conclude that the Cu(II) ioncoordination occurs only by oxygen
atoms from Asp.Figure 4
Distribution diagram for the Cu(II)/Asp/CMP system; percentage of the species refers to total metal. (1)H3Asp; (2) H2Asp; (3) HAsp; (4) Asp; (5) H2(CMP); (6) H(CMP); (7) CMP; (8) Cu(Asp)2;
(9) Cu(Asp)(OH); (10) Cu(Asp)H4(CMP); (11) Cu(Asp)H3(CMP); (12) Cu(Asp)H2(CMP);
(13) Cu(Asp)H(CMP); (14) Cu(Asp)2H(CMP); (15) Cu(Asp)(CMP)(OH);
CCu2+=1×10−3M; CAsp=2.5×10−3M; CCMP=2.5×10−3M.The composition (in particular the
number of hydrogen atoms blocking the coordination) of the Cu(Asp)H4(CMP)
complex and the pH range of its occurrence suggest that it is a molecular
complex. Conclusions concerning the mode of interactions could be achieved on the basis of spectroscopic
studies. The UV-Vis and EPR data (λmax=780nm,g∥=2.409,
and A∥=138, Table 4) indicate bonding of copper(II) ion only via one oxygen atom (coordination
Cu-O in an inner sphere) with the involvement in metallation of a deprotonated
carboxyl group C(1) from the amino acid (the second carboxyl group is
protonated and blocked for coordination). On the basis of this essential
finding, the next stages of the analysis were performed. In the C13 NMR
spectrum of this species (pH=3), the change in the chemical shift of C(1) from Asp is 1.371 ppm. Moreover, the changes in the signals assigned to
the Asp carbon atoms C(2) (0.460 ppm) and C(4) (1.094 ppm) and originated from the nucleotide carbon atoms C(2) and C(4)
by 0.463 and 0.528 ppm, respectively, and the change in the P31 NMR
of CMP by 0.166 ppm confirm clearly the hypothesis relating to the
intermolecular interactions between the protonated complex of Cu(II) with Asp
and the protonated CMP molecule located in the outer coordination sphere. The noncovalent
interaction centre becomes the protonated Asp amine group, the CMP phosphate
group, and N(3)H.With increasing pH and deprotonation of the carboxyl groupC(4) in the Asp molecule, the Cu(Asp)H3(CMP) species is formed. The
protonated nucleotide with the donor atoms blocked is probably involved in
noncovalent interactions with the anchoring CuH(Asp) species. The pH range of
formation of this species overlaps the ranges of formation of other ones (Figure 4), which makes it impossible to perform spectral studies and to explain their
mode of coordination.The
Cu(Asp)H2(CMP) complex begins to form from a pH close to 3 in parallel with the deprotonation
of the nitrogen atom N(3) of the nucleotide, and at pH 4.5, it binds about 70%
of Cu(II) ions. The position of the absorption band in the UV-Vis spectrum in the pH range of the complex domination λmax=710nm and the values of the EPRparameters g∥=2.275 and A∥=179 (Table 4) [48, 51] indicate formation of the {N,Ox} type chromophore and it provides
the basis of information concerning the character of interaction. In the NMR
spectrum of Cu(Asp)H2(CMP), the changes in the chemical shifts of
the carbon atoms C(1) and C(4) from Asp (0.069 and 0.080
ppm, resp.) and the carbon atoms C(2) and C(4) from the nucleotide
(1.042 and 0.932 ppm, resp.), together with the changes in the P31 NMR
signal (0.080 ppm), point to the involvement in metallation of the
deprotonated N(3) atom, the phosphate group of CMP, and the oxygen atoms from
the carboxyl group of Asp. As follows from the value of logKe=6.08 for Cu(Asp)H2(CMP)
(so for the reaction of HCMP attachment to the anchoring CuHAsp), much higher
than that for Cu(CMP) formation logKe=2.71 [20], there is a noncovalent intramolecular ligand-ligand interaction in
the Cu(Asp)H2(CMP) complex that additionally stabilises it (the
presence of weak interactions is confirmed by changes in the chemical shift of C(2) from Asp (0.643 ppm) located in the proximity of the NH3+ group). The equilibrium constant of the
Cu(Asp)H2(CMP) complex formation is by about 3 orders of magnitude
higher than that of the adduct (Asp)H2(CMP), which is a result of
significant differences in the character of the bond (in the metal-free system
the bond is weak, noncovalent, but in the copper complex both ligands bind the
metal ions with additional intramolecular interaction).The Cu(Asp)H(CMP) complex begins to
form from a pH close to 4, and at pH = 6, it binds 80% of the Cu(II) ions. In the
pH range of its dominationλmax=700nm, the EPR parameters are g∥=2.269 and A∥=182, which implies the involvement of
one donor nitrogen atom and oxygen atoms from the ligand molecules in the
coordination ({N,Ox} chromophore). The NMR spectra reveal changes in the
positions of signals coming from the carbon atoms of the Asp carboxyl groups (C(1) 0.769 ppm and C(4) 0.809 ppm), the phosphorus atom of CMP
(P31 NMR 0.190 ppm), and the carbon atom located close to
the Asp amine group (C(2) 0.782 ppm). Moreover, changes in
positions of the signal originated from the CMP carbon atoms located in the proximity
of N(3) (C(2), 0.486 ppm and C(4), 0.772 ppm) are observed. The question
of which nitrogen atoms (either from the Asp amine group or the endocyclic N(3)
atom) from CMP are involved in the metallation has been solved by comparison of
the thermodynamic stability of the two possible species. The first species
corresponds to the formation of a stable system coupled with five- and
six-membered rings (Scheme 3), while the second corresponds to the formation of an unstable
seven-membered ring and a macrochelate structure with a coordination via the –PO42− group and N(3) atom from CMP.Scheme 3
Tentative mode of interaction in the Cu(Asp)H(CMP) complex.In the pH range 7–8, the dominant species isCu(Asp)2H(CMP).
The positions of the d-d bands and the EPR parameters (pH = 8, Table 4) imply the
formation of an {N2,Ox} type chromophore. The Cu(Asp)H(CMP) molecule accepts
another amino acid molecule. As follows from the changes in NMR signals assigned
to the carbon atoms of the carboxyl group (C(1) 0.809 ppm and C(4) 0.689 ppm) and from those in the proximity of the Asp amine group (C(2) 0.749 ppm) as well as the changes in the chemical shifts assigned to the C(2)
and C(4) atoms from the neighbourhood of N(3) in CMP by 0.860 and
0.858 ppm, respectively, and changes in the chemical shifts in the P31 NMR
spectrum of the nucleotide (0.129 ppm), the coordination involves the
oxygen atoms of the three carboxyl groups and the amine group from the Asp
molecule and the endocyclic N(3) atom together with the phosphate group from
CMP (monofunctional character of the second Asp ligand). The hydroxocomplex
Cu(Asp)(CMP)(OH) begins to form from a pH close to 8, and at
pH = 10.5, this species dominates (the equilibrium Cu(Asp)2H(CMP)+OH−⇆Cu(Asp)(CMP)(OH)+H(Asp). The maximum of the
band in the UV-Vis spectrum is at 630 nm, and the EPR parameters are g∥=2.258 and A∥=183, which corresponds to the {N2,Ox} chromophore
with coordination via the nitrogen atom N(3), the phosphate group from CMP, the
oxygen atom from the Asp amine group, oxygen atoms from the carboxyl groups of
the amino acid, and oxygen atoms from the OH group.


## 3.1. Asp/Nucleotide Metal-Free
Systems

Table1 presents the composition, overall stability constants (logβ), and the equilibrium constants of the
formation (logKe) of
molecular complexes appearing in Asp/CMP systems, determined from the computer
analysis of the potentiometric titration data.Table 1
Overall stability constants(logβ) and equilibrium constants (logKe) of adducts formation in
Asp/CMP system.
SpecieslogβlogKeH3Asp15.42 (3)2.06H2Asp13.36 (1)3.73HAsp9.63 (1)9.63H2CMP10.90 (1) [20]4.47HCMP6.42 (2) [20]6.42(Asp)H4(CMP)27.13 (4)2.87(Asp)H3(CMP)23.37 (3)2.84(Asp)H2(CMP)18.79 (3)2.74(Asp)H(CMP)12.01 (3)2.38As it was described earlier [28, 29, 36],
the occurrence of noncovalent interactions between the ligands and the formation
of molecular complexes in the system studied is indicated by the coincidence of
the titration curves obtained experimentally and those obtained by computer
simulation (with the use of the determined β values) as given in Figure 1.Figure 1
Experimental and simulated titration curves for the Asp/CMP system:
dotted line: experimental curve; solid line: simulated curve (an adduct formation was taken into account); dashed line: simulated curve (an adduct formation was not taken into account).Deprotonation of the phosphate group
of the nucleotide begins at low pH (logK3~0.4, [37, 38]), beyond
the range of the study. Subsequent stages of deprotonation correspond to the
abstraction of a proton from the endocyclic nitrogen atom N(3) from CMP and of
another proton from the phosphate group. The deprotonation of the aspartic acid
molecule begins with the proton abstraction from the carboxyl group C(1),
followed by dissociation of the –C(4)OOH
and the –NH3+ group [39, 40].Because of the different stoichiometric
compositions of particular species, the overall stability constantslogβ cannot be directly applied in analysis
of the character of interactions. Therefore, the efficiency of bonding was
estimated on the basis of the equilibrium constants calculated, for example,
for the species (Asp)H2(CMP): logKe=logβ(Asp)H2(CMP)−logβ(HAsp)−logβH(CMP).
On the basis of the protonation constants of the ligands (Table 1) and the pH
ranges of occurrence of individual species (Figure 2), the substrates in the
adduct formation reactions were identified.Figure 2
Distribution diagram for the Asp/CMP system; percentage of the species refers to total ligands. (1)H3Asp; (2) H2Asp; (3) HAsp; (4) Asp; (5) H2CMP; (6) HCMP; (7) CMP; (8) (Asp)H4(CMP);
(9) (Asp)H3(CMP); (10) (Asp)H2(CMP); (11) (Asp)H(CMP); CAsp=6×10−3M; CCMP=6×10−3M.The complex (Asp)H4(CMP)
appears at pH below 4.0 (Figure 2) in the range in which one of the
carboxyl groups of Asp and partly the –PO42− group from CMP are deprotonated [41–44]. In the C13 NMR spectra, the
chemical shifts of the signals assigned to C(2) and C(4) from the vicinity of N(3)
of the nucleotide changed by 0.982 and 0.760 ppm (pH 3.0), respectively, (Table 2) indicate that the protonated N(3)H from CMP is a positive centre of weak
interactions.Table 2
Differences betweenC13 NMR and P31 NMR chemical shifts for the ligands in the Asp/CMP system in relation to the free ligands [ppm].
AspCMPpHC(1)C(2)C(3)C(4)C(2)C(4)C(5)C(6)C(5′)P3.00.0490.0250.0220.0020.9820.7600.0570.0480.0440.0184.20.0000.0130.0700.0540.8610.6610.0400.0470.0600.0255.50.0530.0470.0270.0400.4400.5930.0680.0540.0990.0408.00.4541.0480.2271.3220.1910.1980.0680.0640.1110.105The lack of significant changes in
theP31 NMR spectrum suggests that the partly protonated (however
negative) phosphate group is not active, which is a result of the repulsion
from the negative carboxyl group present in the neighbourhood of –NH3+ group of Asp. The change in the chemical shift of the signal assigned to C(1) by 0.049 ppm suggests that the negative centre of interaction is thedeprotonated carboxyl group from Asp. (As established earlier, the energy of
the noncovalent interactions does not correspond directly with the chemical
shift value [27, 29, 34]). The protonated carboxyl group is not involved in the
interactions as evidenced by the lack of changes in the chemical shifts of the
NMR signals assigned to the carbon C(4) atom. In the (Asp)H3(CMP)
adduct, dominant at a pH close to 4, the N(3)H group of CMP remains a positive
centre of interactions as indicated by the changes in the NMR chemical shifts,
while the group –C(4)OO− becomes a
negative centre of interaction (Table 2), which corresponds to the
deprotonation of the second carboxyl group. The phosphate group remains
inactive. The involvement of only one centre from each ligand in the
interactions is confirmed by similar values of the equilibrium constants of the
tri- and tetraprotonated complexes of logKe=2.84 and 2.87, respectively.With increasing pH, the
deprotonation of the N(3)H group from the pyrimidine ring of CMP takes place.
The (Asp)H2(CMP) adduct starts forming from a pH close to 4 and
reaches its maximum concentration at a pH of about 5.5. The changes in the
chemical shifts of the carbon atoms C(2) and C(4), neighbouring the N(3) atom
of the nucleotide at pH 5.5, in the region of domination of the (Asp)H2(CMP)
adduct, are 0.440 and 0.593 ppm. This observation indicates that N(3) is a
centre of interactions, however, since it is already deprotonated, an inversion
of interactions takes place and the N(3) atom becomes a negative centre of
interaction. The P31 NMR spectrum of (Asp)H2(CMP) at
pH 5.5 does not show any significant changes in the chemical shift of the
phosphorus atom (Table 2), which indicates a low efficiency of the phosphate
group.Above the physiological pH, the
formation of (Asp)H(CMP) begins and it dominates at a pH of about 8. In these
complexes, the phosphate group from the nucleotide is engaged in noncovalent
interactions with amino acid as proved by the changes in the position of the
signal ofP31 NMR, by 0.105 ppm at pH=8. Changes in the
chemical shifts of the C(2) and C(4) atoms from CMP by 0.191 and
0.198 ppm, respectively, indicate that the deprotonated N(3) atom is
another negative centre of interaction. On the other hand, as follows from the changes
in the chemical shift of the signal assigned to C(2) (1.048 ppm), the protonated amine group from Asp (positive, logK1=9.6) takes part in the
interaction. In the molecule of the nucleotide, there are no positive reaction
centres, thus the intermolecular interactions of –COO− are impossible, (similar to the intramolecular interactions). Because of the
lack of another active centre in the Asp molecule, no increase in the logKe value of the
monoprotonated complex, relative to that of diprotonated one, is observed. The
unexpected changes in the chemical shift of the signals assigned to C(1) and C(4) from Asp in the C13 NMR spectrum (Table 2) are a consequence of the interaction of the carboxyl
groups (hard base) with Na+ ions (hard acids) present in the systems
studied. In the additional C13 NMR spectrum taken for the
(Asp)H(CMP) adduct in the system containing (C2H5)4NOH
instead of NaOH, the changes were insignificant (at pH 8: 0.007 and 0.020 ppm for C(1) and C(4), resp.). The positions of the
signals assigned to C(1) and C(4) from Asp in the spectra
of the Na+ free system Asp/CMP were compared to those in the spectra
of Asp, without sodium ions. (This results also explains the unexpected shifts
in the signals assigned to carbon atoms from carboxylate groups in some other
studied systems.) The above conclusions are confirmed by analysis of IR spectra
recorded in the same conditions as NMR ones. A comparison of the position of
the IR band assigned to –COO− of the amino acid (1615 and 1584cm−1) and the (Asp)H(CMP)
adduct (1617 and 1586cm−1) shows that these groups are not
involved in the ligand-ligand interactions (Figure 3), because no changes in
the band positions were observed.Figure 3
IR spectra of Asp and (Asp)H(CMP) adduct pH = 8;CAsp=0.2M, CCMP=0.2M.In all adducts studied above,
noncovalent interaction occurs with the inversion of interaction sites at a pH
close to 3.5 and close to 7, as illustrated in Scheme2. The pH values of the
inversion correspond to the values of the protonation constants, and changes in
the mode of interaction are a result of the deprotonation of the second
carboxyl group of Asp, endocyclic N(3)H, and the phosphate group of CMP. No
significant changes are noted in the acid-base equilibria of the ligands as is
usually the case  when metal-ligand bonds
are formed, which confirms that the interactions are weak, noncovalent.Scheme 2
Noncovalent interaction in the Asp/CMP system.


## 3.2. Cu/Asp Binary Systems

The
stability constants of Cu(II) complexes with Asp were determined in the same
conditions in which the heteroligand complexes in the ternary systems are
formed (Table3). The results are in agreement with the earlier reported data
[39, 40, 45, 46].Table 3
Overall stability constants(logβ) and equilibrium constants (logKe) for the complexes of Cu(II)
ions with Asp or CMP and Cu(II) ions with Asp and CMP.
EquilibriumlogβlogKeCu + CMP⇆ Cu(CMP)2.71 (7) [20]2.71Cu + CMP +H2O⇆ Cu(CMP)(OH) + H+−4.26 (6) [20]Cu +H+ + Asp ⇆ CuH(Asp)12.78 (5)3.15Cu + Asp⇆ Cu(Asp)8.76 (3)8.76Cu + 2Asp⇆ Cu(Asp)215.35 (4)6.59Cu + Asp +H2O⇆ Cu(Asp)(OH) + H+0.79 (9)Cu + 4H+ + Asp + CMP ⇆ Cu(Asp)H4(CMP)32.86 (5)Cu + 3H+ + Asp + CMP ⇆ Cu(Asp)H3(CMP)29.14 (5)5.46Cu + 2H+ + Asp + CMP ⇆ Cu(Asp)H2(CMP)25.28 (4)6.08Cu +H+ + Asp + CMP ⇆ Cu(Asp)H(CMP)20.12 (4)4.94Cu +H++ 2Asp + CMP ⇆ Cu(Asp)2H(CMP)29.87 (4)Cu + Asp + CMP +H2O⇆ Cu(Asp)(CMP)(OH) + H+4.69 (4)In the pH range from 2.5 to 6, a protonated species
CuH(Asp) is formed, while at pH 5.5, the dominant complex is Cu(Asp), binding
about 80% of copper ions. The species Cu(Asp)2 dominates  at a pH from 7 to 10, while the
hydroxocomplex Cu(Asp)(OH) begins forming from a pH close to 6 reaching a
maximum concentration above pH 10.5.The UV-Vis and EPR spectral
parameters (Table4) at pH 3, at which the CuH(Asp) complex dominates: λmax=756nm,g∥=2.413 and A∥=134, indicate
that the oxygen atoms from the Asp carboxyl groups are exclusively involved in the
coordination of the copper ions [26, 32, 47, 48], while the protonated amine group –NH3+ is blocked to coordination and
does not take part in the interactions ({Ox} chromophore). Conclusions concerning
the mode of coordination were drawn on the basis of an analysis of the relation
between the spectral parameters and the number of donor atoms in the inner
sphere of Cu(II) coordination. For
tetragonal and square pyramidal species, the ground state is normally dx2−y2 or rarely dxy. As earlier established for Cu‐Nx(x=1–6) and Cu‐NxOy(x=0–4,y=0–6) in planar bonding, the value
of g∥ decreases and that of A∥ increases [47, 49]. In
general, the value of g∥ is changed in the order CuO4>CuO2N2>CuON3>CuN4. Weak bonding of the donor atoms at
the axial position is a result of the interactions between the 4s and 4p metal
orbitals and the ligand orbitals. The hyperfine coupling constant was
experimentally observed to decrease with increasing electron density of the 4s
orbital [47].Table 4
Vis and EPR spectral data for Cu(II)/Asp and Cu(II)/Asp/CMP systems.
SpeciespHλmax [nm]ε[M−1cm−1]EPRg∥=(dm3/mol⋅cm3)A∥(10−4cm−1)CuH(Asp)3756272.413134Cu(Asp)5700402.301171Cu(Asp)28630762.257187Cu(Asp)H4(CMP)3780252.409138Cu(Asp)H2(CMP)4.5710292.275179Cu(Asp)H(CMP)6700302.269182Cu(Asp)2H(CMP)8660492.261182Cu(Asp)(CMP)(OH)10.5630962.258183The carboxyl group involvement is
confirmed by the changes in theC13 NMR chemical shifts of the carbon atoms from the two carboxyl groups: C(1) and C(4) by 1.536 and 1.163 ppm, respectively (Table 5).Table 5
Differences betweenC13 NMR and P31 NMR chemical shifts
for the ligands in the Cu(II)/Asp and
Cu(II)/Asp/CMP systems in relation to metal-free systems [ppm].
SystemsAspCMPpHC(1)C(2)C(3)C(4)C(2)C(4)C(5)C(6)C(5′)PCu(II)/Asp3.01.5360.1290.1221.163——————5.50.0500.0700.0200.050——————8.00.5950.6020.1690.755——————Cu(II)/Asp/CMP3.01.3710.4600.1471.0940.4630.5280.0750.0850.0570.1664.50.0690.6430.1130.0801.0420.9320.0520.0470.0510.0806.00.7690.7820.1420.8090.4860.7720.0620.0460.0760.1908.00.8090.7490.1520.6890.8600.8580.0710.0640.0620.129The increase in the equilibrium
constant of the formation of Cu(Asp) by approximately 5.5logKe unit relative to the value
for the protonated complex (Table 3) points to the involvement of the
deprotonated amine group –NH2 (for pH above 5) in the coordination of Cu(Asp) species, which is consistent
with the model proposed by Chaberek and Martell, later confirmed in [39, 50].
Moreover, in the electronic absorption spectrum, the λmax is shifted toward higher energy—from
756 to 700 nm, which points to the engagement of the nitrogen atom from
Asp in metal coordination, (besides the oxygen atoms). The {N,Ox} type
coordination was also confirmed by the EPR results as at pH = 5, g∥=2.301
and A∥=171. The attachment of a subsequent molecule of
amino acid to the anchoring Cu(Asp), according to the equation Cu(Asp)+Asp⇆Cu(Asp)2,
leads to a decrease in the equilibrium constant by about 2 logKe, units, so by a value suggesting the same mode of
coordination in the 1:1 and 1:2 complexes, taking into consideration the
statistical relations. The spectral parameters obtained from the UV-Vis and EPR
spectra for the Cu(Asp)2 complex are λmax=630nm,g∥=2.257nm, and A∥=187 (Table 4) indicating (in  agreement
with Gampp et al. [48]) the formation of the {N2,Ox} chromophore with the participation
of the deprotonated nitrogen and oxygen atoms from the carboxyl groups of the
amino acid, which also follows from the changes in the C13 NMR
spectra. At pH 8, at which the Cu(Asp)2 species reaches a maximum
concentration, the changes in chemical shifts of the carbon atoms C(1),C(2), and C(4) are 0.595, 0.602, and 0.755 nm,
respectively.The Cu(Asp)(OH) complex
occurs in the same pH range as that of the dominantCu(Asp)2 species
and, therefore, no spectra could be taken.


## 3.3. Cu(II)/Asp/CMP System

Analysis
of the equilibria in the ternary systems wasperformed using the protonation
constants and overall stability constants(logβ) of the complexes forming in the binary systems of Cu(II)/CMP
[20]. In the system Cu(II)/Asp/CMP, the following complexes were found: the
protonated ones Cu(Asp)H4(CMP), Cu(Asp)H3(CMP), Cu(Asp)H2(CMP),
Cu(Asp)2H(CMP), and the hydroxocomplex Cu(Asp)(CMP)(OH). Table 3
presents the results of a computer analysis of titration curves in ternary
systems with Cu(II) ions, Asp, and CMP. The Cu(Asp)H4(CMP) species
was already observed to form at a pH below 2, binding over 80% of Cu(II) ions
at pH 3 (Figure 4). Taking into regard the number of hydrogen atoms in the
species studied, the position of the d-d bands, and the EPR parameters (at pH
3, λmax=780nm,g∥=2.409, and A∥=138,
Table 4), one can conclude that the Cu(II) ioncoordination occurs only by oxygen
atoms from Asp.Figure 4
Distribution diagram for the Cu(II)/Asp/CMP system; percentage of the species refers to total metal. (1)H3Asp; (2) H2Asp; (3) HAsp; (4) Asp; (5) H2(CMP); (6) H(CMP); (7) CMP; (8) Cu(Asp)2;
(9) Cu(Asp)(OH); (10) Cu(Asp)H4(CMP); (11) Cu(Asp)H3(CMP); (12) Cu(Asp)H2(CMP);
(13) Cu(Asp)H(CMP); (14) Cu(Asp)2H(CMP); (15) Cu(Asp)(CMP)(OH);
CCu2+=1×10−3M; CAsp=2.5×10−3M; CCMP=2.5×10−3M.The composition (in particular the
number of hydrogen atoms blocking the coordination) of the Cu(Asp)H4(CMP)
complex and the pH range of its occurrence suggest that it is a molecular
complex. Conclusions concerning the mode of interactions could be achieved on the basis of spectroscopic
studies. The UV-Vis and EPR data (λmax=780nm,g∥=2.409,
and A∥=138, Table 4) indicate bonding of copper(II) ion only via one oxygen atom (coordination
Cu-O in an inner sphere) with the involvement in metallation of a deprotonated
carboxyl group C(1) from the amino acid (the second carboxyl group is
protonated and blocked for coordination). On the basis of this essential
finding, the next stages of the analysis were performed. In the C13 NMR
spectrum of this species (pH=3), the change in the chemical shift of C(1) from Asp is 1.371 ppm. Moreover, the changes in the signals assigned to
the Asp carbon atoms C(2) (0.460 ppm) and C(4) (1.094 ppm) and originated from the nucleotide carbon atoms C(2) and C(4)
by 0.463 and 0.528 ppm, respectively, and the change in the P31 NMR
of CMP by 0.166 ppm confirm clearly the hypothesis relating to the
intermolecular interactions between the protonated complex of Cu(II) with Asp
and the protonated CMP molecule located in the outer coordination sphere. The noncovalent
interaction centre becomes the protonated Asp amine group, the CMP phosphate
group, and N(3)H.With increasing pH and deprotonation of the carboxyl groupC(4) in the Asp molecule, the Cu(Asp)H3(CMP) species is formed. The
protonated nucleotide with the donor atoms blocked is probably involved in
noncovalent interactions with the anchoring CuH(Asp) species. The pH range of
formation of this species overlaps the ranges of formation of other ones (Figure 4), which makes it impossible to perform spectral studies and to explain their
mode of coordination.The
Cu(Asp)H2(CMP) complex begins to form from a pH close to 3 in parallel with the deprotonation
of the nitrogen atom N(3) of the nucleotide, and at pH 4.5, it binds about 70%
of Cu(II) ions. The position of the absorption band in the UV-Vis spectrum in the pH range of the complex domination λmax=710nm and the values of the EPRparameters g∥=2.275 and A∥=179 (Table 4) [48, 51] indicate formation of the {N,Ox} type chromophore and it provides
the basis of information concerning the character of interaction. In the NMR
spectrum of Cu(Asp)H2(CMP), the changes in the chemical shifts of
the carbon atoms C(1) and C(4) from Asp (0.069 and 0.080
ppm, resp.) and the carbon atoms C(2) and C(4) from the nucleotide
(1.042 and 0.932 ppm, resp.), together with the changes in the P31 NMR
signal (0.080 ppm), point to the involvement in metallation of the
deprotonated N(3) atom, the phosphate group of CMP, and the oxygen atoms from
the carboxyl group of Asp. As follows from the value of logKe=6.08 for Cu(Asp)H2(CMP)
(so for the reaction of HCMP attachment to the anchoring CuHAsp), much higher
than that for Cu(CMP) formation logKe=2.71 [20], there is a noncovalent intramolecular ligand-ligand interaction in
the Cu(Asp)H2(CMP) complex that additionally stabilises it (the
presence of weak interactions is confirmed by changes in the chemical shift of C(2) from Asp (0.643 ppm) located in the proximity of the NH3+ group). The equilibrium constant of the
Cu(Asp)H2(CMP) complex formation is by about 3 orders of magnitude
higher than that of the adduct (Asp)H2(CMP), which is a result of
significant differences in the character of the bond (in the metal-free system
the bond is weak, noncovalent, but in the copper complex both ligands bind the
metal ions with additional intramolecular interaction).The Cu(Asp)H(CMP) complex begins to
form from a pH close to 4, and at pH = 6, it binds 80% of the Cu(II) ions. In the
pH range of its dominationλmax=700nm, the EPR parameters are g∥=2.269 and A∥=182, which implies the involvement of
one donor nitrogen atom and oxygen atoms from the ligand molecules in the
coordination ({N,Ox} chromophore). The NMR spectra reveal changes in the
positions of signals coming from the carbon atoms of the Asp carboxyl groups (C(1) 0.769 ppm and C(4) 0.809 ppm), the phosphorus atom of CMP
(P31 NMR 0.190 ppm), and the carbon atom located close to
the Asp amine group (C(2) 0.782 ppm). Moreover, changes in
positions of the signal originated from the CMP carbon atoms located in the proximity
of N(3) (C(2), 0.486 ppm and C(4), 0.772 ppm) are observed. The question
of which nitrogen atoms (either from the Asp amine group or the endocyclic N(3)
atom) from CMP are involved in the metallation has been solved by comparison of
the thermodynamic stability of the two possible species. The first species
corresponds to the formation of a stable system coupled with five- and
six-membered rings (Scheme 3), while the second corresponds to the formation of an unstable
seven-membered ring and a macrochelate structure with a coordination via the –PO42− group and N(3) atom from CMP.Scheme 3
Tentative mode of interaction in the Cu(Asp)H(CMP) complex.In the pH range 7–8, the dominant species isCu(Asp)2H(CMP).
The positions of the d-d bands and the EPR parameters (pH = 8, Table 4) imply the
formation of an {N2,Ox} type chromophore. The Cu(Asp)H(CMP) molecule accepts
another amino acid molecule. As follows from the changes in NMR signals assigned
to the carbon atoms of the carboxyl group (C(1) 0.809 ppm and C(4) 0.689 ppm) and from those in the proximity of the Asp amine group (C(2) 0.749 ppm) as well as the changes in the chemical shifts assigned to the C(2)
and C(4) atoms from the neighbourhood of N(3) in CMP by 0.860 and
0.858 ppm, respectively, and changes in the chemical shifts in the P31 NMR
spectrum of the nucleotide (0.129 ppm), the coordination involves the
oxygen atoms of the three carboxyl groups and the amine group from the Asp
molecule and the endocyclic N(3) atom together with the phosphate group from
CMP (monofunctional character of the second Asp ligand). The hydroxocomplex
Cu(Asp)(CMP)(OH) begins to form from a pH close to 8, and at
pH = 10.5, this species dominates (the equilibrium Cu(Asp)2H(CMP)+OH−⇆Cu(Asp)(CMP)(OH)+H(Asp). The maximum of the
band in the UV-Vis spectrum is at 630 nm, and the EPR parameters are g∥=2.258 and A∥=183, which corresponds to the {N2,Ox} chromophore
with coordination via the nitrogen atom N(3), the phosphate group from CMP, the
oxygen atom from the Asp amine group, oxygen atoms from the carboxyl groups of
the amino acid, and oxygen atoms from the OH group.


## 4. Conclusions

The main reaction
centres in complexes formed as a result of noncovalent interactions in
metal-free systems at low pH are the nitrogen N(3)H group from CMP and carboxyl
groups from Asp. With increasing pH, the efficiency of the interactions of the
Asp carboxyl group decreases, while that of the amine group increases. For a pH
up to about 5, the protonated N(3)H from the nucleotide is the positive centre
of weak interactions and at a higher pH, an inversion occurs and it becomes a
negative centre. Above a pH of about 7, the involvement of the phosphate group occurs
in the interaction. The change in the mode of interactions corresponds to the
pH ranges of protonation of particular ligands. No significant acid-base shifts
are observed in the systems studied, which confirms the hypothesis that the
interactions are weak. Introduction of the metal ion into the binary system
changes the character of the reaction in the ternary system relative to that in
the metal-free systems. Particularly important is a significant increase in the
efficiency of the phosphate group from CMP in the noncovalent interactions in
the Cu(Asp)H4(CMP) molecular complex. In the (Asp)H4(CMP)
adduct which forms in the same pH range, the –PO4H− group is ineffective.In the ternary systems,
Cu(II)/Asp/CMP coordination is realised through the carboxyl groups from Asp,
and starting from a pH close to 4, also the phosphate groups from CMP and the
endocyclic N(3) atom are involved in interactions. At low pH, the carboxyl
groups from the amino acid are the main metal binding site, but with increasing
pH, their efficiency decreases to the advantage of the phosphate groups, as
follows from the analysis of thelogKe values. The presence of metal ions changes the character of noncovalent
interactions, whereas the introduction of an additionalligand CMP into the
binary system Cu(II)/Asp changes the mode of coordination. For instance, in the
Cu(Asp) complex, the coordination is realised via the oxygen atoms from the
carboxyl groups and the amine group (five-membered ring), while in the ternary
complex Cu(Asp)H(CMP) occuring in the same pH range, the involvement of the
phosphate group from CMP in metal binding leads to a change in the mode of
coordination and the formation of a structure with a system  of coupled five- and six-membered rings.



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*Source: 253971-2008-07-30.xml*