Abstract
Ternary systems of copper(II) ions complexes with glucuronic acid and uridine or uridine derivatives (Urd, UMP, UDP, UTP) and binary systems of tested ligands have been investigated. Potentiometric measurements in aqueous solution were performed using computer analysis. Potentiometric studies allowed the determination of the composition of the complex compounds studied and the overall stability constants. UV-Vis, EPR, 13C NMR and 31P NMR spectroscopic studies allowed the composition of the internal coordination sphere to be determined and the types of chromophores to be identified. MLL’, ML(H)L’ and MLL‘(OH) (L – Urd, UMP, UDP or UTP; L’ – GluA) complexes were observed in the systems studied.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-025-17498-w.
Keywords: Copper(II) ions, Uridine, Uridine derivatives, Complex compounds, Potentiometric measurements, Spectroscopic measurements
Subject terms: Coordination chemistry, Inorganic chemistry
Introduction
Metal ions are essential for the maintenance of life. They can be divided into two basic categories: macronutrients and micronutrients. Metal ions like sodium, potassium, magnesium, calcium, iron, zinc, and copper play a crucial role in numerous life processes, such as the transport of oxygen by the blood, the enzymatic activities, and the structural integrity of biomolecules. Furthermore, they serve as active centers for various enzymes and proteins, including hemoglobin and vitamin B12. Metal ions also have applications in the medical and pharmaceutical fields, functioning as magnetic resonance contrast agents and active compounds in cancer treatment1–6. Copper(II) ions, in particular, are essential for proper body functioning, as they act as active centers of numerous enzymes, such as cytochrome C oxidase, peroxidase, and lysyl oxidase7–9. Additionally, these ions are vital for fundamental life processes, like free radical elimination, energy production, respiration, and oxygen and iron metabolism7,10. Due to their strong complexing properties, Cu-EDTA complexing agents are used to remove heavy metal ions from the human body. Furthermore, copper(II) complexes with chelating agents such as 1,10-phenanthroline exhibit anticancer activity11,12. Abnormal copper(II) ion concentrations in the body can lead to a variety of conditions and diseases, e.g.: osteoporosis, Parkinson’s disease, hernias, Menkes syndrome, Hodgkin’s disease, anemia, leukemia, and general weakness7,10,13,14.
Nucleotides and nucleosides are essential components that contribute significantly to the normal physiological functioning of the human body. These compounds serve as the fundamental building blocks of nucleic acids, like DNA and RNA, and play a pivotal role in numerous metabolic processes, encompassing lipid and carbohydrate metabolism, as well as the biosynthesis of polyamines within the body. Additionally, they function as carriers of energy and as constituents of redox coenzymes, which are indispensable for cellular function. These types of compounds are highly significant during early development; Their abundant concentrations are observed in milk and colostrum, supporting nutrition and immune regulation in infants15–19. Additionally, nucleotides function as signaling molecules, with cyclic adenosine monophosphate serving as a second messenger in various signaling pathways that modulate physiological responses20. Disruptions in nucleotide metabolism can lead to significant clinical implications. Elevated uric acid levels from purine degradation are associated with conditions like gout and metabolic syndrome. Although pyrimidine metabolism is less frequently associated with severe disorders, its dysregulation can lead to conditions such as orotic aciduria, characterized by growth and hematological problems21–23. Nucleotide metabolism is also a target for cancer therapies, with chemotherapeutic agents like 5-fluorouracil designed to disrupt nucleotide synthesis pathways, highlighting the potential for therapeutic interventions in cancer treatment24. As research continues, understanding the complex roles of nucleotides and nucleosides may provide insights into novel approaches to managing various health conditions and improving therapeutic strategies.
Uridine (Urd), a vital pyrimidine nucleoside, plays a crucial role in human metabolic processes, cellular functions, and neurological well-being. It contributes to the synthesis of nucleotides, energy regulation, modulation of metabolic pathways, and the production of glycogen and RNA. Furthermore, uridine serves as a precursor for numerous biological molecules, such as uridine phosphates25–27. The dual availability of uridine highlights its importance in maintaining cellular health and responding to metabolic demands. Additionally, uridine derivatives have garnered attention for their potential therapeutic applications in the treatment of neurodegenerative diseases and metabolic disorders, generating interest in their clinical relevance and underlying mechanisms, including potential uses in the treatment of cancer and viral infections. Uridine and its derivatives can also function as signaling molecules within the central nervous system. The ongoing exploration of uridine and its derivatives emphasizes its multifaceted role in human health, including implications for cognitive function, energy metabolism, and the management of neurological conditions27,28.
D-glucuronic acid (GluA), a member of the uronic acid group, is produced through the oxidation of a hydroxyl group within the glucose molecule, converting it to a carboxyl group. This compound is formed in the body via the dehydrogenation of UDP-glucose. In particular, D-glucuronic acid plays a crucial role in the detoxification processes of the body, facilitating the removal of substances such as bilirubin, oxidized fatty acids, and steroid hormones29–31. The presence of both carboxyl and hydroxyl functional groups allows D-glucuronic acid to form numerous complexes with metal ions, allowing it to be used as a masking agent for various metals, including heavy metals29,32,33.
Potentiometric and spectroscopic studies of the complex compounds formed in the system uridine or its derivative, glucuronic acid, and copper(II) ions are described in the following manuscript. Additionally, the overall stability constants (logβ) and the composition of the internal coordination sphere were determined.
Results and discussion
A series of potentiometric and spectroscopic studies were carried out to determine the mode of coordination in the systems studied: copper(II) ions/uridine/glucuronic acid, copper(II) ions/uridine-5’-monophosphate/glucuronic acid, copper(II) ions/uridine-5’-diphosphate/glucuronic acid and copper(II) ions/uridine-5’-triphosphate/glucuronic acid. On the basis of the results obtained, the overall stability constants and equilibrium constants of the formation reactions were determined, as well as the composition of the internal coordination sphere and the types of chromophores in the studied systems.
The ligands studied contain several potential coordination sites in their structure. In the case of glucuronic acid, a potential coordination site for the metal ion could be the oxygen atom of the carboxyl group. On the other hand, in the case of uridine and its derivatives, coordination may occur via the endocyclic nitrogen N(3) atom and, in the case of phosphate derivatives, via the oxygen atom of the phosphate group. The structural formulae of the studied ligands are shown in Fig. 1.
Fig. 1.
Structural formulae of ligands: (a) glucuronic acid, (b) uridine or uridine derivatives (R: -OH(Urd); -O-PO32−(UMP), -O-(PO3)2H3−(UDP); -O-(PO3)3H4−(UTP)).
The protonation constants of Urd, UMP and GluA, the hydrolysis constant for copper(II) hydroxide (
= − 13.13) and the stability constants of binary systems with copper(II) ions were taken from our previous publications29,34,35. The protonation constants for UDP and UTP were determined for the purposes of this paper.
Protonation of UDP and UTP
Analysis of the results obtained from potentiometric (Table S1; Figure S1, Supplementary Materials) tests allowed the determination of the protonation constants of UDP (HUDP and H2UDP) and UTP (HUTP). The protonation constants for the tested ligands are listed in Table 1.
Table 1.
The protonation constants (logβ) of UDP and UTP and the equilibrium constants (logKe) of formation (standard deviations are given in brackets).
The deprotonation of the two studied ligands, UDP and UTP, starts at a pH value lower than the range of the test scale. The –O-PO3H group (H2UDP: logβ = 16.76, logKe = 7.08; HUTP: logβ = 6.19, logKe = 6.19) predominates in the systems until around a pH value of about 6.0. In both systems, such forms are almost completely present, at about 100%. At a pH of about 6.5, the fully deprotonated form of UTP begins to dominate in these system. For UDP, there is an extra deprotonation step involving the –N(3)H group in the pyrimidine ring (HUDP: logβ = 9.65, logKe = 9.65). The HUDP species dominates in the system at a pH value of about 8.0, making up about 90% of all species. At pH value above 10.0, the system is entirely in the fully deprotonated form (Fig. 2).
Fig. 2.
Distribution diagrams of protonation of: (a) UDP (
=1 × 10−3 mol/dm3), (b) UTP (
=5 × 10−4 mol/dm3).
Binary systems of tested ligands
Potentiometric studies of binary systems of tested ligands: Urd/GluA, UMP/GluA, UDP/GluA and UTP/GluA were carried out (Table S2; Figure S2, Supplementary Materials). The analysis demonstrated that the investigated systems Urd/GluA, UMP/GluA, and UTP/GluA each contain two (L)Hx(L’) configurations, while the UDP/GluA system includes two (L)Hx(L’) and one LL′ configurations (Table 2).
Table 2.
Overall stability constants (logβ) and the equilibrium constants (logKe) of formation of complex compounds in the studied systems (standard deviations are given in brackets).
| Species | logβ | Reactions | logKe |
|---|---|---|---|
| Urd | |||
| (Urd)H2(GluA) | 15.27(9) | HUrd + HGluA⇄ (Urd)H2(GluA) | 2.87 |
| (Urd)H(GluA) | 11.89(7) | HUrd + GluA− ⇄ [(Urd)H(GluA)]− | 2.67 |
| UMP | |||
| (UMP)H2(GluA) | 19.11(3) | GluA− + H2UMP⇄ [(UMP)H2(GluA)]− | 3.98 |
| (UMP)H(GluA) | 13.89(3) | GluA− + [HUMP]− ⇄ [(UMP)H(GluA)]2− | 7.70 |
| UDP | |||
| (UDP)H2(GluA) | 19.55(9) | GluA− + [H2UDP]− ⇄ [(UDP)H2(GluA)]2− | 2.82 |
| (UDP)H(GluA) | 14.08(7) | [HUDP]2− + GluA− ⇄ [(UDP)H(GluA)]3− | 4.43 |
| (UDP)(GluA) | 4.41(9) | GluA− + UDP3− ⇄ [(UDP)(GluA)]4− | 4.41 |
| UTP | |||
| (UTP)H2(GluA) | 12.38(9) | [HUTP]3− + HGluA ⇄ [(UTP)H2(GluA)]3− | 2.99 |
| (UTP)H(GluA) | 9.15(7) | [HUTP]3− + GluA− ⇄ [(UTP)H(GluA)]4− | 2.97 |
In all examined systems, the process of complex formation begins under the test scale (Fig. 3). For the Urd/GluA and UTP/GluA systems, up to a pH value of approximately 2.5, the L(H2)L’ form is dominant, binding roughly 55% of Urd and 50% of UTP. In the UMP/GluA system, the (UMP)H2(GluA) species dominates up to a pH value of 4.0, binding 70% of UMP. In contrast, the UDP/GluA system up to a pH value of about 6.0 is dominated by the protonated form of H2UDP. In the middle range of pH values, the Urd/GluA and UTP/GluA systems are dominated by the protonated forms of the ligand, while the UMP/GluA and UDP/GluA systems are dominated by the dual L(H)L’ forms. The UDP/GluA system exhibits an LL’ structure that becomes dominant in the system at pH values higher than 10.0, allowing the binding of up to 70% UDP. In contrast, other systems at high pH values contain free ligand. UTP begins to dominate at pH 8.0, while UMP becomes dominant at pH values higher than 10.5.
Fig. 3.
Distribution diagrams for the systems: (a) Urd/GluA, (b) UMP/GluA, (c) UDP/GluA (ratio 1:1; 
=1 × 10−3 mol/dm3), (d) UTP/GluA (ratio 1:1; 
=5 × 10−4 mol/dm3).
Ternary system of Copper(II) ions/uridine or uridine derivatives/glucuronic acid
Analysis of data obtained from potentiometric measurements of ternary systems (Table S3; Figure S3, Supplementary Materials) of uridine (Urd), uridine-5’-monophosphate (UMP), uridine-5’-diphosphate (UDP) and uridine-5’-triphosphate (UTP) with glucuronic acid (GluA) and copper(II) ions has shown the formation of similar types of complex forms. The formation of complexes of the types: CuLL’, CuL(H)xL’ and CuLL‘(OH)x were observed in the systems studied. The overall stability constants and equilibrium constants of the formation reactions are presented in Table 3. The equilibrium constants were determined using the proposed reactions for the formation of the individual complex compounds in the systems studied: oM + pL + qL’ + rH ⇄ MoLpL’qHr38. The accuracy of the selected models was confirmed by comparing the experimental and theoretical curves.
Table 3.
Overall stability constants (logβ) and the equilibrium constants (logKe) of formation of complex compounds in the studied systems (standard deviations are given in brackets).
| Species | logβ | Reactions | logKe |
|---|---|---|---|
| Urd | |||
| Cu(Urd)H2(GluA) | 21.94(4) | Cu2+ + HUrd + HGluA⇄ [Cu(Urd)H2(GluA)]2+ | 9.54 |
| Cu(Urd)H(GluA) | 17.07(5) | Cu2+ + HUrd + GluA− ⇄ [Cu(Urd)H(GluA)]+ | 7.85 |
| Cu(Urd)(GluA)(OH) | 3.31(6) | Cu(GluA)(OH) + Urd− ⇄ [Cu(Urd)(GluA)(OH)]− + H+ | 5.66 |
| Cu(Urd)(GluA)(OH)3 | −17.73(9) | [Cu(GluA)(OH)2]− + Urd− + H2O ⇄ [Cu(Urd)(GluA)(OH)3]3− + H+ | 5.63 |
| UMP | |||
| Cu(UMP)H3(GluA) | 27.22(4) | Cu2+ + HGluA + H2UMP⇄ [Cu(UMP)H3(GluA)]2+ | 8.91 |
| Cu(UMP)H2(GluA) | 24.67(3) | [Cu(GluA)]+ + H2UMP ⇄ [Cu(UMP)H2(GluA)]+ | 9.54 |
| Cu(UMP)(GluA) | 12.20(3) | Cu2+ + UMP2− + GluA− ⇄ [Cu(UMP)(GluA)]− | 12.20 |
| UDP | |||
| Cu(UDP)H(GluA) | 21.99(5) | Cu2+ + GluA− + [HUDP]2− ⇄ [Cu(UDP)H(GluA)]− | 12.34 |
| Cu(UDP)(GluA) | 14.95(5) | [Cu(UDP)]− + GluA− ⇄ [Cu(UDP)(GluA)]2− | 9.74 |
| Cu(UDP)(GluA)(OH) | 6.95(5) | Cu(GluA)(OH) + UDP3− ⇄ [Cu(UDP)(GluA)(OH)]3− | 9.30 |
| Cu(UDP)(GluA)(OH)2 | −2.66(5) | Cu(GluA)(OH)2 + UDP ⇄ Cu(UDP)(GluA)(OH)2 | 6.93 |
| UTP | |||
| Cu(UTP)H(GluA) | 17.34(7) | [Cu(HUTP)]− + GluA− ⇄ [Cu(UTP)H(GluA)]2− | 3.55 |
| Cu(UTP)(GluA) | 13.07(6) | [Cu(UTP)]2− + GluA− ⇄ [Cu(UTP)(GluA)]3− | 5.58 |
| Cu(UTP)(GluA)(OH) | 5.26(7) | [Cu(UTP)(OH)]3− + GluA− ⇄ [Cu(UTP)(GluA)(OH)]4− | 6.12 |
| Cu(UTP)(GluA)(OH)2 | −3.05(6) | [Cu(UTP)(GluA)(OH)]4− + H2O ⇄ [Cu(UTP)(GluA)(OH)2]5− + H+ | 5.46 |
In each of the studied systems, the complexcation process begins at pH below 2.5 (Fig. 4). In the Cu/Urd/GluA (Fig. 4a) and Cu/UMP/GluA (Fig. 4b) systems, protonated ternary forms dominate at low pH values: Cu(Urd)H2(GluA) dominates at pH around 2.5 binding 90% of Cu2+ ions; Cu(UMP)H3(GluA) pH dominance reaches beyond the studied pH range, and at pH around 2.5 binds about 50% of copper(II) ions. For Cu/UDP/GluA(Fig. 4c) and Cu/UTP/GluA (Fig. 4d) systems, Cu(GluA) (dominant pH value: 3.0; 60% of Cu2+ ions) and Cu(HUTP) (dominant pH value: 2.0; 80% of Cu2+ ions) systems dominate at low pH values. The occurrence of CuLL’ forms was confirmed for Cu/UMP/GluA and Cu/UDP/GluA systems. At pH around 7.0 the Cu(UMP)(GluA) is the dominant species and binds nearly 80% of copper(II) ions, while the Cu(UDP)(GluA) form predominates at pH around 7.5, binding approximately 50% of copper(II) ions. At high pH values in the Cu/UDP/GluA and Cu/UTP/GluA systems MLL‘(OH)x hydroxocomplexes are the dominate species. The Cu(UDP)(GluA)(OH)2 and Cu(UTP)(GluA)(OH)2 complexes dominate in the systems at pH around 10.5 binding nearly 90% of Cu2+ ions. The other systems are dominated by the Cu(GluA)(OH)2 form, which reaches its maximum at pH around 10.0 for both systems.
Fig. 4.
Distribution curves for the systems: (a) Cu/Urd/GluA; (b) Cu/UMP/GluA, (c) Cu/UDP/GluA (ratio 1:1; 
=1 × 10−3 mol/dm3), (d) Cu/UTP/GluA (ratio 1:1; 
=5 × 10−4 mol/dm3).
Spectroscopic measurements
To confirm the formation of complex compounds in the systems studied and to determine the composition of their internal coordination spheres, a number of spectroscopic studies were carried out. Measurements were carried out at the dominant pH values, which provide the highest percentage of copper(II) ions in the complex forms studied.
UV-Vis and EPR spectroscopy
Absorbance shifts towards shorter wavelengths were observed in the studied systems, which is related to a change in the internal coordination sphere of the central atom. The attachment of oxygen or nitrogen atoms to the internal coordination sphere of the copper(II) ion is observed by changing the spectral parameters of the complex compounds studied (Fig. 5). The spectral parameters obtained for the systems studied are summarised in Table 4.
Fig. 5.
UV-Vis spectrum for systems: (a) Cu/Urd/GluA; (b) Cu/UMP/GluA, (c) Cu/UDP/GluA (ratio 1:1; 
=1 × 10−3 mol/dm3), (d) Cu/UTP/GluA (ratio 1:1; 
=5 × 10−4 mol/dm3).
Table 4.
UV-Vis and EPR spectroscopic parameters for complexes formed in the studied systems.
| Species | pH | λmax [nm] | ε [M−1cm−1] |
AII [cm−1] |
gII | Chromophores | |
|---|---|---|---|---|---|---|---|
| Urd | |||||||
| Cu(Urd)H2(GluA) | 2.5 | 810 | 38 | 145∙10−4 | 2.41 | {1O} | |
| Cu(Urd)H(GluA) | 6.0 | 800 | 40 | 135∙10−4 | 2.38 | {1-2O} | |
| Cu(Urd)(GluA)(OH) | 8.0 | 700 | 68 | - | - | {1N, 2O} | |
| Cu(Urd)(GluA)(OH)3 | 10.5 | 687 | 160 | - | - | {1N, 3-4O} | |
| UMP | |||||||
| Cu(UMP)H3(GluA) | 2.5 | 820 | 22 | 143∙10−4 | 2.40 | {1O} | |
| Cu(UMP)H2(GluA) | 4.5 | 800 | 24 | 138∙10−4 | 2.39 | {2O} | |
| Cu(UMP)(GluA) | 7.0 | 720 | 17 | - | - | {1N, 2O} | |
| UDP | |||||||
| Cu(UDP)H(GluA) | 6.0 | 790 | 60 | 141∙10−4 | 2.41 | {1O} | |
| Cu(UDP)(GluA) | 7.5 | 750 | 86 | 139∙10−4 | 2.39 | {3O} | |
| Cu(UDP)(GluA)(OH) | 9.0 | 720 | 103 | 134∙10−4 | 2.38 | {1N, 2O} | |
| Cu(UDP)(GluA)(OH)2 | 10.5 | 710 | 100 | 127∙10−4 | 2.37 | {1N, 3O} | |
| UTP | |||||||
| Cu(UTP)H(GluA) | 3.5 | 808 | 22 | 142∙10−4 | 2.40 | {1O} | |
| Cu(UTP)(GluA) | 6.0 | 803 | 24 | 139∙10−4 | 2.39 | {1-2O} | |
| Cu(UTP)(GluA)(OH) | 8.0 | 780 | 28 | 137∙10−4 | 2.39 | {1N, 1O} | |
| Cu(UTP)(GluA)(OH)2 | 10.5 | 718 | 40 | 137∙10−4 | 2.38 | {1N, 2O} | |
On the basis of the analysis of data obtained from UV-Vis spectroscopic studies, it was determined that the composition of the internal coordination sphere of the studied complex compounds changes with a change in the pH value. At lower pH values, coordination in the complex compounds studied occurs via oxygen atoms from the glucuronic acid molecule or oxygen atoms of phosphate residues from UMP, UDP or UTP molecules. As pH increases, the nitrogen atom (N3) in the pyrimidine ring is deprotonated and this atom is incorporated into the internal coordination sphere of the compounds studied.
Analysis of the obtained EPR spectra confirmed the presence of monomeric forms in the studied systems. The obtained spectra are characteristic for the compounds containing copper(II) ions in their structure (Fig. 6). The obtained EPR spectroscopic parameters indicate the participation of oxygen atoms from the -COOH group of D-glucuronic acid and oxygen atoms from the phosphate residues of UMP, UDP and UTP in the formation of coordination bonds at lower pH values [Cu(Urd)H2(GluA): AII=145∙10−4, gII=2.41; Cu(UMP)H3(GluA): AII=143∙10−4, gII=2.40]. As the pH increases, the spectral parameters change, indicating the inclusion of N(3) nitrogen atoms from the uridine ring of the analyzed ligands in the composition of the internal coordination sphere [Cu(UDP)(GluA)(OH): AII=134∙10−4, gII=2.38; Cu(UTP)(GluA)(OH): AII=137∙10−4, gII=2.39].
Fig. 6.
EPR spectrum for systems: (a) Cu/Urd/GluA; (b) Cu/UMP/GluA, (c) Cu/UDP/GluA (ratio 1:1; 
=5 × 10−3 mol/dm3), (d) Cu/UTP/GluA (ratio 1:1; 
=5 × 10−3 mol/dm3).
For the Cu(Urd)(GluA)(OH), Cu(Urd)(GluA)(OH)3 and Cu(UMP)(GluA) forms, EPR spectroscopic studies were not performed due to the presence of precipitate in the prepared samples.
13C and 31P NMR spectroscopy
Analysis of the data obtained from 13C and 31P NMR studies allowed confirmation of the mode of coordination in the systems studied. The results are summarized in Table 5. The obtained 13C NMR and 31P NMR spectra for the analyzed complex compounds are included in the supplementary materials (Figure S4-S10).
Table 5.
Differences in 13C NMR and 31P NMR between the signal positions of ligands in complex compounds in relation to the free ligands [ppm].
| Species | pH | Chemical shifts [ppm] | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Urd | GluA | P | |||||||
| C(2) | C(4) | C(6’) | C(8’) | C(9’) | P(1) | P(2) | P(3) | ||
| Cu(Urd)H2(GluA) | 2.5 | −0.02 | + 0.01 | + 0.12 | + 0.01 | + 0.02 | - | - | - |
| Cu(Urd)H(GluA) | 6.0 | −0.01 | + 0.06 | −0.33 | −0.21 | −0.03 | - | - | - |
| Cu(Urd)(GluA)(OH) | 8.0 | + 0.23 | + 0.15 | −0.42 | + 0.05 | −0.26 | - | - | - |
| Cu(Urd)(GluA)(OH)3 | 10.5 | −7.34 | 0.00 | + 0.13 | + 0.19 | + 0.27 | - | - | - |
| UMP | GluA | P | |||||||
| C(2) | C(4) | C(6’) | C(8’) | C(9’) | P(1) | P(2) | P(3) | ||
| Cu(UMP)H3(GluA) | 2.5 | + 0.02 | −0.01 | 3.62 | + 0.83 | + 0.07 | + 0.85 | - | - |
| Cu(UMP)H2(GluA) | 4.5 | −0.01 | −0.01 | −1,36 | −0.13 | + 0.88 | + 2.7 | - | - |
| Cu(UMP)(GluA) | 7.0 | + 0.18 | −0.23 | + 0.36 | + 0.04 | + 0.42 | + 0.76 | - | - |
| UDP | GluA | P | |||||||
| C(2) | C(4) | C(6’) | C(8’) | C(9’) | P(1) | P(2) | P(3) | ||
| Cu(UDP)H(GluA) | 6.0 | + 0.01 | + 0.01 | + 2.52 | + 0.25 | + 0.28 | + 1.43 | + 2.90 | - |
| Cu(UDP)(GluA) | 7.5 | −0.08 | + 0.09 | + 0.52 | + 0.42 | + 0.29 | −0.57 | + 2.86 | - |
| Cu(UDP)(GluA)(OH) | 9.0 | + 0.42 | −0.38 | −0.48 | −0.13 | + 0.10 | + 0.18 | + 0.25 | - |
| Cu(UDP)(GluA)(OH)2 | 10.5 | −0.64 | −0.07 | + 0.50 | −0.02 | −0.01 | −0.35 | + 0.23 | - |
| UTP | GluA | P | |||||||
| C(2) | C(4) | C(6’) | C(8’) | C(9’) | P(1) | P(2) | P(3) | ||
| Cu(UTP)H(GluA) | 3.5 | + 0.00 | −0.01 | + 3.03 | + 0.46 | −0.24 | + 0.06 | + 0.35 | + 1.52 |
| Cu(UTP)(GluA) | 6.0 | −0.01 | 0.00 | + 0.99 | −0.68 | + 0.34 | + 0.01 | −0.31 | + 0.26 |
| Cu(UTP)(GluA)(OH) | 8.0 | −0.39 | + 0.05 | + 0.97 | + 0.35 | + 0.83 | −7.42 | + 0.30 | −1.23 |
| Cu(UTP)(GluA)(OH)2 | 10.5 | −1.13 | −0.10 | + 1.02 | + 0.68 | −0.25 | −0.09 | −0.27 | −0.02 |
NMR spectroscopic analyses of the investigated complexes and ligands confirmed the composition of the inner coordination sphere. At low pH values, changes in chemical shifts were observed, including the C(6’) carbon from the D-Glucuronic acid molecule (e.g.: 3.62 ppm for Cu(UMP)H3(GluA) and 3.03 ppm for Cu(UTP)H(GluA)) and the phosphorus atoms of phosphate residues (e.g.: 2.7 ppm for Cu(UMP)H2(GluA), 1.43 ppm and 2.90 ppm for Cu(UDP)H(GluA) and 0.06 ppm, 0.35 ppm and 1.52 ppm for Cu(UTP)H(GluA)). The observed chemical shift values indicate that these groups participate in the formation of coordination bonds. As the pH values increases, the activity of the nitrogen atom -N(3) from the uridine ring increases, and this atom is incorporated into the inner coordination sphere. This is evidenced by the differences in the chemical shift values of the ligand compared to the corresponding complex form at the C(2) and C(4) carbon atoms (e.g.: 0.18 ppm and − 0.23 ppm for Cu(UMP)(GluA) or 0.23 ppm and 0.15 ppm for Cu(Urd)(GluA)(OH)). The difference in chemical shifts only at C(2) carbon atom (e.g.: −7.34 ppm for the Cu(Urd)(GluA)(OH)3 complex) may be related to the occurrence of lactam-lactim tautomerism39.
Conclusion
A protonation study was conducted for UDP and UTP ligands, as well as studies of Urd/GluA, UMP/GluA, UDP/GluA and UTP/GluA systems. Potentiometric studies of ternary systems of copper(II) ions, uridine or its derivative, and glucuronic acid were also carried out. Based on the analysis of the potentiometric data obtained, the occurrence of ML(H)xL’, MLL’ and MLL‘(OH)x type complexes in the studied systems was observed. Changing the pH of the environment affected the composition of the internal coordination sphere of the studied complex compounds. Chromophore types were determined by UV-Vis, EPR and NMR spectroscopic studies. At low pH values, the composition of the internal coordination sphere includes oxygen atoms of the -COOH group from the glucuronic acid molecule and oxygen of phosphate residues from UMP, UDP or UTP molecules. As the pH of the studied systems increases, the -N(3) nitrogen atom in the uridine ring is deprotonated and this atom is incorporated into the internal coordination sphere.
Materials and methods
Materials
Glucuronic acid, uridine-5’-triphosphate trisodium salt (UTP) and copper(II) nitrate were obtained from Sigma-Aldrich. Uridine, uridine-5’-monophosphate disodium salt (UMP) and uridine-5’-diphosphate disodium salt (UDP) were obtained from Alfa Aesar. All materials were used without purification.
Equilibrium study
Potentiometric titrations were carried out using a Titrando 702 Methrom equipped with an autoburette with a Methrom 6.0233.100 combined glass electrode, which was calibrated before each series of measurements. The pH-meter was calibrated before each series of titrations using two standard buffer solutions of pH = 4.002 and pH = 9.225. All potentiometric titrations were carried out under constant conditions: inert gas atmosphere (He 5.0 Ultra High Purity; 9.1 m3; 200 bar), temperature (20 ± 1 °C) and ionic strength (0.1 M KNO3), pH range from 2.5 to 11.0, and using as a titrant CO2-free NaOH8,34,39–41. In the Cu(II)/Urd/GluA, Cu(II)/UMP/GluA and Cu(II)/UDP/GluA systems, the metal and ligand concentration was 0.001 M, and in the Cu(II)/UTP/GluA system, the metal and ligand concentration was 0.0005 M. The molar ratio of metal to ligands in all samples was 1:1:1. The protonation constants and overall stability constants for the complex compounds were determined using the HYPERQUAD2008 programme.
The correctness of the selected model was confirmed by standard deviation analysis and convergence of the experimental curve with the computer-generated theoretical curve. The distribution curves for each system were obtained using the HySS programme8,29,42.
The following stability constants were determined: (1) binary system without a metal ion
(where L and L’ = ligands) and (2) ternary system 
(where M = Metal ion). The following equations were used to calculate them42:
 |
1 |
 |
2 |
The 
values for hydroxocomplexes were determined using the following equations:
 |
3 |
where M = metal ion, L and L’ = ligands, H+ = proton and OH− hydroxide ion43.
UV-Vis measurements
The UV-Vis spectroscopy studies were performed at room temperature on the SHIMADZU UV-1900 spectrophotometer using a Plastibrand PMMA cell with a 1 cm path length. Measurements were made in the wavelength range of 550 to 900 nm. The concentration of the metal ion was 0.001 mol/dm3 for Cu(II)/Urd/GluA, Cu(II)/UMP/GluA and Cu(II)/UDP/GluA systems and [Cu2+] concentration for Cu(II)/UTP/GluA system was 0.0005 mol/dm3. The metal to ligands molar ratios were 1:1:1. All tests were carried out using UV Probe software.
EPR spectroscopy
EPR studies were performed at −196 °C using glass capillary tubes (130 µl). Measurements were recorded on an SE/X2457 Radiopan spectrometer. The samples were prepared in a mixture of water: glycol (3:1) and the metal concentration in the sample was 0.005 M. The metal to ligands molar ratio in each systems was 1:1:1.
NMR spectroscopy
13C and 31P NMR measurements were recorded using 400 MHz (9.39 T) AVANCE II Bruker NMR spectrometer and 13C NMR measurement for Cu/UTP/GluA system were performed using 700 MHz (16.44 T) AVANCE III Bruker NMR spectrometer. The samples were obtained by dissolving the test ligands and copper(II) ions in a D2O. The pD of the solutions was determined with NaOD and DCl, given that pD = pH + 0.444. The ligand concentration in the prepared samples was 0.05 M for Cu(II)/Urd/GluA, Cu(II)/UMP/GluA and Cu(II)/UDP/GluA systems and 0.01 M for Cu(II)/UTP/GluA system. The metal concentration was 100 times lower due to the paramagnetic nature of the copper(II) ions (metal to ligands molar ratio: 1:100:100).
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Author contributions
K. Stachowiak: Conceptualization, Data curation, Investigation, Methodology, Supervision, Visualization, Writing – original draft. M. Zabiszak: Conceptualization, Data curation, Methodology, Formal analysis, Validation, Supervision, Writing – review and editing, Project administration. A. Teubert: Investigation. R. Jastrzab: Methodology, Validation, Resources, Writing – review and editing, Supervision, Funding acquisition.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- Knobloch, B. et al. Cu2+, or Zn2+, and Histamine, as well as Adenosine 5′-Triphosphate (ATP4–) or Uridine 5′-Triphosphate (UTP4–): An Intricate Network of Equilibria. Chem.---Eur. J.17, 19, 5393–5403. 10.1002/chem.201001931 (2011). Stability and Structure of Mixed-Ligand Metal Ion Complexes That Contain Ni2+. [DOI] [PubMed]
Supplementary Materials
Data Availability Statement
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.