New cation sensors based on eugenol-derived azo dyes

Academic Editors: Gerald Anthony Murphy and Liudmil Antonov Received: 13 May 2025 Revised: 24 June 2025 Accepted: 25 June 2025 Published: 28 June 2025 Citation: Coelho, J.R.A.; Pacheco, A.R.F.; Domingues, D.C.; Rodrigues, A.R.O.; Temitope, A.A.; Coutinho, P.J.G.; Fernandes, M.J.G.; Castanheira, E.M.S.; Gonçalves, M.S.T. New Cation Sensors Based on Eugenol-Derived Azo Dyes. Molecules 2025,30, 2788. https://doi.org/10.3390/ molecules30132788 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article New Cation Sensors Based on Eugenol-Derived Azo Dyes José R. A. Coelho 1, Ana Rita F. Pacheco 2,3, Diogo C. Domingues 1, Ana Rita O. Rodrigues 2,3 , Akani A. Temitope 1, Paulo J. G. Coutinho 2,3 , Maria José G. Fernandes 1, Elisabete M. S. Castanheira 2,3 and M. Sameiro T. Gonçalves 1,* 1Chemistry Centre of the University of Minho (CQ-UM), Department of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (J.R.A.C.); [email protected] (D.C.D.); [email protected] (A.A.T.); [email protected] (M.J.G.F.) 2Physics Centre of Minho and Porto Universities (CF-UM-UP), Department of Physics, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] (A.R.O.R.); [email protected] (P.J.G.C.); [email protected] (E.M.S.C.) 3Associate Laboratory LaPMET, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal *Correspondence: msameir[email protected] Abstract Eugenol-based azo dyes illustrate how bio-sourced compounds like eugenol can be transformed through synthetic processes into functional and colorful compounds. The main purpose of the present work was to develop new responsive colorimetric sensors for metal cations based on eugenol-derived azo compounds. The incorporation of the azo group into the eugenol framework allows for strong electronic interactions with metal cations, leading to distinct color changes observable to the naked eye. These azo-eugenol dyes exhibit shifts in their UV-Vis absorption spectra upon complexation with metal cations such as copper (Cu 2+ ) and lead (Pb 2+ ), making them effective sensors for environmental and analytical applications. The eugenol-based azo dyes were subjected to photophysical studies to understand selectivity, response time, and stability in relation to metal cations, which will be a starting point for the monitoring of toxic metal contaminants in aqueous environments. Keywords: eugenol; phenolic compounds; azo dyes; cation detection; colorimetric sensors 1. Introduction Azo dyes, known for their diversity of appealing colors and stability, have been widely used in various applications such as textiles, food, and analytical chemistry, due to their ease of synthesis, structural variability, and unique chromogenic tonalities to the naked eye, where reds, oranges, and yellows predominate [ 1 – 6 ]. The exact coloration of the respective dyes is based on the existence of a chromophore group (-N=N-), associated with the nature and position of the substituents connected to the aromatic systems, where the amplification of the degree of conjugation of the π system enhances the absorption of light at longer wavelengths [ 7 , 8 ]. These dyes can undergo significant color changes in response to the binding of specific cations, enabling rapid, visual detection of metal ions [ 9 – 11 ]. Among these, azo dyes derived from natural sources, like eugenol, may offer a promising eco-friendly alternative to full synthetic dyes, where it is possible to combine the physicochemical and biological properties inherent in phenolic compounds, known to have significant antioxidant activities [12,13]. Molecules 2025,30, 2788 https://doi.org/10.3390/molecules30132788 Molecules 2025,30, 2788 2 of 17 Eugenol, the major secondary metabolite found in clove oil (Syzygium aromaticum) and present in other essential oils, provides a versatile basis for the synthesis of azo dyes due to its reactive functional groups and aromatic structure. Azo dyes synthesized from eugenol are not only potential environmentally benign, but also possess unique photophysical properties, making them ideal candidates for colorimetric sensing applications [ 14 – 16 ]. From a structural perspective, eugenol is a phenolic compound, meaning that the existence of a hydroxyl group makes it a suitable ligand for complexing with metals. Another crucial aspect is the role of the methoxyl group in the orientation of the ligand in relation to the metal center, which positions the donor atom in the metal’s coordination sphere [ 17 ]. As a ligand, eugenol can form complexes with important biometals such as Fe 2+ and Fe 3+ cations. The eugenol moiety has been already reported as a colorimetric probe for iron(III), where it displays complexes that have a blue-green color ( λmax = 653 nm) [ 18 ]. Thus, eugenol-derived azo dyes present a novel, sustainable, and effective approach for developing selective and sensitive colorimetric sensors for cation detection. This feature is particularly valuable in environmental monitoring, medical diagnostics, and industrial processes, where detecting toxic or essential cations with high sensitivity and selectivity is crucial. Metals present a dichotomy based on their dose and their biological importance [ 19 ]. Some metals are essential in metabolic processes, where excess or deficiency can lead to severe damage. Other metals are real poisons that tend to bioaccumulate in the environment. Copper is a biologically relevant element; however, prominent levels of Cu 2+ can contribute to the development of neurodegenerative diseases (such as Alzheimer’s disease, Menkes’ syndrome, or Wilson’s disease), prion disease, hypoglycemia, and dyslexia, and also to the progression of cancer [ 20 – 22 ]. On the other hand, lead (Pb 2+ ) is described as a poison that can affect many organs in the human body, damaging the brain, nervous system, blood, digestive system, and also the reproductive system, due to its ability to mimic and, in some cases, inhibit the action of calcium as a regulator of cellular function [23,24]. In recent years, scientific research efforts have been directed toward significant advances in the detection of various metal cations, particularly through the development of chemosensors for Cu 2+ and Pb 2+ ions, which have recently been described in detail [ 25 , 26 ]. Organic colorimetric chemical sensors, such as thioureas, Schiff bases (imines), pyridines, thiophenes, thiazoles, indoles, calixarenes, porphyrins, crown ethers, and BODIPYs, are promising candidates [ 27 – 31 ]. Furthermore, the valorization of natural products has been an emerging field in the design of new ligands for the detection of metal cations. Taking these facts into consideration, the present work focuses on broadening the scope of application of eugenol-based azo dyes—including previously reported structures developed for other purposes and applications [ 14 , 16 ]—by evaluating their use in the detection of various metal cations, with promising results in the colorimetric sensing of lead(II) and copper(II) ions. 2. Results and Discussion 2.1. Synthesis of Eugenol Azo Dyes 3a–e Azo dyes incorporating eugenol (4-allyl-2-methoxyphenol) into their structures have been synthesized following a conventional methodology. In this process, eugenol was coupled with diazonium salts of aniline-based amines to form the azo compounds. Aniline 1a, 3-methoxyaniline 1b, 3-bromoaniline 1c, 4-chloroaniline 1d, and 3-amino2-bromobenzoic acid 1e were reacted in an acidic medium with sodium nitrite, at low temperature, producing diazonium salts, which were added to eugenol 2in a basic medium, to yield 4-allyl-2-methoxy-6-(phenyldiazenyl)phenol 3a [ 14 ], 4-allyl-2-methoxy6-((3-methoxyphenyl)diazenyl)phe-nol 3b [ 16 ], 4-allyl-2-((3-bromophenyl)diazenyl)-6- Molecules 2025,30, 2788 3 of 17 methoxyphenol 3c, 4-allyl-2-((4-chlorophenyl)diazenyl)-6-methoxy-phenol 3d [ 14 ], and 3-((5-allyl-2-hydroxy-3-methoxyphenyl)diazenyl)-2-bromobenzoic acid 3e (Scheme 1). Scheme 1. Synthesis of eugenol azo dyes 3a–e. Compounds 3a–ewere obtained as orange and red solids in moderate yields (14–44%). These yields result due to the fact that eugenol has very limited solubility in water, meaning that the reaction tends not to be complete and requires purification of the crude product obtained using chromatographic techniques, which also leads to significant losses. Considering the electronic nature of the substituent groups of eugenol, as well as the possible steric impediments associated with the allyl side chain, the site for azo-functionalization will occur in the ortho position relative to the hydroxyl group. The respective compounds were fully characterized by using the usual analytical techniques: 1 H NMR, 13 C NMR, FTIR spectroscopy, and HRMS (see Figures S1–S22, Supplementary Materials). In the 1 H NMR spectra, it is possible to distinguish different signals for aliphatic protons, namely, methylenic ( δ 3.37–3.43 ppm) and methoxyl ( δ 3.84–3.95 ppm) groups, in addition to the expected protons of the eugenol’s double bond as multiplets CH 2 ( δ 5.06–5.19 ppm) and CH ( δ 5.96–6.07 ppm). With the occupation of the most activated position for the occurrence of azo coupling, the respective aromatic signals of eugenol (H-3 and H-4 or H-4 and H-6, in the case of 3e) will correspond to two doublets (J1.6 or 2.0 Hz) or be included in a multiplet (H-5 in 2b) ( δ 6.80–7.47 ppm). For amines, the aromatic protons will appear at specific chemical shifts, depending on the electronic nature and position of the substituents present. The 13 C NMR spectra of all compounds showed signals of the aliphatic carbons from the CH 2 ( δ 38.81–39.55 ppm) and OCH 3 groups ( δ 56.14–56.48 ppm), in addition to carbons of the eugenol’s double bond CH 2 ( δ 116.03–116.28 ppm) and CH ( δ 136.96–137.42 ppm). The carbons of the aromatic rings were also shown ( δ 112.15–160.38 ppm). For compound 3e, it is possible to assign the carbon in the carbonyl group of the carboxylic acid function at δ 168.04 ppm. The FTIR spectra also confirmed the presence of a CO 2 H group for compound 3e, showing the corresponding C=O stretching vibration bands at 1695 cm−1. 2.2. UV/Vis Absorption of the Eugenol Azo Dyes 3a–e The eugenol derivatives 3a–eexhibit absorption in the UV/visible region, and their absorption spectra are illustrated in Figure 1, showing an intense absorption band in the UV range, at around 345 nm, and another band in the blue region, justifying the yellowish color. The strong red shift of the UV band relative to the eugenol UV/Vis absorption spectrum (maximum at ~280 nm) [ 32 ] points to an extended conjugation between the aromatic moieties, including the azo group. The band in the visible region is probably due to an n→π* transition involving N atoms. Molecules 2025,30, 2788 4 of 17 Figure 1. Absorption spectra of compounds 3a–ein neutral ethanolic solution (2 ×10−5M). The compounds were compared, and small variations are detected in the UV band position and intensity, reflecting the influence of the substituents in the phenyl ring linked to the azo moiety. Maximum absorption wavelengths ( λmax ) and molar absorption coefficients ( ε ) are indicated in Table 1. Compound 3a, with no substituents in the phenyl ring, display the highest absorption coefficient. Compound 3e, possessing two substituents in this ring (–Br and –COOH groups in the ortho position relative to each other), presents a lower absorption coefficient and a significant bathochromic shift, probably due to the extension of the conjugation of the aromatic ring to the carbonyl group of –COOH. Table 1. Maximum absorption wavelengths ( λmax ) and molar absorption coefficients ( ε ) for compounds 3a–ein neutral ethanolic solution. Compound λmax (nm) ε(M−1cm−1) 3a 340 1.14 ×105 3b 338 8.27 ×104 3c 343 1.02 ×105 3d 345 9.40 ×104 3e 353 7.48 ×104 Ground state equilibrium geometries for cis and trans conformers of compound 3a, as well as its hydrazone tautomer, are shown in Figure 2, together with electron density variation upon excitation to the first excited state (Cartesian coordinates in Table S4 of Supplementary Materials). Vibrational analysis confirmed, through the absence of imaginary frequencies, that no metastable molecular geometries were obtained; this allowed us to place the free energies of the cis conformer and the hydrazone tautomer, respectively, at 19.5 kcal/mol and 1.83 kcal/mol above the trans form of compound 3a. This result predicts that 4.4% of compound 3a exists in its hydrazone form at room temperature (25 ◦C). Table 2shows the obtained transition wavelengths and oscillator strengths for the first eight excited states for both conformers and for the hydrazone tautomer. Considering the low oscillator strength for the first excited state and the electron density variations in Figure 2, it is possible to conclude that, in both cis and trans conformers, the first electronic transition is mainly of the n →π * type, as a decrease occurs along the azo backbone and an increase occurs in the π electron system above the molecular plane. The cis conformer shows higher oscillator strength as non-planarity results in a better overlap between nand π * molecular orbitals. The hydrazone tautomer, which is planar like the azo tautomer, clearly Molecules 2025,30, 2788 5 of 17 shows a π→π * transition, corresponding to a higher oscillator strength. Comparing the experimental spectrum (Figure 1) in the region above 440 nm with the oscillator strengths in Table 2(7.4 × 10 −4 for the trans conformer vs. 0.049 and 0.13 for the tautomer), it is possible to conclude that the absorption spectrum mainly corresponds to the trans conformer, as expected from its lower free energy. A  B  C  D Figure 2. Ground state equilibrium geometries and electron density variation upon excitation to the first excited state of trans (A) and cis (B) conformers, the hydrazone tautomer (C), and the Pb 2+ complex (D). The green and purple areas correspond to decreases and increases in electron density, respectively. Table 2. Results of TD-DFT calculations of compound 3a in its trans and cis conformers, as well as its hydrazone tautomeric form and its complex with Pb 2+ : differences in energy between electronic states and corresponding oscillator strengths (see Section 3.4.3 for calculation details). Transition Wavelength (nm) Oscillator Strength (f) trans cis Tautomer Pb2+ Complex trans cis Tautomer Pb2+ Complex S0→S1448 476 472 537 7.40 × 10 −4 4.02 × 10 −2 4.94 × 10 −22.51 ×10−3 S0→S2417 395 445 468 8.15 × 10 −2 6.80 × 10 −3 1.29 × 10 −11.30 ×10−1 S0→S3328 319 348 396 1.06 2.11 × 10 −2 9.02 × 10 −11.05 ×10−2 S0→S4302 288 313 347 1.76 × 10 −2 4.96 × 10 −3 4.05 × 10 −38.59 ×10−2 S0→S5296 280 311 335 3.26 × 10 −4 1.32 × 10 −1 1.50 × 10 −27.84 ×10−4 S0→S6278 275 287 325 5.96 × 10 −2 5.28 × 10 −3 9.03 × 10 −45.03 ×10−2 S0→S7256 271 278 324 1.97 × 10 −3 1.19 × 10 −1 8.14 × 10 −32.99 ×10−1 S0→S8253 258 257 318 1.10 × 10 −2 7.25 × 10 −3 8.93 × 10 −22.17 ×10−1 Prediction of the absorption spectral shape requires vibrational analysis of both ground and excited states in their equilibrium geometries. The geometry optimization of the first excited state resulted in a very high geometry variation, so that the S 0→ S 1 energy difference became very small (~1 eV). Thus, a simplified method was used, in which each electronic transition is considered to be a Gaussian function in wavenumber, with a fixed standard deviation corresponding to 0.4 eV (3226 cm −1 ). In Figure 3, a reasonable accordance is shown between the predicted spectrum and the experimental one, although the molar absorptivity of the S 0→ S 2 band is lower than the experimental value. The higher absorptivity in that region could be due to the contribution of the hydrazone tautomer that has a significant molar absorptivity in that spectral region. Molecules 2025,30, 2788 6 of 17 Figure 3. Calculated absorption spectra (in log scale) of compound 3a (3a calc), its hydrazone tautomer (3a_tautomer calc), and its complex with Pb2+ (3a_Pb calc). 2.3. Colorimetric Assays of Compounds 3a–eas Ion Sensors The compounds 3a–ewere preliminary tested as sensors for several cations in order to obtain a colorimetric assay, which is especially useful for detecting pollutants and poisoning metals. The cations (15 equiv.) were added to compounds’ solutions, and the colors were compared between the neat cation solutions and the compound/cation solutions (Figures 4and 5). K+ Fe2+ Fe3+ Cs+ Zn2+ Cd2+ Ni2+ Cu2+ Al3+ Pb2+ Na+ Cd2+ Co2+ Figure 4. Solutions of the salts of several cations. Cd 2+ was tested as a perchlorate salt (left) and a nitrate salt (right). Considering the objective of obtaining a colorimetric assay for the detection of cations, we find, in Figure 5, evidence that the compounds can be useful for the detection of copper(II) and lead(II), which display a clear change in color, despite compound 3e seeming not useful for this determination. Particularly, compound 3c seems to be the most useful for Cu 2+ , demonstrating a clearly distinguishable and intense pink color in the presence of this cation. This color change can be observed at concentrations as low as 10 µ M of cations, while for the other compounds, higher cation concentrations are needed. Here, the possibility of the detection of Pb 2+ is especially valuable, because lead is described as a poison that can affect a large Molecules 2025,30, 2788 7 of 17 number of organs in human body. Therefore, the five compounds’ solutions were titrated with lead(II) cations. Compound3aCompound3bCompound3cCompound3dCompound3e Figure 5. Solutions of compounds 3a–eafter the addition of 15 equiv. of the salts. Cd 2+ was tested as a perchlorate salt (left) and a nitrate salt (right), and no differences were obtained. Molecules 2025,30, 2788 8 of 17 2.4. Titration of Compound 3c with Cu2+ Spectrophotometric titrations in ethanol were performed to further investigate the ability of compound 3c to detect copper(II) ion. Therefore, increasing amounts of the cation were added to the compound solution, and the corresponding absorption spectra were obtained. A new absorption band, centered at around 530 nm, is observed (Figure 6), the maximum absorption of this band being attained at 10 equivalents of the cation, pointing to a high sensitivity. The presence of three clear isosbestic points indicates a complex interaction between the compound and Cu2+. Figure 6. Absorption spectra of compound 3c (1 × 10 −5 M) with the addition of Cu 2+ . The spectra of 10 and 12 equiv. of Cu2+ are superposed. In these results, a linear relationship between the cation concentration and the absorbance of the new band at 540 nm (where the absorption of the neat compound is negligible) was observed up to 60 µ M of cations (Figure 7A). From this plot, a limit of detection (LOD) as low as 0.16 µ M was estimated. This value shows that compound 3c can be very useful for the detection of Cu 2+ in effluents and other samples, considering that the maximum level of copper in drinking water is 20.5 µ M (WHO, 2008 [ 33 ]), well above the LOD and in the linear range of the calibration curve (Figure 7A). Moreover, the average concentration of copper in blood should not exceed 15.7–23.6 µ M in normal conditions [ 34 ], also in the detectable range. This LOD value is lower than the ones reported for other sensors of copper(II) [ 35 – 37 ], making compound 3c highly advantageous for this purpose. To calculate the binding constant, the Benesi–Hildebrand equation was used, the K a value being determined from the ratio of the intercept to the slope of the Benesi–Hildebrand plot (Figure 7B). A linear relationship was obtained only for n= 2 (while for n= 1, the plot is not linear), indicating a binding stoichiometry of 1:2 between the compound and the copper (II) cation, with a corresponding binding constant of K a = 1.21 × 10 9 M −2 . Azo dyes based on eugenol (without the substituents R, R 1 , and R 2 ) incorporated in silicone films have also shown the capability to detect Cu 2+ metal ions, represented by a change in color of the film, similarly exhibiting a 1:2 stoichiometry [38]. Molecules 2025,30, 2788 9 of 17 Figure 7. (A) Linear plot of absorbance at 540 nm versus Cu 2+ concentration. (B) Benesi–Hildebrand plot for the spectrophotometric titration of compound 3c with Cu2+ at 540 nm. 2.5. Titration of Compounds 3a–ewith Pb2+ Considering the proof-of-concept obtained with copper(II) cations, having a low LOD value, we explored the possibility of the synthesized compounds acting as lead(II) sensors. For this purpose, each of the compounds 3a–ewas titrated with lead(II), taking into account the significant harmful environmental and health effects from high concentrations of lead [ 39 ]. Lead exposure can have serious consequences for health, especially with children. The WHO has established “International Lead Poisoning Prevention Week” (every year in October) to prevent childhood lead exposure and to stop the use of lead in paint [40]. As predicted by the less prominent variation in color upon Pb 2+ addition (comparing with copper), the spectral effects of the presence of the ion are much less pronounced, compound 3e not being sensitive to the presence of Pb 2+ in the titration (as inferred from the color invariance in Figure 5). Moreover, the titration of compound 3d also shows very small spectral changes with the addition of lead cations. Therefore, the titration of compounds 3a, 3b, and 3c with Pb 2+ were analyzed to determine the binding constants and LOD values (Figures 8–10). The parameters for the interaction of each compound, 3a–c, with lead cations are summarized in Table 3. It must be pointed out that for compounds 3a, 3b, and 3c, the Benesi–Hildebrand plots (Figures 8C, 9C and 10C) only display a good correlation for n= 1, indicating a 1:1 stoichiometry for the interaction of lead(II) with the azo compounds. In fact, the spectra for the titration of compounds with lead(II) are completely different from that of compound 3c with copper(II), pointing to a different kind of interaction of the azo dyes with the two cations. Table 3. LOD values and binding constants for the interaction of compounds 3a–cwith lead(II) cations. Compound LOD (µM) Ka(M−1) 3a 0.19 2.80 ×104 3b 0.14 1.33 ×104 3c 0.16 1.02 ×104 Molecules 2025,30, 2788 16 of 17 6. 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