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17 March 2026

An Investigation into Carnosine as a Coordinating Ligand of Essential Metals, Copper, Zinc and Iron, and Some of Its Biological Activity

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Department of Chemistry, Institute of Chemistry, University of São Paulo, São Paulo 05508-000, SP, Brazil
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Author to whom correspondence should be addressed.
Inorganics2026, 14(3), 85;https://doi.org/10.3390/inorganics14030085
This article belongs to the Special Issue Synthesis, Structural Analysis and Biological Activity of Metal Complexes
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Abstract

Carnosine (or β-alanyl-L-histidine) is an endogenous compound playing very important roles in human organisms as antiglycation and antioxidant agents, and, in addition, helping to mitigate illnesses such as cancer and neurodegenerative diseases. Aiming to explore the chelating ability of carnosine, based on its coordinating possibilities, we started to investigate the metal complexes of essential copper(II), zinc(II), and iron(II) ions coordinated to this dipeptide. Different compounds were isolated in the solid state by adding stoichiometric amounts of metal salts to carnosine at controlled pH or under a controlled atmosphere, with the formation of mono-, bi- and polynuclear species. These complexes were subsequently characterized mainly by spectroscopic techniques (UV–Vis, IR, EPR), in addition to elemental analysis. A binuclear species was isolated with copper(II) and had its structure determined by X-ray diffraction, improving previously reported data in the literature. Two insoluble correlated trinuclear species were isolated with zinc(II) ions, using perchlorate or chloride as counter-ions. In the case of iron, a mononuclear species was verified with Fe(II) ions, obtained under an inert atmosphere. Further, the antioxidant properties of free carnosine and the copper–carnosine complex were verified by their scavenging activity toward the ABTS•+ radical, using Trolox as a reference, showing significant activity. The carnosine–metal complexes were also tested as potential antineoplastic agents, in comparison to the free ligand, after 24 h of incubation at 37 °C, using malignant HeLa, SKMEL 28 and SKMEL 147, and non-tumor fibroblast cells. Results indicated neglected or poor anti-proliferative properties of these metal complexes, when compared to other similar compounds described in the literature.

1. Introduction

Different coordination compounds, based on exogeneous or essential metal ions have been planned, isolated and investigated as potential antiproliferative agents [1,2], motivated by the successful results with the metallodrug cisplatin, used in diverse cancer treatments [3]. Among those compounds based on essential ions, copper(II) and zinc(II) species have deserved special interest [4,5,6], with a wide-ranging type of ligands. Many of these studies focused on the structural and spectroscopic properties, as well as the reactivity characteristics of such complexes.
Carnosine (or β-alanyl-L-histidine) is a dipeptide found in many human tissues, particularly in skeletal muscles and in the olfactory bulb at higher concentrations (in the mM range) [7], and in other organs such as the liver, at lower concentrations (in the μM array) [8]. It plays an important role in living organisms as an antiglycation [9] and antioxidant agents [10]. Many of its properties, and applications of its derivatives have been described, including metabolic effects [11], anti-inflammatory, anti-aging [12], and additionally anticancer activities. In the literature, carnosine was reported as a potential antineoplastic agent, especially against colorectal cancer [13], in a few studies about liver [14], breast [15], and ovarian [16] cancer cells. Recently, carnosine has also been applied to bladder cells [17], acting by suppressing angiogenesis, and to prostate [18] cancer cells, decreasing adenosine triphosphate content and acting through reactive species formation. These studies indicated a multimodal mechanism of action of carnosine, including its ability to modulate cell proliferation, cell cycle, apoptosis, and glycolytic energy metabolism [19]. Further, its ability as a detoxicant of reactive carbonyl compounds (RCS) [20] and in the activation of neural cells [21] has also been investigated, possibly targeting its therapeutic activity in oxidative illnesses such as type 2 diabetes, cardiovascular and neurodegenerative disorders [22,23].
Its valuable effects in these applications have been particularly associated with its antioxidant and anti-inflammatory properties, as well as with the anti-formation of advanced glycation products (AGEs) and anti-formation of advanced lipoxidation products (ALEs) [24]. Investigations suggested that there is a correlation between the antioxidant action of carnosine and its ability to react with reactive carbonyl species (RCS), formed in the oxidation of lipids and/or glucose [25]. Therefore, it seems that carnosine inhibits the formation of AGEs and ALEs in reactions of RCS with proteins [12].
Additionally, the coordinating ability of carnosine has already been explored in previous studies, mostly in aqueous solution [26,27], forming stable species with metal ions in different oxidation states, dependent on pH. Carnosine presents different acid dissociation constant (pKa) values [28,29], regarding the deprotonation of its diverse groups: carboxylic (2.76), imidazole (6.72), and amine at the alanine residue (9.32) [30,31]. Therefore, carnosine shows diverse possibilities of metal ion binding, as found in a few examples in the literature. Zinc carnosine microparticles, composed of a one-dimensional coordination polymer, were reported as exhibiting efficacy in inducing an immune response against influenza [32]. Some dinitrosyl iron-carnosine complexes (DNICs) were able to intercept free radicals, reacting with the superoxide radical formed in the xanthine/xanthine oxidase system [33]. In vitro studies on hepatocellular carcinoma HepG2 cells suggested that carnosine may inhibit the cytotoxic action of the anticancer oxalin platinum metallodrug, possibly through the formation of less cytotoxic ternary complexes [34]. An old publication described binuclear Cd(II) complexes, where carnosine acts as a tetradentate ligand, and in which the imidazole moiety of the ligand undergoes a tautomeric change during complex formation [35].
Herein, we explored some essential metal ion complexes with carnosine, formed in aqueous solution, and verified their possibilities of action as anti-proliferative and antioxidant agents, compared to those of the free ligand.

2. Results

2.1. Copper(II)-Carnosine Complex

Different essential metal complexes were prepared, having carnosine as the ligand. With copper(II), a binuclear species was isolated and had its crystallographic structure determined, as shown in Figure 1a. Corresponding structural parameters are displayed in Table S1 (Supporting Materials). A similar structure has been previously reported by Freeman and Szymanski (1967) in the literature [36], but here it is described with improved resolution and detail. A comparison of the selected Cu–ligand bond distances with those previously reported shows a very similar coordination environment around the Cu(II) center (Table 1). The copper atom adopts a distorted square–pyramidal geometry, with the amino nitrogen, peptide nitrogen, imidazole nitrogen and carboxylate oxygen atoms defining the basal plane. The corresponding Cu–N and Cu–O distances are in good agreement with the previously reported values. The axial position is occupied by a water molecule, displaying a longer Cu–O bond consistent with the Jahn–Teller distortion expected for Cu(II) complexes.
Figure 1. (a) The crystal structure of the binuclear copper–carnosine complex [Cu2(car)2]; (b) asymmetric unit of the complex; (c) crystal employed in the single-crystal X-ray diffraction experiment. (d) Full interaction maps (FIMs) highlighting strong donor (blue) and acceptor (red) regions around the functional groups, and (e) XRDdata for single-crystal and powder sample of complex [Cu2(car)2].
Table 1. Comparison of selected Cu–ligand bond distances (Å) for the present structure and the structure reported by Freeman and Szymanski (1967) [36].
The asymmetric unit of the crystal (Figure 1b) corresponds to half of the binuclear complex (Z′ = 0.5), consistent with the trigonal space group P3121. Each copper ion adopts a distorted square–pyramidal geometry, in which coordination involves the carboxylate, amide, and amino groups of carnosine. Essentially, the imidazole ring bridges the two copper ions through its N1 atom, stabilizing the binuclear core. In the supramolecular arrangement, an additional lattice water molecule interacts via hydrogen bonding with the coordinated aqua ligand, reinforcing the packing and contributing to the stability of the crystalline network. The quality of the specimen employed in the single-crystal X-ray diffraction experiment is shown in Figure 1c, highlighting the crystal used for structure solutions. Additionally, XRD measurements indicated very similar results, both for the single crystal and the powder sample, attesting to the crystallinity and phase purity of complex 1, as shown in Figure 1e.
Complementing the structural description, EPR spectroscopy confirmed the local environment around the copper centers, as discussed later in this work. The spectra displayed rhombic symmetry that matches the distortions observed in the crystallographic data, supporting the square-pyramidal description of the coordination sphere. The agreement between X-ray and EPR results underscores the robustness of the binuclear structure in both solid-state and frozen-solution conditions, validating the stability of the supramolecular assembly.
The analysis of the full interaction maps (Figure 1d) provides further insights into the supramolecular stabilization of the copper–carnosine complex [37]. Strong blue regions, corresponding to hydrogen bond donor sites, were observed in proximity to the N–H groups of the peptide backbone, while intense red regions, indicative of hydrogen bond acceptor sites, were located around the carbonyl oxygens and coordinated water. This complementary distribution highlights the propensity of the system to establish robust intermolecular hydrogen bonding networks, consistent with the stabilization observed in the crystal packing. These features confirm that the peptide-like nature of carnosine provides an effective framework for directional hydrogen bonding, driving the three-dimensional assembly of the complex in the solid state. In addition, the presence of multiple donor and acceptor sites may facilitate intermolecular recognition processes, which are often associated with biological interactions. Therefore, these structural features may contribute to the observed biological efficiency and provide useful insights into possible structure–activity relationships (SAR) for this class of copper–peptide complexes.

2.2. Zinc(II)-, and Iron(II)-Carnosine Complexes

Analogous complexes were isolated in the solid state by using zinc(II),or iron(II) salts, as described in the Experimental Section. Elemental analysis data are shown in Table 2. In the case of zinc, two similar white trinuclear compounds were isolated when using different salts. With perchlorate, complex [Zn3(car)2(OH)(H2O)3]ClO4 2 was obtained, while complex [Zn3(car)2Cl2] 3 was formed using chloride. Both are very insoluble species. On the other hand, a mononuclear reddish-brown complex with iron(II) ions, [FeII(car)3](ClO4)2 4, was synthesized under an inert atmosphere. Results of elemental analyses for all these metal complexes are shown in Table 2.
Table 2. Elemental analysis data for the studied metal complexes (1–4). In parentheses, % expected according to the structures proposed based on experimental data.
These results indicated that carnosine forms preferentially bi- or trinuclear species in vitro, due to their multiple coordinating groups, but mononuclear complexes can similarly be obtained. In the case of iron, under an inert atmosphere, a mononuclear species was isolated, but it was partially oxidized at RT, as attested by EPR measurements. On the contrary, in vivo, a mononuclear species should be predominant.

2.3. Spectroscopic Characterization of the Metal Complexes

Spectroscopic measurements were used to better characterize these isolated complexes. FTIR spectra were registered for all of them, as shown in Figure S2 Supporting Materials. Based on these data, the main bands and corresponding attributions are displayed in Table 3.
Table 3. The main spectroscopic bands observed in the FTIR spectra of the ligand carnosine and the corresponding metal complexes studied (1–4).
FTIR technique allows for the identification of the main carnosine functional groups and the study of its coordination sphere when comparing the spectra of the corresponding complexes with the free ligand L-Carnosine (or β-alanyl-L-histidine). In those spectra, it was possible to observe the displacement of some bands to lower wavenumbers and frequencies due to the weakening of intramolecular ligand bonds and strengthening of metal–ligand (M-L) bonds. The effect of the counter-ion was also verified, as the perchlorate ion (ClO4−) lowers the frequency of the respective vibration, causing the overlap of some bands in the fingerprint region. In aliphatic coordination compounds, the M-L bond tends to shift to a lower wavenumber, but the opposite occurs with aromatic ligands. In these complex spectra, the metals also coordinate to the imidazole ring of carnosine and the respective bands appear at higher wavenumbers in contrast to the free ligand spectrum, on account of the hybridization of the aromatic ring.
It is also possible to observe, in the coordination compounds studied, the decrease in band intensities as well as their smothering that can indicate a binding between the metal and the ligand in the respective functional group, or proximity to the coordination site, which can be the imidazole, carboxylate, secondary amide and tertiary amine groups. The free L-Carnosine spectrum is well-known in the literature [27] and its band attribution is also described in Table 3. The observed band displacements regarding the free ligand suggest that coordination occurs at the amine terminal, carboxylate, amide and imidazole groups. Carnosine, as a dipeptide, displays a stable zwitterionic form, showing the n (NH3+) band related to the amine terminal, located in the lower-energy region, and represented by two peaks in 3239 and 3051 cm−1.
Characteristic amide band ν ( C=O) occurs at 1643 cm−1 and the carboxylate is represented by ν(COO−1) at 1403 cm−1 in the free carnosine spectrum, and around 1400 cm−1 in the spectra of the metal complexes. The imidazole band is characterized by ν(N-H) at 1432 cm−1, ν(HC-N) at 1253 and 1227 cm−1, and a further ring distortion at 1270 cm−1 due to the coordination to metal ions.
In Table 3, observed OH bands from water coordination or hydration are very intense, and therefore it was not possible to notice the amine stretching vibrations because of overlapping. In zinc complexes, an overlap occurs with the bands of ν(C=O), at 1643 cm−1, and the vibration ν(C=C) of the imidazole ring, near 1563 cm−1, which also indicates coordination in amide and imidazole groups. Coordination is also indicated by a decrease in band intensity, in cases where a displacement to lower or higher wavenumbers does not occur. This phenomenon happens to the following bands in copper and iron complexes: ν(HC-N) of imidazole, 1253 and 1227 cm−1, and ring distortion, 1270 cm−1, which showed a lower intensity. In most syntheses of coordination compounds, perchlorate salts were used, acting as counter-ions as attested by a characteristic band in the wavenumber range from 1100 to 1070 cm−1.
By using UV–Vis spectroscopy, an enhanced characterization of the complexes under investigation was achieved. With the aim of comprehending and analyzing the compounds in different conditions, UV–Vis spectra were recorded in different solvents, water, ethanol, and acetonitrile, and at different pH values. As observed in the spectrum registered in Figure 2, carnosine usually exhibits shoulders in the 200–300 nm range of spectra. This same behavior happens in the corresponding complexes, as shown in the following figures.
Figure 2. UV–Vis spectra of carnosine (a) in different solvents (0.53 μM): water, acetonitrile:water (1:1), and ethanol:water (1:1); (b) in aqueous solution (0.70 μM) at different pH.
Figure 2 shows the carnosine spectrum in three solvents, at a 0.53 µM concentration. It is possible to observe three shoulders at the same wavelength, 213 nm, indicating that the solvent does not affect the electronic carnosine transitions, and suggesting a π → π * orbital transition. Meanwhile, to study how this compound reacts under different conditions, especially at pH 7.4 in PBS buffer, the experiment was repeated in aqueous solution at different pH levels. These spectra show a peak at 214 nm and shoulders around 278 at pH 7.4, and at 222 nm at pH 3.0, respectively, indicating π → π * transitions related to the ligand.
Figure 3a illustrates the shoulders of [Cu-car] 1 in the selected solvents, which correspond to π → π * transitions originating from the ligand. Due to the solubility of [Cu-car] in DMSO (Figure 3b), a spectrum was obtained in this solvent at higher concentrations, and a peak referring to the copper d-d band appears at 577 nm. Additionally, varying the medium pH value reveals peaks and shoulders from the ligand around 217 nm and between 254 nm and 276 nm. It was observed that the d-d band is more intense at basic pH, indicating that the complex is formed and more stable in an alkaline medium (Figure 3c). Indeed, the acid pH spectrum lacks this band, suggesting that the compound is not formed in these acidic conditions.
Figure 3. UV–Vis spectra of complex [Cu2(car)2(H2O)2] 1 (a) in different solvents: water, acetonitrile:water (1:1), and ethanol:water (1:1); (b) the d-d band in DMSO at diverse concentrations and (c) in aqueous solution at different pH values.
Iron complex [FeII(car)3](ClO4)2 4 was also investigated, as shown in Figure 4, exhibiting a shoulder around 212 nm (see Figure 4a), and at 214 nm (Figure 4b), indicating a π → π * transition attributed to the carnosine ligand. At 325 nm, a metal-to-ligand charge-transfer (MLCT) transition is observed due to the low spin occasioned by the strong field of the ligand.
Figure 4. UV–Vis spectra of iron complex [FeII(car)3](ClO4)2 4, (a) in different solvents: water, acetonitrile:water (1:1), and ethanol:water (1:1); (b) in aqueous solution, at diverse pH values.
The spectrum, in the range from 200 to 800 nm, of all compounds described in this work, is attached to the Supplementary Materials (Figures S2–S4).
For the copper and iron complexes, EPR spectra were also recorded, allowing for the detection of different magnetic species. The spectra of complex [Cu2(car)2(H2O)2] 1 (see Figure 5) in DMSO solution at 77 K exhibit two axial Cu2+ species, in accordance with the structure determined in Figure 1. The first species has g-values equal to g┴ 2.165 and g// 2.238, with a hyperfine coupling constant A// = 84 G. The second species has g-values of g┴ 2.139 and g// 2.332, with a hyperfine coupling constant A// = 91 G.
Figure 5. EPR spectra of [Cu2(car)2(H2O)2] complex 1 (a) in the solid state at RT and 77 K, and (b) in DMSO solution at 77 K (experimental and simulated).
By using the Sakaguchi approximation [38], the geometry around the two copper centers in frozen DMSO solution can be estimated. For the first species, the calculated Sakaguchi parameter (g///A//) is 255, and for the second species, this parameter is 235, indicating a tetragonal geometry with a little tetrahedral distortion for both copper ions. The low intensity of the spectrum in Figure 5a, referred to as the signal in solution at 77 K, is probably due to antiferromagnetic coupling between the two copper centers (electronic configuration d9 and ground orbital dx2-y2).
For the iron complex 4, the elucidation of the spectra was more difficult, probably due to the presence of both oxidation states of iron.
In the solid state at 77 K (Figure 6), compound 4 shows diamagnetic behavior, being a low-spin species with a very weak isotropic signal at g = 2.026, ascribed to an iron(II) ion surrounded by N-coordinating atoms of the ligands. In DMSO solution at 77 K, a similar diamagnetic signal was also observed, with an isotropic signal at g = 2.621. However, in the solid state at RT, a typical iron(III) spectrum with two signals was verified, one at g = 2.026 and the second one at g = 6.204, probably due to the partial oxidation of iron(II) to iron(III) ions by dioxygen [39]. A mixed-valence species, possibly [Fe(III)LFe(II)LFe(III)], could be formed. Further studies are needed to elucidate its structural features.
Figure 6. EPR spectra of the iron complex [FeII(car)3](ClO4)2 4, in the solid state at RT and 77 K, and in DMSO solution at 77 K.

2.4. Proposed Structures

Based on those experimental data, the probable structures for the complexes studied are proposed, shown in Figure 7.
Figure 7. Proposed structures of free carnosine and some essential metal complexes isolated and characterized (1–5), drawn by ChemDraw software.

2.5. Biological Activities

2.5.1. Anti-Proliferative Activity

In the literature, carnosine is well-reported as a potential antineoplastic agent against different types of cancer cells [13,14,15,16,17,18]. Its mechanism of action against malignant cells remains unclear, but it is related to decreasing oxidative stress and, as a result, reducing the production of reactive oxygen species due to some biological properties of carnosine and derivatives [19].
In this work, it was intended to explore carnosine as a potential anticancer agent for melanoma and cervical cancer cells, using the MTT methodology [40]. Cell viability in the presence of carnosine and complexes 1 and 4 was tested, in the range from 5 to 100 μM, using 24 h incubation at 37 °C. In Table S4 in the Supporting Materials, data obtained in those tests are displayed.
As shown in Figure 8, the higher concentration (100 μM) and incubation time used in the experiments (24 h) were not enough to achieve an IC50 value. This demonstrates only poor anti-proliferative activity of carnosine and the synthesized complexes. Only the iron complex 4 showed some low activity in inhibiting the proliferation of melanoma cells (SK-MEL 28 and SK-MEL 147). Regarding the copper complex 1, it was indeed observed that there was a proliferation protection of those cells up to 100 μM, especially SK-MEL 28 and HeLa cells.
Figure 8. Cell viability of carnosine, metal complexes [Cu2(car)2(H2O)2] 1 or [Cu-car], and [FeII(car)3](ClO4)2 4 or [FeII-car] against tumor cells, SK-MEL and HeLa, and non-tumor fibroblast cells. For all compounds, concentration = 100 μM.
In the literature, carnosine has been reported as an anti-proliferative compound in diverse studies [13,14,15,16,17,18]. However, in all these investigations, the concentrations used are very high, usually in the range of mM, and after long incubation times (48 to 96 h), as shown in Table S3 in the Supporting Materials. As a few examples, against bladder EJ malignant cells, the IC50 reported is 50 mM after 24 h of incubation [17]; for PC3 metastatic prostate cells, the reported value is 40.36 mM after 48 h of incubation [18]; and for HCT-116 colon cells, the determined value is 33 to 37 mM for carnosine inserted in nanoparticles after 96 h of incubation [13].
Our studies, on the contrary, showed that carnosine or the corresponding metal complexes investigated do not exhibit remarkable anti-proliferative properties in comparison with other compounds already tested and reported in the literature.

2.5.2. Antioxidant Properties

Carnosine has been designed to prevent and treat neurodegenerative diseases due to its well-known properties such as acting as an anti-inflammatory agent, reducing oxidative stress and preventing the formation of advanced glycation end products (AGEs), as well as advanced lipoxidation products (ALEs) [24]. These disorders are related to oxidative stress, similarly to cancer. One way to evaluate the performance of compounds for a potential treatment against this illness is to verify their capacity to react with ABTS or [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] through the free radical ABTS•+ scavenging assay [41,42].
Supported by the competence of cell protection observed through the cell viability assay, it is worthwhile to study carnosine and its potential role against neurodegenerative disorders. The assay adopted to analyze the ability of this dipeptide as an antioxidant agent is the Trolox equivalent antioxidant capacity (TEAC) [43]. This methodology measures spectroscopically the capacity of a compound to scavenge the free radical cation relative to the antioxidant ability of Trolox, widely used as an antioxidant compound [44]. The results are reported as inhibition in terms of percentage and as TEAC values that indicate the concentration of Trolox having an equivalent antioxidant capacity of the sample studied, in this case carnosine.
In reaction with ABTS•+ radical, the dipeptide is oxidized through electron and/or hydrogen transfer reactions [45], according to:
2 ABTS•+ + carnosine → 2 ABTS + oxidation products
Figure 9 shows the scavenging activity of carnosine in terms of inhibition (%) against the concentration (µM). Measurements were made 6 min after mixing the tested compound with the free radical, and after 24 h of incubation. The results indicated that carnosine does not respond very well in a short period of time, but after 24 h it was possible to achieve EC50 equal to 22 µM of carnosine.
Figure 9. ABTS•+ inhibition due carnosine solution in different concentrations during two periods of time: 0 h, and after 24 h.
Using the standard curve of ABTS•+ inhibition from 0 to 20 µM of Trolox in PBS at pH 7.4, attached as Figure S5 in the Supplementary Materials, it was possible to determine the TEAC values for carnosine as an antioxidant agent. In the Supplementary Materials (Table S3), the corresponding Trolox equivalent antioxidant capacity (TEAC) values are displayed for copper(II) ions and the carnosine-copper(II) complex.
According to the literature, carnosine has a potential anti-neurodegenerative activity toward diseases related to oxidative stress and aggravation by free radicals. This activity can be improved by analogs of this dipeptide, or by reacting carnosine with other molecules showing antioxidant properties [41].
As observed in Figure 9, just after mixing, an inhibition of 44% and TEAC values of approximately 11 µM were obtained using 200 µM of carnosine. After 24 h for the same concentration, the observed inhibition was 97%, corresponding to a 24 µM Trolox solution (TEAC). Similar experiments were performed with [Cu-car] complex 1 (see Figure 10) and, for comparison, with copper(II) perchlorate hydrate salt, and with a mixture (1:1) of carnosine plus copper(II) salt, trying to mimic a more probable species formed in vivo.
Figure 10. Antioxidant activity of [Cu-car] 1 complex, copper(II) perchlorate salt, and mixture of carnosine—copper(II) (1:1).
Different dipeptides had their ABTS•+ scavenging ability evaluated, indicating EC50 values (corresponding to 50% inhibition) in the range from 7 to 14.5 μM [46]. Therefore, our results indicated moderate antioxidant activity of carnosine and the corresponding copper(II) complex 1 relative to other dipeptides already tested [45,46], and that this property can be improved in the presence of copper(II) ions.

3. Experimental Section

Materials and Methods—Carnosine 99% was purchased from Sigma-Aldrich (São Paulo, Brazil). Metal salts, copper(II) perchlorate hexahydrate, zinc(II) chloride and perchlorate, iron(II) perchlorate hydrate and iron(III) perchlorate hydrate were P.A. grade, from Merck/Sigma. The solvents used were purchased from Sigma Aldrich or Synth. ABTS [or 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] and Trolox were also obtained from Sigma Aldrich.
UV–Vis spectra were recorded on a Shimadzu spectrophotometer (Shimadzu, Kyoto, Japan), mod. 1650, using quartz cells with a 1.000 cm width. Cell viability data were analyzed using GraphPad Prism (version 8), and general results were analyzed by Origin software (2024). Compound structures (Figure 7) were drawn with ChemDraw 2022 (v. 22.0.0.22).
FTIR spectra were recorded on an Agilent Cary 630 spectrometer (Santa Clara, CA, USA). EPR measurements were recorded on an EMX Bruker instrument (Billerica, MA, USA), operating at X-band (9.5 GHz, 100 kHz, 20 mW power), using quartz tubes, at RT and at 77 K. Simulations were obtained by Easy Spin software [47], on a MatLab platform. Elemental (C, N, H) and ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) (Central Analitica, IQ-USP, São Paulo, Brazil) analyses were obtained at the Central Analítica facility in our institution (http://ca.iq.usp.br/), using a Perkin Elmer 2400 series analyzer (Perkin-Elmer, São Pauloo, Brazil) or a Spectro spectrometer, mod. Arcos. Crystallographic structure determination was carried out on a Rigaku Synergy-S diffractometer (Tokyo, Japan), using a CuKα (λ = 1.54184) radiation source, coupled to an HPC area detector, at the Institute of Physics, University of São Paulo, Campus São Carlos (IFSC-USP). Measurements were done under liquid nitrogen (T = 100 K).
Syntheses of the metal complexes (1–4)—A general method to prepare the metal complexes consisted of dissolving 0.6790 g of carnosine (3 mmol) in ~5 mL of deionized water and adding it to 3 mmol of an adequate metal salt dissolved in 5 mL of deionized water. By adjusting the pH of this solution to 8–9 via the addition of a few drops of NaOH 1 M, the formation of a precipitate was observed in all cases. For copper(II), a blue complex [Cu2(car)2(H2O)2] 1 was isolated. In the case of zinc(II) ions, two white complexes, [Zn3(car)2(OH)(H2O)3]ClO4 2 and [Zn3(car)2Cl2] 3, were obtained. Synthesis of [Fe(car)3](ClO4)2 4 required an inert atmosphere. Each of the obtained products was then washed with cold ethanol and ethyl ether and dried in a desiccator at low pressure. For the copper(II) complex 1, the obtained yield was 72.4%, and the compound was soluble in water, DMSO, ethyl ether, ethanol and acetone. From the filtrate, adequate blue crystals were separated and analyzed by X-ray diffraction measurments. Yields for the carnosine complexes [Zn3(car)2(OH)(H2O)3]ClO4 2, [Zn3(car)2Cl2] 3, and [Fe(car)3](ClO4)2 4 were 46.0, 82.0, and 15.4, respectively. The iron(II) complex 4 is soluble in water. On the other hand, zinc complexes were very insoluble in water and common solvents (ethanol, methanol, DMF, CH3CN, and DMSO).
Cell culture—Human melanoma SK-MEL 147 (CVCL 3876) cells were kindly donated by Professor Roger Chammas from the Instituto do Câncer do Estado de São Paulo (ICESP, São Paulo, Brazil). Human melanoma SK-MEL 28 cells were donated by Professor Silvya Stuchi Maria-Engler, from the School of Pharmaceutical Sciences at the University of São Paulo (FCF-USP). Human non-tumor CCD-1072Sk fibroblast cells and cervical HeLa (ATCC CCL-2) cells were purchased from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil, https://bcrj.org.br/). SK-MEL cell lines and HeLa cells were cultured in DMEM supplemented with 10% FBS, while CCD-1072Sk cells used IMDM culture media, supplemented with 10% FBS. All the cell lines were maintained in an incubator with a humidified 5% CO2 atmosphere at 37 °C.
Viability Assay—For SK-MEL cell lines, 1.0 × 104 cells/well were seeded in 200 μL in 96-well plates containing DMEM supplemented with 10% FBS and incubated for 16 h at 37 °C in a 5% CO2 atmosphere. Then, the culture medium was withdrawn, the cells were washed with PBS and DMEM containing the complexes in a concentration ranging from 5 to 100 μM was added to each well. The plates were left in the incubator for 24 h at 37 °C, in a 5% CO2 atmosphere. Another step of the culture medium withdrawal and the washing of the cells was employed, followed by the addition of DMEM containing MTT at a 0.3 mg MTT/mL concentration. Cells were incubated for 3 h and cell viability was measured by MTT reduction in living cells [40]. The formazan crystals were solubilized in DMSO (100 μL/well) with each well being measured at 570 nm, in a Tecan Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). The IC50 value was obtained by analyzing absorbance data from three independent experiments. A similar procedure was used with fibroblast cells, using IMDM culture medium supplemented with 10% FBS.
ABTS•+ Radical Scavenging Assay—The ABTS•+ radical was produced by reacting 7 mM ABTS in aqueous solution with 2.45 mM potassium persulfate for 12–16 h in the dark at room temperature [48]. For this study, the absorbance of ABTS•+ was measured on a Shimadzu spectrophotometer, mod. 1650, using quartz cells with a 1.000 cm width, and adjusted to (0.70 ± 0.2 ) at 734 nm by dilution with PBS at a pH of 7.4. A 96-well plate was used to mix the samples, in the desired concentrations, and the diluted ABTS•+ stock solution, maintaining the volume of 200 µL in each well. The prepared plate was kept in the dark for 6 min to allow the occurrence of a complete mixture and reaction, and the measurements were recorded on a Spectra Max Paradigm multi-mode detection platform (Molecular Devices, San José, CA, USA)—molecular devices. The Trolox inhibition of ABTS•+ was used as standard reference data for the construction of the concentration–response curve, to obtain the line equation and, afterward, calculate the TEAC values for the compounds under study (see the Supporting Materials). All experiments were conducted in sextuplicate, and the ABTS•+ radical scavenging activity (%) was calculated based on the following formula:
s c a v e n g i n g   a c t i v i t y   ( % ) =   A b l a n k − A s a m p l e A b l a n k × 100
The appropriate solvent, in this case PBS at a pH of 7.4, was used as a blank in each assay.

4. Conclusions

Different complexes of carnosine coordinated to essential metal ions copper(II), zinc(II), or iron(II) were isolated and characterized. Elemental analysis and spectroscopic results are consistent with the structures proposed. For copper(II), a blue binuclear neutral complex [Cu2(car)2 (H2O)2] 1 was isolated and had its crystal structure refined by X-ray diffraction and XRD measurements. The two copper centers are bridged by two carnosine ligands, coordinated by NNO bonds, from amine, amide, imidazole and carboxylic groups. For zinc(II), two dissimilar white complexes were prepared, [Zn3(car)2(OH)(H2O)3]ClO4 2 and [Zn3(car)2Cl2] 3, both very insoluble in most common solvents. In complex 2, the central zinc ion is bonded to the imidazole groups of two ligands, and its coordination sphere is completed by a water molecule and a hydroxyl anion. On the contrary, the terminal zinc centers are coordinated to amine (N), amide (N), carboxylic groups (O) and a water molecule. In complex 3, the terminal zinc ions are bonded to imidazole (N), amine (N) and amide groups (O), in addition to a chloride ion. In the case of iron, a mononuclear reddish-brown species [FeII(car)3](ClO4)2 4 was isolated under an inert atmosphere. The carnosine acts as a bidentate ligand, coordinating the metal by amine (N) and amide (N) groups in an octahedral environment. This iron complex 4 was verified to be diamagnetic in the solid state or in frozen DMSO solution at 77 K, probably having all spins paired (low-spin, electronic configuration d6). However, in the solid state at RT, EPR measurements detected an isotropic signal of low intensity, probably due to the partial oxidation of iron(II) to iron(III) by dioxygen on the surface of the powder sample.
Regarding their biological activity, carnosine showed only poor or negligible anti-proliferative activity up to a 100 μM concentration towards melanoma and HeLa cells, after 24 h of incubation at 37 °C. Nevertheless, in the literature, some previous studies reported better performance toward different malignant cells [13,14,15,16,17,18]. In such studies, higher concentrations, usually in the mM range, were used after longer periods of incubation (48–96 h).
For the corresponding carnosine–metal complexes studied, the measured anti-proliferative activity was also poor, although in the case of iron(II) complex 4, some decrease in cell viability was verified in comparison to free carnosine. In contrast, the copper(II) complex 1 indeed showed some protection of the cells compared to free carnosine. In the literature, there are many metal complexes of copper, zinc or iron, with an extensive selection of nitrogen ligands (especially imines, hydrazones, pyridines, etc.) which have been described as remarkable anticancer compounds [2,4,5,6], showing much better anti-proliferative properties, in the range from 5 to 100 μM.
Finally, carnosine and copper(II) complex 1 were also tested for their antioxidant properties, regarding their scavenging ability toward ABTS•+ radicals compared to Trolox. Free carnosine showed only 40% inhibition at a 200 μM concentration immediately after mixing with ABTS•+ radicals. However, after 24 h of incubation, 50% inhibition was achieved at 22 μM and complete inhibition was observed at 200 μM. The copper complex 1 shows similar activity to aqueous copper(II) ions, while a mixture of aqueous copper ions and carnosine showed 60% inhibition up to 150 μM.

5. Future Perspectives

Our studies indicated the possible formation of mono-, bi- and trinuclear carnosine complexes with essential metal ions implicated in neurodegenerative diseases. These investigations can help detect the additional roles of carnosine in biological systems, showing its interactions with copper, zinc and iron ions.
As shown in our reported investigations, free carnosine is a good antioxidant agent, up to 200 μM, but a poor anti-proliferative compound in the range from 5 to 100 μM, incubated for 24 h at 37 °C. The corresponding copper(II) complex 1 seems to be, on the contrary, a pro-proliferative agent toward malignant cells at a 100 μM concentration, increasing its viability.
Further studies with carnosine and [Metal–carnosine] 1:1 species are in development in our lab, with the aim of better understanding their biological properties. Carnosine’s anti-proliferative properties are limited and do not comprise reactive species formation, as usual in the mechanism of action of promising anticancer agents. On the other hand, its antioxidant properties seem to be correlated with oxidative stress and essential metal dyshomeostasis, implicated in neurodegenerative pathologies such as Parkinson’s or Alzheimer’s diseases.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/inorganics14030085/s1.

Author Contributions

Conceptualization, G.C.d.L., A.P.A.d.O. and A.M.d.C.F.; methodology, G.C.d.L., A.P.A.d.O. and J.H.d.A.-N.; investigation, G.C.d.L., A.P.A.d.O., J.H.d.A.-N. and M.C.P.; validation, G.C.d.L., A.P.A.d.O., J.H.d.A.-N. and A.M.d.C.F.; formal analysis, G.C.d.L., A.P.A.d.O., J.H.d.A.-N., M.C.P. and A.M.d.C.F.; writing—original draft preparation, G.C.d.L., A.P.A.d.O., J.H.d.A.-N., M.C.P. and A.M.d.C.F.; writing—review, and editing, G.C.d.L. and A.M.d.C.F.; project administration, and resources, A.M.d.C.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the São Paulo State Research Foundation (FAPESP, Redoxoma project, grant 2013/07937-8) and the National Council for Scientific and Technological Development (CNPq, grant 408593/2025-3).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data are available from the authors under request.

Acknowledgments

All the authors are thankful to the São Paulo State Research Foundation (FAPESP, Redoxoma project, grant 2013/07937-8) and the National Council for Scientific and Technological Development (CNPq, grant 408593/2025-3) for financial support. APA de Oliveira and GC de Lima are grateful to FAPESP for their fellowships, grant 2020/06719-0 and grant 2023/11511-8, respectively. Authors also thank Mauricio Baptista da Silva (Photobiochemistry and Photochemistry Lab, at IQ-USP) for the use of the cell manipulation room.

Conflicts of Interest

The authors declare no conflicts of interest.

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