Novel Colorimetric Anthracene-Based Thiosemicarbazone Probe for Selective Cyanide Ion Detection: From Synthesis to Real-Time Applications

Abstract

A novel thiosemicarbazone, N’-(anthracen-9-ylmethylene) morpholine-4-carbothiohydrazide (MA), was synthesized and characterized for selective cyanide ion sensing. Interaction between the cyanide ion and sensor MA was examined thoroughly with the aid of absorption, emission, and ¹H NMR spectra, and the binding constant was estimated. The detection limit obtained was much lower than those reported recently. The ¹H NMR spectra titration studies clearly indicated that deprotonation occurs from the N–H group of the receptor. The sensing mechanism was further explored by density functional theory (DFT) and time-dependent DFT (TDDFT) methods using Gaussian16 software. In silico investigation reveals that the added cyanide ion abstracts a proton from the N–H group, which supports experimental results. The synthesized receptor MA was found to be a very good candidate for selective sensing of cyanide.

1. Introduction

The identification of cations and anions and neutral molecules have received a lot of scientific interest in recent years, indicating their importance in biological systems and potential use in sensors and separation technologies. − This selective detection has been achieved using fluorescent organic materials. − Among various ion sensing applications, the detection of cyanide and fluoride ions has received particular attention due to their significant impact on human health. , It has long been known that cyanide is toxic. The cyanide ion is first identified and isolated by Swedish Chemist Scheele in 1782, who died due to cyanide poisoning. Although cyanide is highly toxic, it remains an important reagent in numerous industrial applications. It is extensively used in gold extraction and other metallurgical operations, in the synthesis of nitriles, nylon, and acrylic polymers, and in the production of fertilizers, pesticides, and pharmaceuticals. Additional uses include electroplating, surface hardening of metals, plastics, and gum manufacturing, leather tanning, herbicide formulation, and chelation in water treatment processes. However, effluents from these industries often release significant amounts of cyanide ions, leading to serious environmental contamination. − The presence of cyanide in drinking water poses serious health risks due to its high toxicity, as it inhibits oxygen transport in the body. The World Health Organization (WHO) established 2.7 μM as the maximum amount of cyanide that can be present in drinking water. Cyanide directly interacts with the heme group and results in a cellular respiration problem. Hence, it causes malfunctioning in the nervous system, vascular, cardiac, visual, and metabolic activities. , Moreover, accidental leaking of cyanide into the environment causes more biological and environment issues. Hence, the development of diagnostic techniques for monitoring cyanide ions in biological and environmental materials has generated significant attention. A colorimetric technique is used to detect the cyanide ion because it allows for easy observation and detection without requiring expensive analytical instruments. − Therefore, developing a colorimetric sensor capable of detecting anion by color change in visible light became a crucial research subject. Chemical sensor’s sensitivity and selectivity rely heavily on their signaling unit. The sensor with an effective signaling unit may detect various analytes by adjusting its optical characteristics.

Anthracene and its derivatives are significant fluorophores, owing to their commercial availability, distinctive characteristics, and ease of manufacturing. They are used to detect pH, tiny organic molecules, metal ions, and inorganic anions. − Anthracene has been successfully used as a fluorophore because of its variable emission properties based on surroundings. − Introducing donor/acceptor units into anthracene backbones causes superior excimer and monomer emission differences, which are linked to the spacing between two segments. ,

We have developed a simple yet effective sensor N’-(anthracen-9-ylmethylene) morpholine-4-carbothiohydrazide (MA) using anthracene’s unique physicochemical features for anion, especially for cyanide. In this system, a thiosemicarbazone unit is coupled with an anthracene fluorophore, where the thiosemicarbazone group functions as the recognition site and the anthracene moiety serves as the signaling unit. Cyanide binding perturbs the electron distribution within the receptor, leading to a distinct change in the fluorescence of the anthracene core, enabling selective and sensitive detection. Absorption, emission, ¹H NMR spectral studies, kinetics, fluorescence lifetime decay titration studies, and DFT investigation provided additional evidence to validate the sensing behavior and fluorescence enhancement of the receptor MA toward the cyanide ion.

2. Results and Discussion

2.1. Synthesis and Characterization of MA

A novel thiosemicarbazone (MA) was synthesized by a reported procedure with slight changes (Scheme ). It was characterized by elemental analysis, FTIR, ¹H NMR, ¹³C NMR, and mass spectra. Further, the structure is confirmed by single crystal XRD analysis. The FTIR spectrum (Figure S2) confirmed the presence of functional groups, such as HC=N, N–NH, and −C=S, as evidenced by the characteristic absorption peaks resulting from bond stretching vibrations. The −C=N and −C=S groups of MA were indicated by sharp peaks at 1548 and 1218 cm^{– 1}, respectively. The ¹H NMR spectrum of MA shows singlets at δ 11.40, 9.45, and 8.66 ppm, which are corresponding to −N₂–H, C₁₀–H, and H–C₆=, respectively (Figure S3). Morpholine ring protons show triplet peaks at δ 3.98 and 3.68 ppm. Aromatic protons appear as multiplets and triplets at δ 7.60 and 8.10 ppm, respectively. The ¹³C NMR spectrum shows well-defined peaks at δ 181.2 and 144.6 ppm corresponding to thionyl carbon (C₅=S₁) and azomethine carbon (C₆=N₃), respectively (Figure S4). The aromatic anthracene carbons appear between δ 126.0 and 131.5 ppm, while those attached to the nitrogen atom and oxygen atom appear at δ 50.1 and 66.4 ppm, respectively.

1. (a) Synthesis of Precursor 1a Starting from N-methyl Aniline and (b) Synthesis of Thiosemicarbazone (MA).

2.2. X-ray Crystallographic Analysis

A solitary yellow plate-shaped crystal of MA was chosen via a Leica M80 microscope. The crystal was affixed to a nylon loop and then cooled in a nitrogen stream (Oxford) at a temperature of 100.01(10) K. Crystal screening, unit cell determination, and data acquisition were performed by using the XtaLAB Synergy, Dualflex, and HyPix diffractometer. The diffraction pattern was indexed, and computations using the CrysAlisPro program ascertained the total number of runs and images. Data were collected utilizing ω scans with Cu Kα radiation, achieving a maximum resolution of 79.895° (0.78 Å). The unit cell was refined using CrysAlisPro 1.171.43.98a (Rigaku OD, 2023) on 8323 reflections (58% of observed). Crystallographic along with structural details are given in Table .

1. Crystal Structural Refinement for MA .

compound	MA
empirical formula	C20H19N3OS
D calc/ g cm–3	1.388
m/mm–1	1.818
formula weight	349.44
shape	plate-shaped
size/mm3	0.09 × 0.08 × 0.01
T/K	100.01(10)
crystal system	monoclinic
space group	P21/c
unit cell dimensions (Å)	a = 21.1560(3)
	b = 9.46720(10)
	c = 8.40660(10)
angles (°)	a = 90
	b = 96.5690(10)
	g = 90
V/Å3	1672.69(4)
Z	4
Z’	1
GooF	1.048

The molecular structure of MA is depicted with atom labeling in Figure a, while molecular packing is illustrated in Figure b. A yellow plate-shaped crystal of 0.09 × 0.08 × 0.01 mm³ dimensions (maximum, intermediate, and minimum) was selected from a representative sample of crystals exhibiting the same habit and subsequently analyzed. R value was 5.67%, and the Goodness of fit value was 1.048. The molecule crystallizes in monoclinic, P2₁/c having Z = 4 and Z’ = 1. Important parameters: a = 21.1560(3) Å, b = 9.46720(10) Å, c = 8.40660(10) Å, β = 96.5690(10)°, α = γ = 90°, and V = 1672.69(4) Å (CCDC: 2377250). Bond parameters are given in Table .

1.

(a) Thermal ellipsoid plot of MA (50% probability). (b) Molecular packing showing interactions in MA.

2. Comparison of Bond Parameters, Experimental, and Computational.

bond parameters		experimental	computational
bond length (Å)	S(1)–C(5)	1.686(2)	1.754
	O(1)–C(2)	1.421(2)	1.457
	N(1)–C(1)	1.471(3)	1.487
	N(2)–N(3)	1.384(2)	1.383
bond angles (°)	C(3)–O(1)–C(2)	108.68(15)	110.51
	C(4)–N(1)–C(1)	112.88(16)	112.80
	C(5)–N(2)–N(3)	120.12(17)	122.34
	N(2)–C(5)–S(1)	122.49(15)	121.42
torsion angles (°)	O(1)– C(3)–C(4)–N(1)	–54.6(3)	–54.92
	N(1)–C(1)–C(2)–O(1)	55.6(2)	56.54
	N(3)–N(2)–C(5)–S(1)	–3.9(3)	–12.60
	C(1)–N(1)–C(4)–C(3)	47.8(2)	52.40

2.3. Colorimetric Analysis and UV–Vis Spectroscopy

Using colorimetric analysis, interactions of different anions (such as F^–, Cl^–, Br^–, I^–, CN^–, SO₄ ^2–, PO₄ ^3–, NO₃ ^–, OH^–, AcO^–, ClO₄ ^–, SCN^–, NCS^–, HSO₃ ^–, HS^–, S^2–, ClO^–, and N₂H₄) and cations (such as Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Fe²⁺, Ag⁺, and Na⁺) with MA were analyzed in the solution state at room temperature. Upon the introduction of CN^–, a distinct and noticeable color (a deep pink color) change was observed. In contrast, no discernible color change occurred upon the addition of other anions and cations. The UV–vis spectrum of MA exhibited a single broad peak at 407 nm, with an epsilon value in the order of 10⁴ indicating a strong π–π* transition. When various cations and anions were added, no significant spectral changes were observed, except in the case of the CN^– ion. Upon addition of a cyanide ion, a new broad band appeared at 498 nm (Figure a), indicating the formation of an MA–CN complex. Likewise, the new band showed a hyperchromic shift upon further addition of CN^– ions. The presence of isosbestic points at 317, 361, and 447 nm suggests the coexistence of multiple components in the system such as the MA–CN complex and deprotonated MA. The formation of the new absorption band and the change in the solution’s color can be attributed to the formation of hydrogen bonding as well as the deprotonation of MA. The observed bathochromic shift is a result of these interactions, which confirm both hydrogen bond formation and sensor deprotonation (see Figure c). Absorbance spectra of MA with excess CN^– ions, as well as with combinations of CN^– and various other cations and anions, were recorded (Figure S6a,b). When an excess ion was added, no alteration in peak or intensity was observed. In all combinations, the peak corresponding to CN^– was consistently observed, confirming the selective and competitive binding of CN^– to MA even in mixed analyte environments. No interference from other ions was detected under these conditions, clearly demonstrating the receptor’s high selectivity toward the cyanide ion. Additionally, the stability of sensor MA was examined by recording absorbance spectra repeatedly five times at time interval of 2 days (Figure S6c). Absorbance spectra did not show any shift in wavelength as well as negligible alteration in intensity, which can be caused by personal error or instrumental error. This suggested that our sensor is highly stable over days.

2.

(a) Absorption spectra of MA with different ions, (b) emission spectra of MA with different ions, (c) absorption spectra of MA with addition of varying concentrations of CN^– (up to 1.7 equiv, at which absorbance is saturated), (d) emission spectra of MA with addition of varying concentrations of CN^– (experiment performed as similar as absorbance titration), (e) Benezi–Hilderbrand diagram for MA interacting with CN^– to find out the binding constant, and (f) Job’s plot for MA interacting with CN^– to find out the stoichiometry ratio of binding. Spectra were recorded in DMF/H₂O (5/95, v/v).

Job’s plot suggests a mole fraction ratio of nearly 1:1 between MA and CN^–, as shown in Figure f. Hence, it can be concluded as MA bonded with CN^– in a 1:1 ratio. Although the maximum is observed near 0.65, this slight deviation from the ideal 0.5 position can be attributed to experimental uncertainties such as baseline fluctuations and instrumental limitations. Benezi–Hildebrand equation (eq ) was employed to calculate the association constant K

$$\frac{1}{A-A_0}=\frac{1}{(A_{\infty }-A_0)K[C]}+\frac{1}{A_{\infty }-A_0}$$ 1

where ‘A, A ₀, A _∞, [C], and K denote the absorbance at a specific ion concentration, free substance, excess ion concentration, ion concentration, and the association constant, respectively. The plot of 1/(A – A ₀) vs 1/[C] demonstrates a linear relationship, which is utilized for the calculation of binding constant values (Figure e). The binding constant (K) for CN^– was determined to be 1.38 × 10⁵ M^{– 1}, indicating a strong affinity. This value is higher than those reported for recently developed cyanide sensors, which typically exhibit K values in the 10⁴ M^{– 1} range. −

2.3.1. AIE Behavior of MA

Absorbance spectra of MA were recorded with the gradual addition of water in DMF up to 95% content in the H₂O/DMF binary system. After each addition starting from 0% water content to 95%, the absorbance spectra remained the same, having negligible absorbance intensity difference. In all the cases, absorbance maxima was obtained at 407 nm (Figure S7a). The emission of each of the binary system was examined under UV light. Except for 0 and 10% water content, all percent composition showed the same emission (Figure S7c). The enhancement in emission of MA upon aggregation was confirmed by time-dependent photoluminescence decay measurement studies. The fluorescence lifetime of MA increased progressively with the addition of water and reached a maximum value of 3.28 ns at 95% composition of the binary system, Figure S7b. In general, Schiff base molecules containing hydrophobic groups often exhibit aggregation-induced emission (AIE) as a result of restricted −C=N isomerization. In the case of MA, this phenomenon is clearly supported by the present observations.

2.3.2. Effect of pH

To study the effect of pH on CN^– sensing by MA, absorption spectra was recorded within the pH range of 4–12. The solution pH was adjusted using 1 M HCl and 1 M NaOH prepared in distilled water. A stock solution of the ligand was prepared, and its pH was carefully regulated by the gradual addition of NaOH, with the final value confirmed by using a pH meter. The same procedure was followed to adjust the pH in the acidic region. Absorbance spectra clearly reveal that sensing occurs only within the pH 10–12 range (Figure S8a). As the pH decreases to an acidic medium, no sensing response is observed and the absorbance spectra shift to lower wavelengths. To evaluate the influence of pH on the binding constant, incremental additions of cyanide were performed at pH 11 and 12 (Figure S8b,c) (for pH 10, see Section ). The binding constants were calculated from plots of 1/(A – A ₀) vs 1/[C], yielding values of 3.07 × 10⁴ M^{– 1} at pH 11 (Figure S8d) and 3.6 × 10⁴ M^{– 1} at pH 12 (Figure S8e), both lower than the value obtained at pH 10. These results indicate that sensing is more efficient at pH 10.

2.3.3. Reversibility Studies

To investigate the reversibility of the MA probe toward CN^– ions, trifluoroacetic acid (TFA) was introduced as a protonating agent since protonation can neutralize cyanide binding and regenerate the free MA. , The free ligand had an absorbance band at 407 nm, and upon interacting with cyanide, a new broad band formed at 498 nm followed by the occurrence of dark pink color. Further, the addition of 0.1 equiv of TFA to the MA–CN complex reverts the absorbance maxima to 498 nm, and disappearance of the broad band at 498 nm indicates the regeneration of MA. Out of 10 cycles performed, in all of them, MA was found to have effective reversibility without any sensitivity loss. MA demonstrated to undergo protonation and deportation and can detect CN^– ions selectively in real samples based on multiple cyclic experiments (Figure S9).

2.4. Fluorescence Spectral Studies

Similar to UV–vis spectral titration studies, fluorescence spectral titrations were performed to further investigate the receptor’s sensing capabilities. Emission spectra of MA, excited at 407 nm, were recorded in the presence of all of the aforementioned ions. MA exhibited an emission peak at 499 nm. Upon the addition of various cations, no observable changes in emission were detected. In contrast, among the tested anions, only CN^– induced significant changes (Figure b), leading to enhanced emission intensity accompanied by a hypsochromic shift. The new emission bands appeared at 422, 446, and 473 nm, with the main peak showing a hypsochromic shift of 26 nm relative to that of the parent compound. This enhancement in fluorescence intensity is likely due to the formation of −N–H----NC– hydrogen bonds (Figure d). Nearly 4-fold intensity enhancement was observed upon addition of 1.7 equiv of CN^– at which the emission signal became saturated. It is also noteworthy that, in addition to the enhancement in emission intensity, a gradual decrease in bandwidth was observed with increasing CN^– concentration, indicating the formation of a higher proportion of the free deprotonated form of MA in the solution.

The binding constant was estimated using modified Benezi–Hildebrand equation, eq , and the result is as follows

$$\frac{1}{F_x-F_0}=\frac{1}{(F_{\infty }-F_0)K[C]}+\frac{1}{F_{\infty }-F_0}$$ 2

where F ₀ indicates the emission intensity of MA alone, F _x is the emission intensity of MA with the addition of ion, F _∞ is the emission intensity of MA with the addition of the excess ion. K is the association constant, and [C] is the ion concentration. K is determined from the plot of 1/(F _x– F ₀) vs 1/[C] (refer Figure S10), where the binding constant of MA with CN^– was computed from the intercept to slope ratio as 1.03 × 10⁵ M^–1.

2.4.1. Detection Limit

The detection limit was determined using the formula 3σ/K, where σ represents the standard deviation of the fluorescence intensity obtained from blank measurements of MA, and K is the slope of the plot of emission intensity versus cyanide ion concentration at 446 nm. Calculation details are provided in Figure S11. A better linear relationship between emission intensity and CN^– concentration was observed in the range of 7–15 × 10^–7 M. The detection limit of MA for cyanide ions was determined to be 1.37 nM. This value is significantly lower than those reported for recently developed cyanide sensors, which typically have detection limits in the micromolar range. Moreover, the obtained detection limit is well below the World Health Organization’s standard of 2.7 μM (approximately 70 μg/L) for cyanide in drinking water. − Table lists out some of the recently reported sensors for cyanide ions and their detection limit. Most of them had LODs in the micromolar range, but our sensor has an LOD in the nanomolar range, indicating its efficiency and high advantage over others. With its ultralow detection limit of 1.37 nM and strong selectivity for cyanide ions, our sensor is an excellent candidate for monitoring drinking water safety. Given that cyanide contamination poses a severe hazard to both the environment and human healthparticularly in industrial sectors such as mining, electroplating, and chemical manufacturingMA has great potential for practical application as an effective cyanide monitoring tool.

3. Comparison of Sensor Properties with Recently Reported Cyanide Sensors − .

2.4.2. Real-Time Studies and Food Sample Analysis

To better understand the sensing capability of MA for cyanide detection, real-time images were recorded in solution, on filter paper, and on cloth. Figure a shows the naked-eye detection of MA with cyanide ions, where the solution developed a distinct dark pink color upon addition of CN^–, which exhibited strong luminescence under UV light (Figure b). To further demonstrate real-time application and naked-eye sensing, MA was tested with increasing concentrations of CN^– on filter paper (Figure c,d) and on cloth (Figure e). The very low detection limit of MA enabled clear naked-eye detection in all cases, underscoring the high efficiency of the sensor. Real-time images of MA interacting with various cations are provided in Figure S12a,b .

3.

Real-time images of MA interaction with anions in the order MA, F^–, Cl^–, Br^–, I^–, CN^–, SO₄ ^2–, PO₄ ^3–, NO₃ ^–, OH^–, AcO^–, and ClO₄ ^– (a) under normal light and (b) under UV light (DMF/H₂O, 5/95, v/v). MA interaction with increasing concentration of CN^– ion from left to right on filter paper (c) under normal light and (d) under UV light. (e) MA interaction with increasing concentration of CN^– ion from left to right on cloth and (f) absorbance spectra of MA interacting with food samples; apple seed, sprouted potato, and almond skin, showing efficient sensing of cyanide in food sample by MA.

To evaluate the practical efficiency of the probe for real-time cyanide detection, its sensing ability was examined using naturally occurring sources, such as almond skin, apple seeds, and sprouted potatoes, as these materials are known to contain trace levels of cyanogenic glycosides. The food sample was prepared by the following procedure. A 1 g of quoted food sample was crushed first and mixed with 50 mL of water, and 0.1 g NaOH was added to it. It was kept for fermentation for 5 days. , The fermented solution was collected by filtration and centrifuged to get a clear solution, diluted, and tested for sensing. On MA interacting with the food sample, a broad new band was obtained at 498 nm indicating that a significant portion of cyanide was absorbed at the wavelength, followed by MA–CN complexation (Figure f). Therefore, MA demonstrates potential as a practical and sensitive colorimetric probe for the real-time monitoring of cyanide ions in food matrices. The observed chromatic variations further validate its applicability for the selective detection and quantitative assessment of CN^– in complex environmental samples (Figure S12c,d).

2.5. Chemical Kinetics Aspects

Chemical kinetic studies were conducted to further understand MA’s sensing abilities and to find the rate of response for CN^–. For this purpose, UV–vis spectra were monitored for a fixed time interval. , The plot of A/A _max (normalized absorbance) against time under standardized conditions (10 equiv of cyanide, RT) are given in Figure a and Figure S13. Assuming pseudo-first-order kinetics (massive excess cyanide utilized), linear plots reproduced from the graph yield a rate constant of 8.3 × 10^–3 s^–1 (see Figure b). Further, the chemical kinetics of reaction of the sensor with cyanide ion was examined by increasing cyanide ion concentrations as a variable (Figure a). Study was done at tapering intensity of sensor absorbance at 407 nm and measured as a function of time for a series of kinetic runs, where [CN^–] increased gradually. The initial rate (υi) for each run was estimated by fitting a linear function to the absorbance versus time data. Reaction order with respect to CN^– was determined from the slope of the linear plot of ln­(υi) versus ln­[CN^–], utilizing initial rates method. Figure b illustrates the relevant data. The result shows the order 0.33 with respect to cyanide, which shows a very selective sensing property even at very low concentrations of cyanide ion.

4.

(a) Graph of accepted absorbance A/A _max vs time at 407 nm after the addition of [ ⁿ Bu4N]­CN (10 equiv) to sensor MA and (b) logarithmic plot used to ascertain pseudo-first-order rate constants, DMF/H₂O 5/95 v/v medium.

5.

(a) Absorbance against time graph for tapering of band at 407 nm with different [ ⁿ Bu₄N]­CN concentrations. (b) Black: plot of ln­(υ_i) against ln­[CN^–] for tapering of band at 407 nm with different concentrations of [Bu₄N]­CN, red: linear fit plot. (DMF/H₂O, 5/95, v/v).

2.6. Lifetime Measurement

Both absorption and emission spectra showed notable changes upon the addition of CN^– ions compared to free sensor MA. Furthermore, the average lifetime-based sensing properties of MA were investigated. Time-dependent photoluminescence decay measurements were performed by incrementally adding CN^– ions to the sensor solution using the time-correlated single photon counting (TCSPC) technique with excitation at 450 nm. The device was calibrated using a solution of milk powder in deionized water. From the fluorescence lifetime decay experiments, the average lifetime of the sensor MA was determined to be 3.28 ns (λ_em = 501 nm), calculated using eq

$$A+B_1^{*}\exp \left(-\frac{i}{{\tau }_1}\right)+B_2^{*}\exp \left(-\frac{i}{{\tau }_2}\right)$$ 3

Upon addition of 0.2 equiv of CN^–, the average lifetime increased to 4.02 ns (λ_em = 446 nm). With each successive addition, the lifetime continued to rise, reaching up to 6.39 ns upon addition of 1 equiv of CN^– (Table ). Each measurement involved fitting the decay curves to a double-exponential decay model. These findings confirm a delay of 3.11 ns in the decay process upon interaction of CN^– with MA. The observed fluorescence enhancement was further validated through the formation of the MA–CN complex, as shown in Figure .

4. Average Fluorescence Lifetime at Varying Cyanide Concentration (10^–5 M Probe) .

Sl. no.	equivalence of CN–	τ1 (ns)	τ2 (ns)	τav (ns)
1	0.2	2.17068	10.3267	4.026504
2	0.4	2.63121	11.3201	4.684155
3	0.6	2.93007	12.1457	5.420993
4	0.8	3.35700	12.678	6.233074
5	1.0	3.21701	12.6971	6.398192

^aτ_av represents the average lifetime. τ₁ and τ₂ denote the exponential fitting lifetime values.

6.

Variation in fluorescence decay curve of MA on addition of different equivalents of CN^– ion. (DMF/H₂O, 5/95, v/v).

2.7. ¹H NMR Titration

The presence of acidic hydrogen in the senor −C=N–NH makes it rely on deprotonation rather than nucleophilic substitution. , The method is usually quicker and uses less energy than a nucleophilic substitution reaction at sulfur or carbon centers. The strongly basic CN^– ion can effectively deprotonate this acidic proton, especially in aqueous or polar solvents, where deprotonation is both kinetically and thermodynamically more favorable. This process often leads to electron delocalization across the conjugated system between the −C=N and −C=S groups, resulting in significant changes in the electronic structure that manifest as observable shifts in color or fluorescencemaking it ideal for sensing applications. Moreover, the intrinsic structure of thiosemicarbazones resists substitution reactions, further supporting deprotonation as the dominant sensing mechanism. The sulfur atom in the −C=S group, being less electrophilic and larger, exhibits a reduced reactivity toward CN^–. To investigate the sensing mechanism of MA and confirm the deprotonation pathway, a ¹H NMR titration study was performed with CN^– (using tetrabutylammonium cyanide) in DMSO-d ₆. For free sensor MA (without CN^–), singlet peaks were observed at δ 11.40, 9.45, and 8.66 ppm, corresponding to −N₂–H, C_{1 0}–H, and H–C₆=N protons, respectively.

As shown in Figure b, upon addition of 0.2 equiv of CN^–, the −N₂–H singlet exhibited a downfield shift accompanied by broadening. A slight downfield shift and peak splitting were also observed for the H–C₆=N proton. When one equivalent of CN^– was added, the −NH proton peak completely disappeared, and the C_{1 0}–H peak split into a doublet with a further downfield shift. The peak broadening is attributed to the formation of hydrogen bonds between the cyanide ion and the −N–H group (i.e., −N–H···CN^–). Deprotonation of the −N₂–H proton results in its signal vanishing, as the released proton is captured by CN^–. Additionally, interactions between CN^– and the H–C₆=N proton likely cause the splitting of this peak into a doublet, while the peak at δ 9.45 also shows broadening.

7.

(a) ¹H NMR titration of MA with varying equivalents of CN^– and (b) zoomed ¹H NMR spectra of MA with varying equivalents of CN^–. Spectra recorded in DMSO-d ₆.

Moreover, new broad peaks appeared at δ 6.3 and 6.9 ppm, which subsequently split into doublets. The formation of these peaks indicates hydrogen bonding interactions, such as CN^–···H and H···CN^–. However, deprotonation was observed exclusively at the −N₂–H site. The changes in the ¹H NMR spectra clearly demonstrate the establishment of hydrogen bonds and support the proposed deprotonation mechanism (Figure a). The overall proposed sensing mechanism for cyanide detection by MA is illustrated in Figure .

8.

Potential detection strategy of MA using the cyanide ion.

2.8. Computational Study

All computations were conducted with the licensed Gaussian 16 software package. The electronic absorptions and excited state characteristics of the system were examined by hybrid density functional theory (DFT) and time-dependent DFT (TDDFT). All geometry optimizations were undertaken using the B3LYP functional combined with the polarized 6-31G­(d,p) basis set to ensure accurate structural parameters. CAM-B3LYP functional in conjunction with same basis set were used for TDDFT calculations. Water, with a dielectric constant (ε) of 78.4, served as the solvent throughout all computational operations, and solvent effects were simulated using the conductor-like polarizable continuum model (CPCM).

2.8.1. Geometry Optimization

For the thorough understanding of the sensing mechanism of MA, the ground state, excited state, and deprotonated state of MA with cyanide ion were optimized. The ground state energy of MA is −8.85 × 10⁵ kcal/mol with a polarizability of 403.711 au. The dihedral angle between the anthracene and thiosemicarbazone moieties in the ground state is 154.846°, indicating that the molecule is not planar. Similarly, the angle between the morpholine ring and the thiosemicarbazone moiety is 162.571°. No substantial alteration was seen in the excited state of MA. However, noticeable changes have been observed on MA interaction with CN^–. In the ground state, where CN^– interacts only with the −N–H proton, the dihedral angle between morpholine and thiosemicarbazone ring twisted to 20.121° and that between the anthracene ring and thiosemicarbazone group is 147.442°. In the excited state, where CN^– interacts with both −N–H and −C–H protons, the dihedral angle twisted to 28.563° between the morpholine ring and thiosemicarbazone. Both anthracene ring and thiosemicarbazone also contracted to 133.101°. The energy of MA–CN at ground state was higher than that of MA, i.e., −9.43 × 10⁵ kcal/mol.

In MA, at its ground state, −N–N, −N–H, −C=N, and −C–H bond lengths are shown to be 1.383, 1.017, 1.301, and 1.092 Å, respectively. In the ground state of the MA–CN complex, the −N–N and −C=N bond lengths were decreased to 1.369 and 1.281 Å, respectively. The −N–H bond lengths in MA, ground state MA–CN, and excited state MA–CN* are shown as 1.017, 1.041, and 1.040 Å, respectively. This elongation in bond length confirms the interaction of the receptor with the CN^– ion in the excited state.

2.8.2. Absorption Spectra and Molecular Orbital Analysis

Time-dependent DFT calculations at the TDDFT/CAM-B3LYP/6-31G­(d,p) level were carried out to explore the absorption properties and sensing capabilities of MA, based on its optimized ground state geometry. Figure a and Figure b show the optimized geometries of the MA–CN complex in its ground and excited states, respectively. For free sensor MA, the calculated absorption peak appears at 445.45 nm with an oscillator strength of 0.4247, corresponding well with the experimental absorption band and indicating a primary π–π* transition from the HOMO to the LUMO, with an energy gap of 3.113 eV (Figure c). The second most significant transition for MA occurs at 264.8 nm, with an oscillator strength of 0.9871. In the case of the MA–CN complex, a bathochromic shift is observed, with the absorption peaks shifting to 267.39 nm (oscillator strength: 0.253) and 450.18 nm (oscillator strength: 0.46). In the primary π–π* transition, π-delocalization extends through both the anthracene ring and the thiosemicarbazone moiety in the HOMO and LUMO orbitals. The calculated energy gap between HOMO and LUMO in the MA–CN complex is 5.5328 eV in the ground state (Figure d) and 5.634 eV in the excited state (Figure e). For enhanced visualization, HOMO–LUMO maps are provided in Figure S14.

9.

(a) Optimized ground state geometry of MA–CN and (b) excited state geometry of MA–CN*. HOMO–LUMO energy gap in (c) MA, (d) MA–CN, and (e) MA–CN*.

3. Experimental Section

Here are some chemicals that were acquired from Alfa Aesar and Sigma-Aldrich: carbon disulfide, N-methyl aniline 99%, sodium chloroacetate 98%, hydrazine 98%, morpholine 99%, and 9-anthracene carboxaldehyde 99%. Finar limited supplied anhydrous solvents methanol 99% and acetonitrile 99%. Sigma-Aldrich supplied NMR solvent DMSO-d ₆, which has 98.9 atoms. No further purification was performed on the analytical grade chemicals utilized throughout the procedure. Sensing studies were conducted in an organo-aqueous mixture consisting of dimethylformamide/water (5/95, v/v), which is deionized water. The absorption spectra were acquired using a Shimadzu 1900 UV–visible spectrophotometer, spanning the wavelength range of 200–800 nm. Infrared spectra recorded in the range of 650–4000 cm^{– 1} using an ATIR Cary 630 from Agilent in 2021. ¹H NMR and ¹³C NMR spectra were obtained by using a JEOL 400 MHz spectrometer. Emission spectra were obtained using JASCO FP-8200 instrument, which uses cutoff filters. Lifetime titration studies were conducted using TCSPC Delta-Pro from HORIBA, employing a 450 nm excitation source at room temperature.

3.1. Synthesis of N’-(Anthracen-9-ylmethylene) Morpholine-4-carbothiohydrazide (MA)

Thiosemicarbazone [N’-(anthracen-9-ylmethylene) morpholine-4-carbothiohydrazide] was synthesized according to the reported procedure by Scovill − with slight modifications (Figure S1). A dried round-bottom flask was charged with morpholine-4-carbothiohydrazide, dissolved in hot methanol, followed by addition of methanolic solution of 9-antharcene carboxaldehyde, and allowed to reflux for 2 h. While being in reflux, the reaction mixture was acidified by drops of conc. HCl. Following filtering to remove the yellow precipitate, it was washed with portions of chilled methanol and dried under vacuum. Further compound was recrystallized in the form of CH₃OH:CHCl₃ (1:1) (Scheme ).

MA: (Yellow powder) Yield: 85%. Chemical formula: C₂₀H₁₉N₃OS; elemental analysis calculated (%): C 68.74, H 5.48, N 12.02, S 9.18, found (%): C 68.73, H 5.47, N 12.03, S 9.17; ¹H NMR (400 MHz, δppm DMSO-d ₆): δ 3.65–4.01 (8H, m), 7.59–7.67 (4H, m), 7.92–8.33 (2H, t, J = 10.35), 8.63–8.70 (1H, d, J = 12.52), 8.71–8.97 (2H, t, J = 15.60), 9.15–9.92 (1H, d, J = 15.82), 11.13–11.72 (1H, s, J = 23.08) (Figure S3); ¹³C NMR (100 MHz, δppm DMSO-d ₆) δ 50.1 (C₁–N₁, C₄–N₁), 66.4 (C₃–O₁, C₂–O₁), 126.0, 127.6, 129.5, 130.0, 131.5 (aromatic carbons C₁₀–C₁₁) 144.6 (C₆=N₃), 181.2 (C₅=S₁) (Figure S4); FTIR (cm^–1): 1548 (s, υ, (C=N)), 1218 (s, υ (C=S)), 3182 (s, υ (N–H)), 1116 (S, υ, C–O) (Figure S2); ESI mass: 350.13 (M + H) (Figure S5).

4. Conclusions

A novel anthracene-based thiosemicarbazone (MA) was successfully synthesized and thoroughly characterized using various spectroscopic methods, including FTIR, UV–vis, NMR, and mass spectrometry. MA demonstrated a significant and selective sensing property toward cyanide ions. The sensing behavior of MA was comprehensively investigated through UV–vis and photoluminescence spectral titrations. To further elucidate the underlying sensing mechanism, detailed studies were conducted, including ¹H NMR titration, lifetime studies, and kinetic studies. The presence of isosbestic points clearly indicates the formation of an MA–CN complex within the system. The binding constant was determined to be 1.38 × 10 5 M ^–1 with a 1:1 stoichiometric ratio between MA and cyanide ion obtained from Job’s plot. MA demonstrated an impressive low detection limit of 1.37 nM, which is significantly lower than recently reported values for cyanide ions in aqueous medium. The ¹H NMR titration confirmed the complete disappearance of the −N₂–H proton from the MA receptor, providing insight into the possible sensing mechanism (see Figure ). Kinetic studies allowed for the calculation of the reaction rate constant, revealing that MA can effectively sense even very low concentrations of cyanide ions. The average lifetime of the MA–CN complex was observed to increase from 3.28 to 6.39 ns upon the incremental addition of one equivalent of CN^–. These results collectively indicate the selective and efficient sensing of cyanide ions by probe MA. Moreover, MA was highly reversible over 10 cycles, indicating its high efficiency and selectivity toward CN^–. Furthermore, the experimental findings were cross-checked with theoretical (TDDFT) calculations, which included geometry optimization, UV–vis spectral prediction, and orbital analysis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06461.

• Detailed synthesis procedure and characterization (FTIR, ¹H NMR, ¹³C NMR, and HRMS) results, AIE, pH study, reversibility analysis, and real-time images along with computational results (PDF)

The authors declare no competing financial interest.