Coordination Chemistry of Phosphate Groups in Systems Including Copper(II) Ions, Phosphoethanolamine and Pyrimidine Nucleotides

The activity of phosphate groups of phosphoethanolamine and pyrimidine nucleotides (thymidine 5-monophosphate, cytidine 5-monophosphate and uridine 5’monophosphate) in the process of complexation metal ions in aqueous solution was studied. Using the potentiometric method with computer calculation of the data and spectroscopic methods such as UV-Vis, EPR, 13C and 31P NMR as well as FT-IR, the overall stability constants of the complexes as well as coordination modes were obtained. At lower pH, copper(II) ions are complexed only by phosphate groups, whereas the endocyclic nitrogen atom of nucleotides has been identified as a negative center interacting with the -NH3+ groups of phosphoethanolamine.


Introduction
The binding of metal ions by bioligands and the interactions between them have been the subject of study for years. Nucleic acids are especially important here, and their behavior has been studied most often. Metal ions occurring in living systems form coordination bonds with, e.g., DNA or RNA bases and phosphate groups. Depending on the type of metal and the sequence of nucleotides, the binding of the metal to the bases could destabilize the double helix structure [1][2][3][4]. On the other hand, metal ions are also involved in stabilizing the DNA structure, such as in coordination with the negatively charged phosphate backbone [5][6][7]. Research on the coordination chemistry of nucleic acids and d-block metal ions, such as copper, is extremely interesting-they could lose their water molecules very easily and give inner sphere coordinated complexes [8].
Copper ions are an important microelement in the human body and play a role as a cofactor for enzymes such ascytochrome oxidase, ferrooxidases, superoxide dismutase, and amine oxidases. The total amount of copper ions in the human organism, not only in blood but also in individual organs, is in homeostatic control. Changes in these amounts may indicate disorders and diseases. Excessive amounts of copper have the potential to damage cells and their constituents, especially due to the production of reactive oxygen species and DNA and chromatin damage, and can be the basis for the effects of copper related to cancer and other pathologies [9][10][11][12][13]. Copper complexes are also promising for anticancer treatment-they can interact with DNA, and the mechanism of action is still the subject of research by scientists [14][15][16]. They predominantly form non-covalent interactions with DNA via electrostatic forces of attraction, intercalation or minor groove binding [17][18][19]. For this reason, it is extremely important to study the interactions of metal ions, such as copper(II), especially with phosphorylated compounds that occur in living organisms [20][21][22]. Examples of such compounds are nucleotides, phosphoserine, phosphothreonine, phosphocholine, and phosphoethanolamine. Phosphoethanolamine (enP) plays a key role in the Kennedy pathway-the main metabolic route of synthesis phosphatidylethanolamine (PE) and phosphatidylcholine (PC)-the components of phospholipids which are structural and functional components of biomembranes [23][24][25]. Phosphoethanolamine and its derivatives have a significant influence on living organisms; they are useful for the treatment of cancer and infectious diseases, the tracking and prevention of some mutations, and antibiotic therapy directed to the bacterial membrane [26][27][28][29].
For phosphoethanolamine, the main coordination site in solution at a low pH value is the phosphate group, and at a higher pH value, the amine group becomes the metalation site [30]. Potential non-covalent interactions and metal-ion bonding sites for pyrimidine nucleotides are donor nitrogen atoms (N3) and phosphate groups [31][32][33].
This article presents the results of potentiometric and spectral studies of the complexes of phosphoethanolamine with copper(II) in ternary systems with monophosphorylated pyrimidine nucleotides: cytidine 5-monophosphate (CMP), uridine 5'monophosphate (UMP) and thymidine 5'-monophosphate (TMP).

Results and Discussion
The structures of the studied ligands are presented in Figure 1 and discussed with respect to the atom numbering shown in this picture. The values of the overall protonation constants of the ligands are given in Table 1.
ions, such as copper(II), especially with phosphorylated compounds that occur in living organisms [20][21][22]. Examples of such compounds are nucleotides, phosphoserine, phosphothreonine, phosphocholine, and phosphoethanolamine. Phosphoethanolamine (enP) plays a key role in the Kennedy pathway-the main metabolic route of synthesis phosphatidylethanolamine (PE) and phosphatidylcholine (PC)-the components of phospholipids which are structural and functional components of biomembranes [23][24][25]. Phosphoethanolamine and its derivatives have a significant influence on living organisms; they are useful for the treatment of cancer and infectious diseases, the tracking and prevention of some mutations, and antibiotic therapy directed to the bacterial membrane [26][27][28][29].
For phosphoethanolamine, the main coordination site in solution at a low pH value is the phosphate group, and at a higher pH value, the amine group becomes the metalation site [30]. Potential non-covalent interactions and metal-ion bonding sites for pyrimidine nucleotides are donor nitrogen atoms (N3) and phosphate groups [31][32][33].
This article presents the results of potentiometric and spectral studies of the complexes of phosphoethanolamine with copper(II) in ternary systems with monophosphorylated pyrimidine nucleotides: cytidine 5-monophosphate (CMP), uridine 5'monophosphate (UMP) and thymidine 5'-monophosphate (TMP).

Results and Discussion
The structures of the studied ligands are presented in Figure 1 and discussed with respect to the atom numbering shown in this picture. The values of the overall protonation constants of the ligands are given in Table 1.  For enP, CMP and UMP protonation constants and stability constants for their complexes with copper(II) ions in binary systems were previously described [32,34]. For TMP, we determined two protonation constants of thymidine 5-monophosphate by computer calculations from titration data. The first protonation constant for the N(3) atoms of TMP is logK1 9.73 (a similar value as for thymidine-logK 9.79 [32]), and it is assigned to the protonation of the endocyclic N(3) atom. The second logK2 is 6.04, which corresponds to the -O-PO3 2− group. The values of protonation constants for TMP as well as for UMP are much higher compared to CMP, which significantly changes the efficiency of metal ion   3 --−23.64 -For enP, CMP and UMP protonation constants and stability constants for their complexes with copper(II) ions in binary systems were previously described [32,34]. For TMP, we determined two protonation constants of thymidine 5-monophosphate by computer calculations from titration data. The first protonation constant for the N(3) atoms of TMP is logK 1 9.73 (a similar value as for thymidine-logK 9.79 [32]), and it is assigned to the protonation of the endocyclic N(3) atom. The second logK 2 is 6.04, which corresponds to the -O-PO 3 2− group. The values of protonation constants for TMP as well as for UMP are much higher compared to CMP, which significantly changes the efficiency of metal ion binding. Deprotonation of the first proton of the phosphate group occurs at a relatively low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary system Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and the computer calculation of the potentiometric titration data was carried out taking into account the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (logβ = −13.13 for Cu(OH) 2 ) [35]. The protonated form of the complex and hydroxocomplexes with their equilibrium constants were established based on the proposed reaction of their formation (ion charges were omitted for simplicity): binding. Deprotonation of the first proton of the phosphate group occurs at a relatively low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary system Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and the computer calculation of the potentiometric titration data was carried out taking into account the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (logβ = −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocomplexes with their equilibrium constants were established based on the proposed reaction of their formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. From a pH close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominates at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. From pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at pH close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of the Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measuring scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EPR spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters and changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was established. The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = 2.37 and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coordination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in complexes with respect to these on the free ligand on atom C(5') indicate the activity of the phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) ( Table  3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating the presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chemical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) from 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordination. Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate group is still an active site in the complexation process. A similar coordination mode with an additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 complex (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to precipitation at samples made in higher concentrations.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary system Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and the computer calculation of the potentiometric titration data was carried out taking into ac-count the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (logβ = −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocomplexes with their equilibrium constants were established based on the proposed reaction of their formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. From a pH close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominates at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. From pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at pH close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of the Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measuring scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EPR spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters and changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was established. The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = 2.37 and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coor-dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in com-plexes with respect to these on the free ligand on atom C(5') indicate the activity of the phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) ( Table  3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating the presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chem-ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) from 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordination. Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate group is still an active site in the complexation process. A similar coordination mode with an additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 complex (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to precipi-tation at samples made in higher concentrations.
Cu(TMP)(OH) + H + · · · · · · · · · · · · logK e = 14.16 binding. Deprotonation of the first proton of the phosphate group occurs at a relatively low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary system Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and the computer calculation of the potentiometric titration data was carried out taking into ac-count the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (logβ = −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocomplexes with their equilibrium constants were established based on the proposed reaction of their formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. From a pH close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominates at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. From pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at pH close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of the Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measuring scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EPR spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters and changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was established. The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = 2.37 and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coor-dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in com-plexes with respect to these on the free ligand on atom C(5') indicate the activity of the phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) ( Table  3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating the presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chem-ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) from 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordination. Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate group is still an active site in the complexation process. A similar coordination mode with an additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 complex (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to precipi-tation at samples made in higher concentrations.
Cu(TMP)(OH) 2 + H + · · · · · · logK e = 4.14 At the beginning of the measurement, free copper(II) ions were observed. From a pH close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominates at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. From pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at pH close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of the Cu(TMP)(OH) 2 form begins to increase, and its dominant point is out of the measuring scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EPR spectra, taking into account d-d transition energy as well as g and A parameters and changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was established. The Vis and EPR spectral parameters for the Cu(HTMP) complex (λ max = 801 nm, g = 2.37 and A = 157 · 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coordination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in complexes with respect to these on the free ligand on atom C(5') indicate the activity of the phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) ( Table 3). For the Cu(TMP)(OH) complex, the value of λ max decreases to 711 nm, indicating the presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chemical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) from 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordination. Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate group is still an active site in the complexation process. A similar coordination mode with an additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH) 2 complex (λ max = 673 nm). For this complex, EPR and NMR studies were impossible due to precipitation at samples made in higher concentrations.
binding. Deprotonation of the first proton of the phosphate group occurs at a relatively low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary system Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and the computer calculation of the potentiometric titration data was carried out taking into account the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (logβ = −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocomplexes with their equilibrium constants were established based on the proposed reaction of their formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. From a pH close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominates at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. From pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at pH close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of the Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measuring scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EPR spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters and changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was established. The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = 2.37 and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coordination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in complexes with respect to these on the free ligand on atom C(5') indicate the activity of the phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) ( Table  3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating the presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chemical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) from 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordination. Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate group is still an active site in the complexation process. A similar coordination mode with an additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 complex (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to precipitation at samples made in higher concentrations.   In the copper(II)/phosphoethanolamine/thymidine 5'-monophosphate ternary system, complexes are formed (the overall stability constants are presented in Table 4 ( Figure 2b). In this system, only one complex, Cu(enP)H 4 (TMP), dominates. At pH 2.5, this form binds almost 65% of total copper(II) ions introduced into the solution. According to the λ max = 802 nm value, the EPR parameters of g = 2.39 and A = 135 · 10 −4 cm −1 ( Table 5) and values of the protonation constants logK e = 5.70 and 6.04 for enP and TMP, respectively, and changes between chemical shifts on the 31 P NMR spectra (−4.70 ppm for enP and −1.06 ppm for TMP) in the inner coordination sphere comprises only the oxygen atom of phosphate groups of enP (see Supplementary Materials). That shift in the phosphorus atom of TMP may indicate an interaction with the amine group of enP. Analysis of the FT-IR spectrum confirmed these interactions (antisymmetric stretching band at 1079 cm −1 in the IR spectrum of the complex and at 1084 cm −1 in the spectrum of the free ligand [36]). From the beginning of the measurement to pH 7.0, the Cu(enP)H 3 (TMP) complex appears, the maximum concentration of which overlaps the range of domination of Cu(enP)H 4 (TMP), Cu(enP)H 2 (TMP) and Cu(HTMP), which makes a spectral study of this complex impossible to perform. A similar mode of interaction is observed for the Cu(enP)H 2 (TMP) complex (appears in the pH range 4.5 to 8.0) and Cu(enP(TMP)(OH) 2 . In this system, binary complexes are formed at a relatively high concentration. Table 4. Overall and stability constants as well as equilibrium constants of Cu(II) complexes in the Cu(II)/enP/NMP systems (standard deviation is given in parenthesis).

Species
Overall binding. Deprotonation of the first proton of the phosphate group occurs at a re low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 computer calculation of the potentiometric titration data was carried out taking count the protonation constants of Table 1 and the constant for Cu(II) hydrolysis −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocom with their equilibrium constants were established based on the proposed reaction formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. Fro close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominate close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the me scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramet changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved i dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicat presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C( 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordi Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 c (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a relat low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary sy Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 an computer calculation of the potentiometric titration data was carried out taking int count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It domin at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. F pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates a close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration o Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measu scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establis The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in c dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity o phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (T 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicatin presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ch ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordina Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate g is still an active site in the complexation process. A similar coordination mode wit additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 com (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pre tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a relati low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary sys Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and computer calculation of the potentiometric titration data was carried out taking into count the protonation constants of Table 1 and the constant for Cu(II) hydrolysis (lo −13.13 for Cu(OH)2) [35]. The protonated form of the complex and hydroxocompl with their equilibrium constants were established based on the proposed reaction of t formation (ion charges were omitted for simplicity): At the beginning of the measurement, free copper(II) ions were observed. From a close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It domin at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. F pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration o Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measu scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establis The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in c dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in c plexes with respect to these on the free ligand on atom C(5') indicate the activity o phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (T 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ch ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) f 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordina Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate gr is still an active site in the complexation process. A similar coordination mode wit additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 com (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pre tation at samples made in higher concentrations.  binding. Deprotonation of the first proton of the phosphate group occurs at a rela low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary s Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 a computer calculation of the potentiometric titration data was carried out taking in count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming (Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the meas scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ parameter changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establ The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicatin presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordin Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 co (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pr tation at samples made in higher concentrations.  binding. Deprotonation of the first proton of the phosphate group occurs at a re low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 computer calculation of the potentiometric titration data was carried out taking count the protonation constants of Table 1  . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramet changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved i dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicat presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C( 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordi Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 c (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations.  binding. Deprotonation of the first proton of the phosphate group occurs at a relat low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary sy Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 an computer calculation of the potentiometric titration data was carried out taking int count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It domin at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. F pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates a close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration o Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measu scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establis The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in c dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity o phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (T 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicatin presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ch ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordina Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate g is still an active site in the complexation process. A similar coordination mode wit additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 com (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pre tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a relati low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary sys Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and computer calculation of the potentiometric titration data was carried out taking into count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From a close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It domin at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. F pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration o Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measu scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establis The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in c dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in c plexes with respect to these on the free ligand on atom C(5') indicate the activity o phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (T 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ch ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) f 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordina Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate gr is still an active site in the complexation process. A similar coordination mode wit additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 com (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pre tation at samples made in higher concentrations.  binding. Deprotonation of the first proton of the phosphate group occurs at a rel low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary s Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 a computer calculation of the potentiometric titration data was carried out taking in count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the mea scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramete changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicati presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordin Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 co (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations.  REVIEW 3 binding. Deprotonation of the first proton of the phosphate group occurs at a relati low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary sys Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and computer calculation of the potentiometric titration data was carried out taking into count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From a close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It domin at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. F pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measu scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establish The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in c dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in c plexes with respect to these on the free ligand on atom C(5') indicate the activity of phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (T 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ch ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) f 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordinat Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate gr is still an active site in the complexation process. A similar coordination mode with additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 com (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to prec tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a re low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 computer calculation of the potentiometric titration data was carried out taking count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. Fro close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominate close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the me scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramet changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved i dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicat presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C( 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordi Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 c (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a rel low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 a computer calculation of the potentiometric titration data was carried out taking i count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming (Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the mea scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramete changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved i dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand i plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicat presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordi Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 co (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a rela low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary s Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 a computer calculation of the potentiometric titration data was carried out taking in count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming (Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the mea scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis an spectra, taking into account d-d transition energy as well as g‖ and A‖ paramete changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was estab The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicati presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordin Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 co (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to p tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a rela low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary s Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 a computer calculation of the potentiometric titration data was carried out taking in count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dom at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the meas scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and spectra, taking into account d-d transition energy as well as g‖ and A‖ parameter changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establ The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in plexes with respect to these on the free ligand on atom C(5') indicate the activity phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicatin presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordin Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate is still an active site in the complexation process. A similar coordination mode w additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 co (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to pr tation at samples made in higher concentrations. binding. Deprotonation of the first proton of the phosphate group occurs at a relative low pH value, and this protonation constant was not determined.

Cu/enP/TMP System
In the first step of the Cu(II)/enP/TMP study, the investigation in the binary syste Cu(II)/TMP was performed. Potentiometric titrations in a metal:ligand ratio of 1:1 and t computer calculation of the potentiometric titration data was carried out taking into a count the protonation constants of Table 1  At the beginning of the measurement, free copper(II) ions were observed. From a p close to 3.0, the first form of the complex Cu(HTMP) starts forming ( Figure 2). It dominat at pH 6.0 and binds to around 55% of the total amount of copper ions in solution. Fro pH 5.5 to 11.0, the hydroxocomplex Cu(TMP)(OH) is observed, and it dominates at p close to 8.0, binding over 85% of the copper ions. From pH 8.0, the concentration of t Cu(TMP)(OH)2 form begins to increase, and its dominant point is out of the measuri scale. At pH 11.0, it binds 85% of Cu 2+ . On the basis of the analysis of UV-Vis and EP spectra, taking into account d-d transition energy as well as g‖ and A‖ parameters a changes in the chemical shifts of 31 P and 13 C NMR, the coordination mode was establishe The Vis and EPR spectral parameters for the Cu(HTMP) complex (λmax = 801 nm, g‖ = 2. and A‖ = 157 • 10 −4 cm −1 , Table 2) indicate that only one oxygen atom is involved in coo dination. Significant chemical shifts in the 31 P and 13 C NMR spectra of the ligand in com plexes with respect to these on the free ligand on atom C(5') indicate the activity of t phosphate group in the inner coordination sphere ( 31 P −3.22 ppm, C(5') 0.58 ppm) (Tab 3). For the Cu(TMP)(OH) complex, the value of λmax decreases to 711 nm, indicating t presence of nitrogen atom N(3) in the internal coordination sphere. Changes in the chem ical shifts between free ligand and ligand in the complex in 13 C NMR spectra (C(2) fro 0.23 ppm to −0.08 ppm and C(4) from 0.05 to 0.12 ppm) prove this type of coordinatio Changes in the chemical shifts on C(5') atom (−0.72 ppm) show that the phosphate grou is still an active site in the complexation process. A similar coordination mode with additional oxygen atom from the hydroxyl group is observed for Cu(TMP)(OH)2 compl (λmax = 673 nm). For this complex, EPR and NMR studies were impossible due to precip tation at samples made in higher concentrations.

Cu/enP/UMP System
The complexes forming in the ternary system Cu(II)/enP/UMP are Cu(enP)H 4 (UMP), Cu(enP)H 3 (UMP), Cu(enP)H 2 (UMP), Cu(enP)(UMP) and dinuclear mixed complex Cu 2 (enP) 2 (UMP). The first protonated complex Cu(enP)H 4 (UMP) binds almost 100% of copper(II) ions at the beginning of the measurement (Figure 3). The spectral results (Vis and EPR) indicate the formation of an {O x } chromophore (λ max = 798 nm, g = 2.41 and A = 137 · 10 −4 cm −1 ) (Table 5, Figure 4). As indicated by the changes in the NMR spectrum, in the inner coordination sphere, there is a phosphate group of phosphoethanolamine ( 13 C NMR C(1) −0.94 ppm, 31 P NMR −0.92 ppm) ( Figure 5). The change between shifts on the second carbon of enP C(2) −0.94 ppm neighbouring the protonated amine may be a result of non-covalent interactions with the phosphate group of UMP as a negative center. For UV-Vis, EPR and NMR spectra, see Supplementary Materials). This interaction was confirmed by the IR spectra of the complex related to the free ligand, where the positions of the IR stretching vibration bands (1083 cm −1 for free UMP and 1079 cm −1 for complex) are shifted slightly ( Figure 5).          group of CMP in coordination. Changes in the chemical shift on the 13 C NMR (0.69 ppm) on the C(2) atom of CMP were observed due to the close proximity of the nitrogen N(3) atom. We excluded the participation of the carbonyl group in the complexation process due to the lack of shifts in the IR spectrum (1651 cm −1 for both the complex and free ligand).
The Cu(enP)H 2 (CMP) complex, created from pH 4.0 and dominant at pH 6.0, bound 65% of metal ions in the solution (Figure 3). The value of λ max = 751 nm and the EPR parameters (g = 2.33 and A = 159 · 10 −4 cm −1 ) ( Table 5) indicate that in the inner coordination sphere, there are one nitrogen atom and oxygen atoms. The changes in the chemical shifts in the 13 C NMR of enP (C(1) −0.97 ppm, C(2) −0.95 ppm) indicate that the phosphate group is still active in the coordination of copper ions. The changes in the chemical shifts between the free ligand and the ligand in the complex for CMP on the 13 C NMR (C(2) 0.48 ppm) indicate that the nitrogen atom N(3) was in the inner coordination sphere.
The deprotonated Cu(enP)(CMP) complex was observed in the 5.7 to 11.0 pH range. It dominated at a pH close to 7.2 and bound almost all copper ions in the solution (Figure 3). For this complex, the formation of the stability constant (logK e = 14.50) is higher than for protonated forms and points to different modes of coordination. A significant decrease in the value of the maximum wavelength (λ max = 680 nm) and changes in the EPR parameters (g = 2.30; A = 158 · 10 −4 cm −1 ) means that in the inner coordination sphere, there are two nitrogen atoms. According to the 13 C NMR and the 31 P NMR spectra, we can observe decreasing changes in the chemical shift of the phosphate group of enP and C(1) atom as well as increasing changes in the chemical shift from the C(2) atom, which indicate the involvement of an amine group in complexation.
The hydroxocomplex Cu(enP)(CMP)(OH) begins to form at a pH close to 8.0 and becomes dominant at a pH of 10.0. An analysis of the Vis spectral studies (λ max = 689 nm) indicates that the inner coordination sphere is the same as in the Cu(enP)(CMP) complex, with the addition of one oxygen atom from the hydroxyl group. Because at pH 10.0, we can observe not only this ternary hydroxocomplex but also this binary Cu(enP)(OH) 2 complex, this value of λ max may be overstated.

Materials and Methods
O-phosphoethanoloamine (enP), cytidine 5'monophosphate (CMP) and uridine 5'monophosphate disodium salt (UMP) were purchased from Sigma Aldrich (Steinheim am Albuch Baden-Württemberg, Germany), and thymidine 5'-monophosphate disodium salt (TMP) was purchased from Alfa Aesar (Thermo Fisher, Kandel, Germany) and used without additional purification. Copper(II) nitrate from Merck was purified by recrystallization from water. The concentration of copper ions in the solution was determined by the method of inductively coupled plasma optical emission spectrometry (ICP OES) (Shimadzu, Kyoto, Japan). All the prepared solutions and performed measurements were carried out with the use of demineralized, carbonate-free water.
Potentiometric titrations were performed using a Metrohm system (Titrino 702 equipped with an autoburette with a combined Metrohm glass electrode) (Metrohm AG, Herisau, Switzerland). Before each series of measurements, the pH meter indication was corrected with two standard buffer solutions of pH 4.002 and pH 9.225, and the electrode was calibrated in terms of H + concentration [37]. The concentrations of phosphoethanolamine, nucleotides and copper(II) ions in the potentiometric studies were 1.0 × 10 −3, and the metal ion to ligand ratio was 1:1 for binary systems (Cu(II)/TMP) and 1:1:1 for ternary systems (Cu(II)/enP/NMP). All potentiometric titrations were carried out under strictly defined conditions: under a helium atmosphere (He 5.0) (Linde Gaz, Krakow, Poland), at a constant ionic strength of µ = 0.1 M (KNO 3 ), a temperature 20 ± 1 • C, a pH range of 2.5 to 10.5, and with NaOH without CO 2 at a concentration of 0.2011 M as a titrant. The calculations were performed using 150-350 experimental points for each titration. The data obtained from potentiometric titrations were subjected to computer analysis by the HYPERQUAD program for the determination of protonation constants and stability constants and the HYSS (Hyperquad Simulation and Speciation) program for the calculation of the distribution of particular species [38,39]. For the complexes formed in binary and ternary systems, the stability constants could be evaluated by the following equilibria (the charge was omitted for simplicity): pL + qH + ↔ L p H q pL + qL + rH + ↔ L p L q H r β = L p L q H r The determined ionic product of water was pK w = 13.78, and the hydrolysis constant for copper(II) was taken from our previous publications [40][41][42][43]. Testing began with the simplest hypothesis, and then the model was expanded to include progressively more complex forms [44]. After the improvement process, a set of complexes was established. The accuracy of the model was confirmed by verifying the results described in the papers [30][31][32]. 13 C and 31 P NMR spectra were measured on an AVANCE III Bruker 500 MHz spectrometer (Bruker, Billerica, MA, USA). Dioxane as an internal standard for 13 C NMR and phosphoric acid for 31 P NMR were used. Samples for 13 C and 31 P NMR measurements were performed in D 2 O, and pD was adjusted by the addition of NaOD or DCl solutions, taking into account that pD = pH + 0.40 [45,46]. The concentration of the ligands in the samples was 0.1 M. The M:L molar ratio was 1:100 in binary systems, and the M:L:L' molar ratio was 1:100:100 in ternary systems. The samples for infrared spectra were prepared in D 2 O; the value of M:L was 1:1, and the value of M:L:L' was 1:1:1. Measurements were collected in the range of 400-4000 cm −1 using INVENIO R (Bruker, Bremen, Germany) with the ATR technique.
The UV-Vis spectra were determined using the Evolution 300 UV-VIS ThermoFisher Scientific spectrometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA) equipped with a xenon lamp (range 450-950 nm, accuracy 0.2 nm, and sweep rate 120 nm/min). Samples for the visible studies were prepared in H 2 O in molar ratios of 1:1 and 1:1:1 in concentrations C M = 0.002-0.02 M. The spectra were recorded at 20 • C in a PLASTIBRAND PMMA cell (Brand, Wertheim, Germany) with a 1 cm path length.
The EPR spectra were recorded on a SE/X 2547 Radiopan instrument (Radiopan, Poznan, Poland). The samples were performed in a water:glycol mixture (3:1) at a concentration C M = 0.005, and the measurements were carried out at −196 • C using glass capillary tubes (volume 130 µm 3 ).

Conclusions
In all investigated systems, the formation of protonated complexes has been established, as well as MLL' for Cu(II)/enP/CMP and Cu(II)/enP/UMP systems and the hydroxocomplexes MLL'(OH) x for Cu(II)/enP/TMP and Cu(II)/enP/CMP. It should be noted that in the ternary system with uridine-5'-monophosphate, the mixed-type dinuclear complex Cu 2 (enP) 2 (UMP) is observed, which was confirmed by the disappearance of the signal in the EPR spectra. Analysis of the results presented above allows us to conclude that in the Cu(II)/enP/NMP systems, the reaction centers change depending on pH. At lower pH values, enP and nucleotides coordinate only via the phosphate groups, and with increasing basicity, the efficiency of phosphate groups decreases and the main reaction center becomes the amine group of enP and the endocyclic nitrogen atom N(3) from the pyrimidine ring of nucleotides. Additional non-covalent interactions have been found to occur between the bioligands where adducts are protonated amine groups from phosphoethanolamine and donor nitrogen atoms from the nucleotide. In binary systems, the stability of Cu(II)/NMP complexes increases in the order CMP < UMP < TMP, which corresponds to the values of their protonation constants. For ternary systems, with the presence of copper(II) ions, a nucleotide and a second ligand, which is phosphoethanolamine, we have also observed the lowest stability of ternary complexes of CMP, but for UMP and TMP, this order changes, and the same type complexes in the Cu(II)/enP/UMP system are more stable than complexes in the CU(II)/enP/TMP system. We hope that the results obtained will contribute to a broadening of the knowledge about complex compounds of phosphorylated ligands with copper (II) ions and their interaction with DNA and RNA. They may be crucial for explaining the processes taking place in living organisms, e.g., mutations and neurodegenerative diseases in the brain where phosphorylated lipids are abundant in the myelin sheaths.