Selectively sensing first-row transition metal ions through fluorescence enhancement

Abstract

Transition metal ions, especially the first-row ones are ubiquitous in nature and they perform many biological functions. However, high accumulation of these ions can be quite detrimental to health. Their spatial distribution with high fidelity inside as well as outside cells is, therefore, of paramount importance. In addition to biological sensing, fluorescence enhancement by transition metal ions can be potentially useful in other areas of science. However, most of the transition metal ions are paramagnetic and they very effectively quench fluorescence. Surmounting this problem of fluorescence quenching, a number of systems have been reported which are the topic of the present review.

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Sanchari Pal

Sanchari Pal was born in West Bengal, India. She received her M.Sc. (Gold medalist) from Jadavpur University, in 2010. She is currently pursuing her Ph.D. at the Department of Chemistry, IIT Kanpur under the supervision of Prof. P. K. Bharadwaj. Her research interests focus on Fluorescence signaling, Cryptands and MOFs.

Nabanita Chatterjee

Nabanita Chatterjee was born in West Bengal, India. She received her M.Sc. from University of Mysore, in 2010. She is currently pursuing her Ph.D. at the Department of Chemistry, IIT Kanpur under the supervision of Prof. P. K. Bharadwaj. Her research interests focus on Fluorescence signaling and MOFs.

Parimal K. Bharadwaj

Parimal K. Bharadwaj received his Ph.D. (1979) from the Indian Institute of Technology Kharagpur. After spending a year as a UNESCO fellow (1980) at the Tokyo Institute of Technology, he moved to Rutgers University in NJ where he carried out research in Bioinorganic Chemistry (1980–1985) first as a Postdoctoral Fellow and then as an Assistant Professor (non-tenure track). Thereafter, he spent two years (1986–1987) at the University of California at Davis working on radiopharmaceuticals. He returned to India as an Assistant Professor of Chemistry at the Indian Institute of Technology Kanpur where he became a full Professor in 1995. His current research is focused on fluorescence signaling, FRET, using cryptands as platforms for photoinduced energy and charge separation and on the design and synthesis of functional metal-organic framework materials.

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1. Introduction

Many transition metal ions are ubiquitous in nature and they perform¹ several biological functions while at the same time, excess accumulation of these ions in biosystems can lead to diseases² and ultimately to fatal consequences. Their spatial as well as temporal distribution inside and outside the cells is, therefore, of paramount importance to understand physiological conditions. Fluorescence spectroscopy³ is a sensitive tool to detect and measure concentration of an analyte with high fidelity. The basic design of the signaling system is, however, consists of a signaling (fluorophore) and a guest-binding (receptor) moieties that may be integrated or covalently attached through a spacer (Scheme 1). Out of these two designs, the one with a spacer is preferable as either the fluorophore or the receptor or simultaneously both units can be varied at will. An ideal chemosensor will be the one that can recognize a particular metal ion in presence of many other competing ones. It should be designed with new recognition principles or fine tuning the existing ones, both of which are formidable challenges to chemists. The right donor atoms should be placed at strategic positions to match the coordination preferences of the metal ion. Biologically relevant paramagnetic transition metal ions quench emission quite effectively and in many cases, emission quenching has been adopted⁴ as a signaling mode to detect the presence of an analyte. However, for practical as well as sensitivity reasons, fluorescence enhancement and not quenching should be the preferred mechanism in fabricating a sensor. In simple terms, blocking of the fluorescence quenching by a paramagnetic transition metal ion requires that metal ion–receptor (M–R) interaction should be greater than the metal ion–fluorophore (M–F) communication. This idea can be incorporated in designing the overall architecture of the signaling system. Choice of the fluorophore should be made based on its excitation/emission characteristics, photochemical as well as thermal stability, magnitude of the Stokes shift, solubility properties and so on. In cases where a spacer is used, the nature of the later is also important⁵ in the signal transduction process. Although most of the emission techniques and uses revolve around small organic molecules as receptors, recent years have witnessed a large number of supramolecular systems with interesting photochemical as well as photophysical properties. In addition to the sensory roles, it can also be used for information processing⁶ that opens up a whole new window of opportunities. Finally, accessing a signal via remote control with waveguides and fiber-optics technology has led to widespread use of the fluorescence techniques in chemistry related sensing and switching applications. In the present review, only first-row transition metal ions are covered without Zn²⁺ ion as several excellent review articles⁷ on Zn²⁺ ion sensing are available. Generally, literature from 2005 onwards has been covered. Of course, in some cases chemosensors reported prior to 2005 are included especially for those metal ions for which not many examples are known. However, the literature coverage is not exhaustive and at the outset, we apologize if we have not covered any important work in the present review. There are several reviews available in the literature⁸ on transition metal ion chemosensors using fluorescence.

Scheme 1 Two common designs used in fluorescence signaling unit (a) through spacer signaling unit and (b) integrated signaling unit.

2. Sensing of metal ions

2.1. Scandium chemosensors

Pure organic stereoisomers are much sought after synthetic compounds in pharmaceutical, agrochemical, and other industries. These aspects have spurred research in asymmetric synthesis and the development of analytical techniques for determining their stereochemical purity. Fluorescence enhancement upon binding of Sc³⁺ with stereoisomers has been used as a high throughput technique for determining the purity of stereoisomers. Thus, chemosensor 1,8-bis(3-tert-butyl-9-acridyl)naphthalene N,N′-dioxide (1), has been synthesized from 3-tert-butylaniline and 2-chlorobenzoic acid.⁹ Free 1 does not show any significant emission upon excitation. However, with Sc³⁺ ion, it forms a 2 : 1 complex showing high chelation-enhanced fluorescence enhancement (CHEF) while other competing metal ions like Cu²⁺, Zn²⁺, Yb³⁺, Sn²⁺ and In³⁺ do not exhibit such enhancement. Compound 1 thus serves as a selective fluorescence-on chemosensor for the Sc³⁺ ion. Stereoselective displacement of Sc³⁺ from its complex with concomitant loss of fluorescence output can be used to determine the presence of a particular enantiomer.

The compound 2,3,5,6-tetrakis(2-pyridyl)pyrazine (TPPZ) (2) itself shows negligible fluorescence but in presence of 1 equivalent of Sc³⁺ ion in MeCN affords a strong fluorescence due to the formation of a 1 : 1 complex¹⁰ (Scheme 2) albeit with a low association constant of (1.4 ± 0.1) × 10². Since Zn²⁺ ion strongly interferes, it must be absent for sensing of Sc³⁺ ion.

Scheme 2 Binding mode of 2 with metal ion.

2.2. Vanadium chemosensors

Vanadium is an essential trace element due to its significant roles in various enzyme systems.¹¹ Vanadate ion (VO₄³⁻) is a structural analogue of phosphate; it is also a transition state analogue of phosphate in the penta-coordinated form. So, vanadate interacts with a broad range of phosphate-metabolizing enzymes.¹² Besides, vanadate is also found as the cofactor of vanadium haloperoxidases (V-HPOs),¹³ which are important enzymes that catalyze halogenation of natural compounds.¹⁴ In aqueous medium, however, vanadate ion undergoes complicated hydrolysis and oligomerization reactions that can make it a difficult one to trace. Epidemiological evidences suggest that vanadium may also play a beneficial role in the prevention of heart-disease.¹⁵ However, it is quite toxic and at high concentration level, vanadium is a potentially dangerous chemical pollutant that can play havoc with the agricultural system. It is thus quite important to have a method available for quantitative detection of the metal ion in aqueous medium.

The rhodamine-derived chemosensor 3 has been synthesized by reacting rhodamine hydrazide with 5-chlorosalicylaldehyde in ethanol¹⁶ to provide a N₂O donor set. Metal free 3 does not show any emission when excited at 530 nm in aqueous MeOH (1 : 1, v/v, pH 7.2) as the spirolactam ring remains intact. However, in presence of 10⁻⁹ M VO²⁺ at pH 7–8, a 40-fold enhancement of emission is observed at 583 nm due to spiro-bond cleavage (Scheme 3). On the other hand, Cu²⁺ affords only 3.6 fold enhancement. Other competing metal ions (Bi³⁺, Tb³⁺, Pb²⁺, Cr³⁺, Al³⁺, Eu³⁺, Ru³⁺, Sm³⁺, Sn⁴⁺, Ni²⁺, Co²⁺, Yb³⁺, Hg²⁺, Ga³⁺, Zr⁴⁺, Er³⁺, Ho³⁺, Fe³⁺, Zn²⁺, Mg²⁺, Mn²⁺, La³⁺, Nd³⁺) including heavy metal ions do not give any emission. The VO²⁺ ion which has a preference for O donors, forms a square pyramidal complex with the chemosensor. ESI-MS data supports the formation of a 1 : 1 complex although no stability constant value for the complexation is available.

Scheme 3 Proposed sensing mechanism of 3 to VO²⁺.

A flow method has been developed for the determination of pentavalent V in different media. In this method, continuous fluorescence measurement is done on the complex formed between V⁵⁺ and 1,2-dihydroxyanthraquinone-3-sulfonate (4) sorbed on anionic resin beads.¹⁷ Although no association constant data are available, it is found that the dye shows increased sensitivity by about 4 × 10³ times higher compared to the same system without immobilizing beads. Besides, immobilization of the reagent in the solid support¹⁸ makes possible the production of a more rigid environment which closes deactivation pathway. Both these factors allow nM amounts of V⁵⁺ to be detected in different kinds of samples, viz., water from various sources, urine and serum, as well as mussel tissues.

The receptor 5 has been designed for sensing vanadates and its oligomers.¹⁹ The pyrene groups in 5 with their strong stacking tendency, can make the receptor pre-organized²⁰ and this can open up to accommodate dimer and trimers of vanadate. The chemosensor is found to bind preferentially the vanadate VO₄³⁻ ion in DMSO (Scheme 4). The fluorescence spectrum of 5 in DMSO exhibits pyrene monomer emission bands at 390, 410 and 437 nm as well as a small peak at 607 nm. Upon addition of VO₄³⁻, the monomer emissions along with the emission at 607 nm are quenched while a peak at 508 nm, assigned to the emission of the pyrene excimer²¹ and a shoulder at 564 nm appear, confirming stacking of the pyrene units. With the addition of 7 equivalents of VO₄³⁻, the intensity of the major peak at 513 nm, with a 5 nm red-shift, increased significantly with a 30-fold enhancement. After about 8 h, the peak at 513 nm disappears and a new peak at 564 nm appears showing a 50-fold enhancement due to binding of the oligomers that are in equilibrium with the monomer. This sensor, therefore, represents a design to tackle complicated hydrolysis and oligomerization of the vanadate ion in aqueous medium.

Scheme 4 Proposed binding mechanism of 5 with different vanadate species.

2.3. Chromium chemosensors

Cr³⁺ is an essential trace element in human nutrition. It exerts great impacts on the metabolism of carbohydrates, fats, proteins and nucleic acids by activating certain enzymes and stabilizing proteins and nucleic acids.²² Insufficient dietary intake of Cr³⁺ leads to increased risk factors associated with diabetes and cardiovascular diseases, including elevated levels of circulating insulin, glucose, triglycerides and total cholesterol and impaired immune function.²³ As a contaminant, chromium is found mostly in Cr(VI) form and its bacterial reduction to Cr³⁺ is considered as one of the promising strategies for bioremediation. However, exposure to high levels of Cr³⁺ can negatively affect cellular structures. The environmental protection agency (USA) has set the maximum permissible level of total chromium at 100 ppb. Fluorescence turn-on reagents for monitoring intracellular Cr³⁺ concentration remain underdeveloped especially in aqueous media. In earlier reports on Cr³⁺ sensors, several divalent and trivalent metal ions like Cu²⁺, Zn²⁺, Cd²⁺, Ni²⁺ or Al³⁺ were found²⁴ to interfere with the detection processes. Amongst different designs, spirolactam ring opening in presence of the metal ion has been a popular method of detection of the Cr³⁺ ion.²⁵

The chemosensor 6 is based on rhodamine B where 8-hydroxyquinoline moiety has been attached along with a ferrocene substituent.²⁶ This is found to be highly selective for the Cr³⁺ ion in presence of a host of other biologically relevant metal ions. The association constant for Cr³⁺ is estimated to be 7.5 × 10³ M⁻¹ in the aqueous ethanol (1 : 1, v/v, pH 7.4) solution. On excitation at 530 nm in 50% aqueous ethanol, metal-free 6 shows very low fluorescence at 580 nm since the spirocyclic form of rhodamine prevail. Upon addition of Cr³⁺ ion in the pH range of 5–10 in aqueous ethanol, a 15-fold increase in fluorescence is observed within 1 min, with a peak at 587 nm due to opening of the spirolactam ring. Under similar experimental conditions, only Hg²⁺ exhibits slight enhancement. However, other metal ions do not show any discernible change compared to the metal-free chemosensor. The most likely donor atoms for Cr³⁺ are the conjugated moiety including carbonyl O, imino N, and quinoline N and O atoms (Scheme 5). Due to its instantaneous response and solubility in aqueous ethanol, 6 is used in cell imaging studies. When the HeLa cells are stained with a 20 μM solution in ethanol–PBS buffer (1 : 99, v/v) for 30 min at 25 °C a very faint intracellular fluorescence is observed. Addition of Cr³⁺ at 37 °C, affords a significant fluorescence in the intracellular domain in 2.5 h. Bright-field measurements after the Cr³⁺ treatment indicates that the cells remain viable throughout the period. Moreover, differential pulse voltammetric studies in ethanol gives a significant shift of the ferrocenium/ferrocene potential in absence/presence of Cr³⁺ ion indicating that 6 can be used as a redox chemosensor of Cr³⁺.

Scheme 5 Probable complexation mechanism of 6 with Cr³⁺.

The rhodamine 6G based chemosensor 7 has been designed incorporating a diethylene triamine moiety as the binding site for Cr³⁺ ion at pH 7.2 in aqueous medium.²⁷ The association constant (K_a) with Cr³⁺ is found to be 4.16 × 10⁴ M⁻¹. This sensor gives about 60-fold enhancement upon addition of 5 equivalents of Cr³⁺ due to spiro-ring opening (Scheme 6) while other ions including Cd²⁺, Co²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Mg²⁺, Ba²⁺, Pb²⁺, Na⁺ and K⁺ afford no distinct response. Only Fe³⁺ also forms a 1 : 1 complex giving a stability constant of 4.73 × 10³ M⁻¹ and shows a 17-fold enhancement upon addition of 5 equivalents of Fe³⁺ ion. Iron being much more biologically abundant than chromium, this chemosensor can only be useful in absence of Fe³⁺ ion which seriously limits its utility. This also suggests that cell imaging studies for temporal distribution of Cr³⁺ cannot be made with 7.

Scheme 6 Proposed mechanism for fluorescence enhancement of 7 upon the addition of Cr³⁺.

The rhodamine B derived chemosensor 8 forms a 2 : 1 complex with Cr³⁺ and exhibits appreciable response due to spiro-bond opening in the range, 0–10 μM with a detection limit of 1 ppm. The system is functional within a wide range of pH (4.0–8.0) in 20% aqueous methanolic medium.²⁸ Other competing metal ions that include alkali/alkaline earth, first row transition and heavy metal ions, do not give any significant response. The response towards Cr³⁺ is found to be independent of the nature of anion used. Although response time for enhancement is found to be sluggish (∼10 min), no interference from competing metal ions makes it an excellent sensor for the Cr³⁺ ion. Its usability in aqueous alcohol allows for imaging of living MGC803 cell lines demonstrating its potential applications in biological systems.

For sensing of Cr³⁺ ion in aqueous medium with low detection limit, the rhodamine B derivative 9 has been synthesized.²⁹ This chemosensor gives Cr³⁺ ion induced spiro-bond opened turn-on fluorescence in aqueous ethanol within a wide pH (5–9) range. The detection limit is found to be quite low at 0.023 μM. Besides, the Cr³⁺ ion can be removed using EDTA making the system reversible. However, the metal–chemosensor stoichiometry of 1 : 1 proposed by the authors is not consistent with the strong preference of hexacoordintion for the Cr³⁺ ion (Scheme 7). No association constant value for complexation is available. However, since other competing metal ions do not show any significant emission, this system has been used for intracellular imaging in live cells and determination of the ion in tap water and river water.

Scheme 7 Proposed mechanism for fluorescence enhancement of 8 upon the addition of Cr³⁺.

The chemosensor 10 is structurally quite similar to 9 except that a thiophene moiety has been used in place of a furan to bind a metal ion through the carbonyl O, inamine N and thiophene S donors.³⁰ The sensor forms a 1 : 1 complex with Cr³⁺ giving a moderately high association constant of 2.0 × 10⁴ M⁻¹. Complexing with Cr³⁺ triggers the formation of the highly fluorescent ring-opened form which is pink in colour. It affords an emission centering at 583 nm with about 1200-fold enhancement. However, Hg²⁺ also gives significant emission at 576 nm with this fluorescent probe.

Rhodamine 6G derived chemosensors 11 and 12 are found to bind specifically to Hg²⁺ or Cr³⁺ in presence of large excess of other competing ions with associated changes in their optical and fluorescence spectral properties.³¹ For 11, the detection limit is lower than the permissible [Cr³⁺] or [Hg²⁺] in drinking water as per standard U.S. EPA norms. The chemosensor 12 can be used as a ratiometric sensor for detection of Cr³⁺ and Hg²⁺ based on the Förster resonance energy transfer (FRET) process involving the donor naphthalimide and the acceptor Cr³⁺/Hg²⁺-bound xanthene fragment (Scheme 8). Studies reveal that 12 can be used for recognition and sensing of Hg²⁺/Cr³⁺. Further, confocal microscopic studies confirm that 12 can also be used as an imaging probe for detection of these ions in A431 cells.

Scheme 8 Probable metal ion binding mode of 12.

On the basis of FRET from 1,8-naphthalimide to rhodamine, the dyad 13 has been synthesized as a Cr³⁺ selective fluorescent probe for monitoring Cr³⁺ ion concentration in living cells with ratiometric fluorescence methods.³² When excited at 405 nm in aqueous ethanol (1 : 2 v/v), a yellow fluorescence centered at 536 nm is observed attributable to an intramolecular charge transfer (ICT) band involving the 1,8-naphthalimide chromophore. Addition of 10 equivalents of Cr³⁺ ion causes the appearance of a strong absorption band centered at 568 nm changing the colour of the solution to red-orange from yellow. The Cr³⁺-complex on excitation at 515 nm, shows a 15-fold enhancement of fluorescence at 590 nm due to spirolactam ring opening (Scheme 9). Further, when excited at 405 nm, the ICT band intensity at 536 nm is reduced drastically with a concomitant increase of the intensity at 594 nm due to an efficient FRET from the naphthalimide to the ring-opened spirolactam. This fluorescent probe exhibits excellent selectivity over other metal ions. Competition experiments for Cr³⁺ mixed with excess of competing metal ions does not show any change in the emission band intensity, although Mn²⁺, Ag⁺ and Al³⁺ ions induces slight quenching or increasing of the ratio, F₅₉₄/F₅₃₆. Confocal microscopic studies confirm that 13 can be used for monitoring intracellular Cr³⁺ levels in living cells with general fluorescence and FRET methods. Such systems will be of help in studying the bioactivity of Cr³⁺ in biological systems.

Scheme 9 Probable sensing mechanism of 13.

The absorption maximum of a rhodamine derivative normally appears around 550 nm and typically gives a 25 nm Stokes shift of the emission band. This can limit its application as an effective chemosensor in many biological applications.^33,34 To circumvent this, the chemosensor 14 capable of showing two-photon excited fluorescence (TPEF) upon Cr³⁺ binding has been synthesized.³⁵ In presence of Cr³⁺ ion, 14 changes to the open-ring form giving remarkable fluorescence enhancement both in one and two-photon excitations. The broad excitation wavelength, on/off fluorescence and high selectivity to Cr³⁺ (K_S = 9.4 × 10³ M⁻¹) enable 14 to be a powerful Cr³⁺ cation sensor with potential applications in biological systems.

The photoinduced electron transfer (PET) chemosensor 15 represents one of the few fluorescence chemosensor for Cr³⁺ where a rhodamine is not used.³⁶ The signaling and the receptor moieties are covalently attached. In THF, it exhibits 17-fold chelation enhanced fluorescence (CHEF) in presence of Cr³⁺. The receptor is found to be highly selective for Cr³⁺ ion against the background of environmentally and biologically relevant metal ions forming a 1 : 1 complex with a high association constant of 11.3 × 10⁴ M⁻¹.

The highly selective luminescent chemosensor 16 in aqueous solution has been assembled by a low-selectivity luminogenic receptor with Cu²⁺ as a metal quencher.³⁷ Three tetranitrogen chelating sites are integrated into the multichannel receptor with a tris(1,10-phenanthroline)ruthenium(II) luminophore at the core. This receptor exhibits chelating affinity towards several transition metal cations, among which Cu²⁺ efficiently quenches the emission. Addition of Cr³⁺ into the Cu²⁺-complex of 16 results in a metal-exchange reaction with concomitant recovery of fluorescence. Other transition metal ions do not take part in the metal-exchange reaction. The quencher displacement sensing strategy in this design can be a simple but efficient approach for OFF–ON luminescent sensing of metal cations that inherently lack selective ligands. This strategy of fluorescence sensing is, however, not that useful for biological imaging studies as many side reactions might interfere with the determination of a particular ion.

Two PET chemosensors 17 and 18, incorporating dansyl dye as a fluorophore and various carboxhydrazones as receptors, have been reported.³⁸ Sensor 17 contains a pyridine-carboxhydrazone tridentate coordination site, forming a 2 : 1 complex with Cr³⁺ (K_as = 6.07 ± 0.10 × 10⁷ M⁻¹). Metal-free 17 gives a weak dansyl based emission at 545 nm when excited at 350 nm in DMF–water (9 : 1, v/v). Upon addition of Cr³⁺ it affords a 5-fold enhancement in the pH range, 5–9. No emission is observed in presence of alkali, alkaline earth or competing first-row transition metal ions. Likewise, 18 offers a 4-fold enhancement of dansyl based emission selectively in presence of Cr³⁺ under similar experimental conditions (Scheme 10). The metal ion forms a 1 : 1 complex with 18 giving an association constant value of K_as = 9.59 ± 0.03 × 10³ M⁻¹.

Scheme 10 Possible binding mode of 17 and 18 with Cr³⁺ (X is the coordinating anion or solvent).

Biaryl borondipyrromethene (BODIPY) fluorophore has high photostability with high fluorescence quantum yields. Besides, it is excitable with visible light, has narrow absorption and emission bands and is amenable to easy structural modification that can lead to spectral shifts in the absorption and emission bands to longer wavelengths. In 19, a BODIPY group is covalently connected to a receptor incorporating NO donors. In acetonitrile, metal-free 19 gives almost no fluorescence when excited at 560 nm due to efficient PET from the amino N to the BODIPY.³⁹

Upon addition of Cr³⁺ ion, initially it forms a 2 : 1 complex (Scheme 11) giving weak fluorescence because the uncoordinated amine nitrogens can take part in the PET process to the photoexcited BODIPY moieties. With gradual increase of Cr³⁺ ion concentration, it forms a 2 : 2 complex (Scheme 11) engaging all the N lone-pairs that blocks PET to the BODIPY group. As a result, a strong BODIPY based emission with nearly 2800-fold enhancement factor is observed around 640 nm. The enhancement is observed within 10 s indicating that 19 can detect Cr³⁺ very rapidly. Other competing metal ions do not interfere with Cr³⁺ sensing. However, in presence of more than 1% water in MeCN, Fe³⁺, Cu²⁺ and Hg²⁺ strongly quenches fluorescence and thus seriously limits the use of 19 as a probe. Besides, the detailed coordination mode of Cr³⁺ with 19 requires further studies.

Scheme 11 Possible sequence for coordination between 19 and Cr³⁺.

For ratiometric fluorescence sensing of Cr³⁺ in aqueous medium, a rhodamine derivative (RG1) and a coumarin derivative (CG1) have been used.⁴⁰ Together, they constitute the chemosensor 20 as shown in Scheme 12. Upon addition of Cr³⁺ to a buffered solution (pH = 6) containing equimolar amounts of CG1 and RG1, the metal ion binds to both the components as shown (Scheme 12). When the coumarin derivative is excited at 450 nm, the metal ion acts as a conduit for efficient FRET from the coumarin moiety to the ring-opened rhodamine moiety.⁴¹ Fluorescence measurements also show an excellent selectivity toward Cr³⁺ over metal ions such as alkali, alkaline earth and other first-row transition metal ions. Even the presence of heavy metal ions cannot bring any obvious fluorescence change. Confocal fluorescence microscopic investigation on HeLa cells are found to be capable of detecting subcellular concentration of Cr³⁺ ion.

Scheme 12 Possible sensing mechanism of 20.

The fluorescent probes 21(a–d) having N, O/S/Se donors show donor specific ‘turn-on’ recognition property⁴² towards Cr³⁺, Fe²⁺ and Cu²⁺. Here, the fluorescence signals are controlled by the conformational change of the ligand framework upon binding with the metal ion (Scheme 13). Theoretical studies at the B3LYP/6-31G* level supports the mode of binding. However, they suffer from poor selectivity and only provide the starting point for the development of chemosensors with better selectivity and sensitivity.

Scheme 13 The binding mode of Cr³⁺ ion with the fluorescent probe 21.

The bis-dioxime chemosensor 22 upon excitation at 417 nm gives an emission at 515 nm that shows about 60% CHEF enhancement in presence of Cr³⁺ while quenching in presence of Fe³⁺.⁴³ Therefore, in absence of Fe³⁺, this sensor can be used for detecting Cr³⁺ ion.

The chemosensor 23 with the naphthalimide based fluorophore connected to a BINOL framework shows low emission in THF–H₂O (85/15, v/v) due to an efficient PET from the receptor.⁴⁴ It forms a 1 : 1 complex (Scheme 14) with Cr³⁺ ion with a moderately high association constant of 2.4 × 10⁴ M⁻¹ in THF–H₂O (85/15, v/v). Once a Cr³⁺ ion binds at the receptor it gives a 600-fold emission enhancement with the band centering at 498 nm. Other competing metal ions gives slight quenching although in the competition experiments they do not interfere. The detection limit is calculated to be 0.20 μM and response time is found to be about 10 s making it a fast responsive and highly sensitive probe for Cr³⁺.

Scheme 14 The binding mode of Cr³⁺ ion with the fluorescent probe 23.

A dansyl fluorophore is covalently attached to asparagine to have the fluorescent probe 24.⁴⁵ The probe shows a fluorescence “turn-on” and can detect Cr³⁺ at trace levels (5.2 ppb) in aqueous solution under acidic pH (5.9). When a Cr³⁺ ion binds at the sulphonamide group, the ICT from the sulfonamide to the dansyl is inhibited giving a 1.6-fold fluorescence enhancement. However, first row transition metal ions and few heavy metal ions also exhibit enhancement to a lesser degree and hence 24 should be regarded as a signaling system with poor chemoselectivity.

A spirobenzopyran derivative 25 with an amide moiety as the binding site for Cr³⁺ has been designed for use in MeCN.⁴⁶ The spiro-bond breaking in presence of Cr³⁺ (Scheme 15) gives 25-fold enhancement of fluorescence at 675 nm. The metal ion forms a 1 : 1 complex with the association constant (K_a) of 7.5 × 10⁴ M⁻¹. This is one of the few probes that gives emission in the NIR region although it is workable in organic media and cannot be used in imaging studies. Efforts should be made to modify the system at sites away from the receptor for enhanced water solubility.

Scheme 15 Possible sensing mechanism of 25.

A fairly strong interaction between Cr³⁺ and the PET probe 26 is found in aqueous MeCN at pH 7.4 with 1 : 1 stoichiometry and a binding constant value of 8 × 10⁴ M⁻¹.⁴⁷ The metal-free fluorescent probe does not emit significantly when excited at 409 nm. Upon addition of Cr³⁺, a 50-fold enhancement at 550 nm is observed due to snapping of the PET (Scheme 16). Competition experiments with other biologically relevant metal ions show that none of the metal ions interfere. Cell viability tests for the fluorescent probe is encouraging but use of organic solvent limits its use in live cell imaging studies.

Scheme 16 Possible sensing mechanism of 26.

2.4. Manganese chemosensors

Manganese is an essential metal in all forms of life.⁴⁸ The manganese enzymes that have attracted considerable attention are the O₂ evolving complex of photosystem II and superoxide dismutase of prokaryotes and of the organelles of eukaryotes. This metal is also used widely as a versatile tool for biological studies. For example, high-spin Mn²⁺ is an excellent MRI relaxation agent that has been used in clinical diagnosis and is of widespread interest as a tool in neurobiological research.⁴⁹ Chronic overexposure to manganese can result in movement disorders, mental disturbances and other brain-related toxicities.⁵⁰ Fluorescent probes for detection and quantification of Mn²⁺ in biological systems will be of tremendous importance. However, development of an effective fluorescent probe for Mn²⁺ faces several challenges such as its paramagnetic nature, selectivity over abundant cellular metal ions especially Ca²⁺, aqueous solubility for cell imaging and so on. Overcoming these hurdles, presently there are few Mn²⁺ specific fluorescence signaling systems available. One such system (27) is a conjugate of 1,2-bis(2-pyren-1-ylmethylamino-ethoxy)ethane (NPEY) and graphene nanosheets (GNs) via π–π stacking that can be synthesized by simply mixing the diluted aqueous solutions of both components.⁵¹ The conjugate itself does not show any significant emission due to an efficient PET from NPEY to GN. Upon addition of Mn²⁺ ion that binds to the NPEY blocking the PET to give enhancement of fluorescence. The NPEY–GN combination (27) not only shows good selectivity toward Mn²⁺ with the detection limit as low as 4.6 × 10⁻⁵ M, but also shows “turn-on” response both in vitro and in living cells (Scheme 17). These sensing capabilities of NPEY–GNs in living cells make it a robust candidate for many biological fields, such as intracellular tracking, intracellular imaging, etc.

Scheme 17 Schematic demonstration of fluorescence “Turn-On” mechanism for Mn²⁺ detection by 27.

The receptor (O,O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid) popularly known as BAPTA, is an excellent receptor for Ca²⁺ ion and several Ca²⁺ sensors are built around this moiety. Chemosensors 28 and 29 have been designed to differentiate binding affinities of Mn²⁺ over Ca²⁺ by partially replacing hard carboxylate oxygens of BAPTA by softer pyridines.⁵² Both Mn²⁺ and Ca²⁺ are classified as “hard” metals and, therefore, prefer to bind hard donors. However, Mn²⁺ appears to be more tolerant toward softer donors compared to Ca²⁺.^53,54

The association constant ratio (log K_Mn/log K_Ca) is found to be 2.368 for 28 while it is 2.402 for 29. When excited at 493 nm, metal-free 28 shows a low intensity emission centering at 519 nm in aqueous medium. Upon addition of Mn²⁺, a 4-fold enhancement is observed until saturation after 1 equivalent. On the other hand, metal-free 29 can be excited at 505 nm to obtain an emission maximum at 530 nm. It also affords almost a 4-fold enhancement in presence of 1 equivalent of Mn²⁺ ion. Both the probes have been used for intracellular Mn²⁺ with HeLa cell lines. However, it is to be noted that biological concentration of Ca²⁺ is about thousand times that of Mn²⁺ and hence cell imaging studies for Mn²⁺ will not be suitable especially when it is present in trace quantities.

The two-metal two-dye displacement assay has been adopted for detection of Mn²⁺ ion in 30(a,b) where two commercially available dyes, viz., calcein blue (CB) and fluozin-1 (Fz1) have been used.⁵⁵ It works due to differential binding abilities of the two dyes. Initially, Cd²⁺ is chelated by the stronger ligand CB and the resulting complex is highly fluorescent. The other ligand, Fz1 remain uncomplexed and gives a weak fluorescence. Upon addition of Mn²⁺ to the mixture, the Cd²⁺ is displaced from CB by Mn²⁺ resulting in quenching of fluorescence (Scheme 18). However, Cd²⁺ presently available in the solution, forms a complex with Fz1 accompanied by a strong fluorescence. The Cd²⁺-complex of CB gives a green fluorescence with the emission band at 493 nm upon excitation at 350 nm. On the other hand, Cd²⁺-complex of Fz1 gives an emission with maximum at 518 nm. Thus, it allows indirect detection of Mn²⁺ and also cell imaging studies.

Scheme 18 Possible metal ion binding mode of 30(a,b).

The PET chemosensor 31 forms a 1 : 1 complex with Mn²⁺ with log β ≈ 3.0.⁵⁶ On excitation at 310 nm in aqueous MeCN (1 : 1, v/v, pH 4.0) a 4-fold enhancement of fluorescence peaking at 360 nm is observed upon addition of Mn²⁺ ion due snapping of PET. However, alkaline earth metal ions also show enhancement albeit to lesser degrees.

2.5. Iron chemosensors

Iron is an essential element for many biological processes. But it is also detrimental to health as it causes the generation of highly destructive oxygen species. Accumulated data on iron metabolism suggest that alterations in the intracellular pool of ‘redox-active’ iron (also called ‘chelatable iron’) can contribute substantially to a variety of injurious processes such as ischemia-reperfusion injury, ethanol toxicity and oxidative injury after nutrient deprivation⁵⁷ and so on. On the other hand, chelatable iron is increasingly found to be decisively involved in several intracellular signalling pathways.⁵⁸ Most studies have been focused on detecting the cytosolic pool of chelatable, redox-active iron which is generally regarded as the cellular chelatable iron pool.⁵⁹ However, a ‘transit’ pool of chelatable iron must also be assumed to exist within the mitochondria during haem synthesis. Iron is incorporated into protoporphyrin IX within the mitochondrial matrix. In addition, there is now increasing list of evidences that suggest a definite role of mitochondrial chelatable iron in the pathogenesis of various human diseases.⁶⁰ It follows, therefore, that quantitative knowledge of spatial distribution of different forms of iron is of extreme importance.

Like many first-row transition metal ions, fabrication of iron sensors has been dependent heavily on the spirolactam ring opening in presence of the metal ion. One or two rhodamine derivatives have been used in sensing the metal ion in 2+ and 3+ oxidation states. Thus, chemosensor 32 gives a spirolactam ring-opening fluorescence enhancement at 583 nm by a factor of 112 in aqueous MeCN in presence of Fe³⁺ while an enhancement of 18-fold is observed with Cu²⁺.⁶¹ Other biologically relevant alkali, alkaline earth and transition and heavy metal ions show very weak response. With Fe³⁺, 32 forms a 1 : 1 complex with the stability constant value of 2.3 × 10⁴ M⁻¹. This fluorescent probe has been used for live cell imaging studies although authors are silent about the solvent system used in imaging studies and possible interference from Cu²⁺ ion. It is important that cell imaging as well as cell viability studies are made in pure aqueous or aqueous ethanolic medium.

The rhodamine B hydroxamate fluorescent probe 33 having a biomimetic hydroxamate binding unit is prepared in two steps from rhodamine B.⁶² It binds Fe³⁺ in 1 : 1 stoichiometry in MeOH–MeCN (1 : 1, v/v) mixed solvent with the association constant of 2.3 × 10⁴ M⁻¹. Upon addition of Fe³⁺ to the probe solution, a 112-fold enhancement is observed due to spirolactam ring opening (Scheme 19). Other biologically relevant metal ions do not show any significant response or fluorescence response of Fe³⁺ is not affected by the presence of any other metal ion. The reversible binding of Fe³⁺ with 33 is demonstrated by observing immediate disappearance of fluorescence (and colour) upon the addition of excess of EDTA into the solution of 33 and Fe³⁺.

Scheme 19 Possible binding mode of chemosensor 33.

The imine linkage in the rhodamine 6G based chemosensor 34 is broken in presence of Fe³⁺ ion in aqueous medium and in the process, the spirolactam ring is also broken (Scheme 20).⁶³ As a result, a strong emission band at 551 nm is observed. This Fe³⁺-catalyzed hydrolysis reaction of 34 is not influenced by excess amounts of other biologically active metal ions such as K⁺, Na⁺, Fe²⁺, Ca²⁺, Mg²⁺ or Mn²⁺ ion. Besides, heavy and transition-metals that are potential pollutants do not interfere in the binding of Fe³⁺ and subsequent emission enhancement. The high selectivity of this chemosensor for Fe³⁺ has been used to determine excess, free Fe³⁺ ions deposited into live cells using a ferric citrate-overloaded cell line. The greater concentration of fluorescent cells and the stronger intensities are found to be much more evident for the iron-overloaded cells compared to the normal untreated cells. Thus, this chemosensor can be used for determination of free Fe³⁺ in live liver cells without any detrimental effect on the Fe³⁺-based enzymes.

Scheme 20 Possible mechanism of Fe³⁺-induced Schiff base hydrolysis and the spirolactam ring opening of 34.

The rhodamine 6G–phenylurea conjugate 35 gives a strong fluorescence in the presence of Fe³⁺ in H₂O–CH₃CN (1 : 1, v/v) due to spirolactam ring opening.⁶⁴ In presence of acetate ion, the metal ion is extracted with the formation of spirolactam bond once again thus offering a reversible fluorescence (Scheme 21). In response to acetate ions, the system not only provides remarkable fluorescence quenching but also exhibits a clear colour change from pink to colourless for naked eye observation. The background anions show small or no interference with the detection of acetate ions. This is the first chemosensor based on metal ion complexes that can selectively detect acetate ions over fluoride and dihydrophosphate ions in an aqueous environment. Such systems are expected to find a lot of practical applications.

Scheme 21 Possible binding mode of chemosensor 35.

The chemosensor 36 containing quinoline moiety as the binding site for Fe³⁺ ion has been synthesized.⁶⁵ Upon addition of Fe³⁺ in aqueous ethanol medium, the spirolactam bond is broken (Scheme 22) affording an approximately 190-fold enhancement of fluorescence with a peak at 559 nm following excitation at 505 nm. Fe³⁺ forms a 1 : 1 complex with 36 as shown in Scheme 22 with a high association constant of 1.1 × 10⁶ M⁻¹. While other transition, alkali and alkaline earth or heavy metal ions do not interfere, Cr³⁺ and Al³⁺ give mild enhancement of fluorescence. However, none of these metal ions are essential and hence the probe can be used in imaging studies. Structural studies of 36 indicate that the phthalimide side chain plays an important role in the ion selectivity due to its steric bulk. Upon addition of excess EDTA, Fe³⁺ is removed changing the solution to colourless with 98% quenching of fluorescence. Thus 36 can be classified as a reversible chemosensor for Fe³⁺ ion. Bioimaging studies of EJ cells with 36 shows that it can act as a Fe³⁺ sensor in living cells.

Scheme 22 Possible binding mode of chemosensor 36.

The rhodamine 6G derived chemosensor 37 has been synthesized that provides a definite binding site for Fe³⁺ (Scheme 23).⁶⁶ The chemosensor forms a 1 : 1 complex with Fe³⁺ and the association constant is moderately high at 1.04 × 10⁴ M⁻¹. The free 37 gives only a weak emission at 550 nm when excited at 505 nm. A 23-fold enhanced green-yellow fluorescence is observed with the peak at 550 nm upon addition of Fe³⁺ in aqueous MeCN (1 : 1) at pH = 7.2. No interference from other competing ions is observed. The detection limit is found to be in the 10⁻⁸ M range. Gradual addition of Na₂–EDTA to the Fe³⁺-complex causes decrease in fluorescence intensity and upon addition of 10 equivalents of EDTA²⁻ ion, Fe³⁺ is totally removed with complete quenching of the fluorescence making it a reversible chemosensor for Fe³⁺ ion. However, its lack of solubility in aqueous ethanolic medium does not allow its use in live cell imaging studies.

Scheme 23 Possible binding mode of chemosensor 37.

For increasing water solubility, rhodamine B hydrazide derived chemosensor 38 has been synthesized. This binds Fe³⁺ with 1 : 1 stoichiometry (Scheme 24) with the association constant K_a showing 7.52 × 10⁴ M⁻¹.⁶⁷ The fluorescent probe gives spiro-bond opened 10-fold enhancement of fluorescence in presence of Fe³⁺ in water containing less than 1% organic co-solvent. Other biologically relevant metal ions exerted negligible effect even when present in excess. However, Cr³⁺ also gives enhancement to a lesser extent although that may not be a problem as compared to chromium, iron is present in large amount in biosystems. This probe is also found to be cell permeable and suitable for monitoring intracellular Fe³⁺ with low cytotoxicity in living cells by confocal microscopy.

Scheme 24 Possible sensing mechanism of 38.

To improve selectivity towards Fe³⁺, a different coordination moiety has been added to rhodamine B to have 39.⁶⁸ Indeed, this probe offers a spiro-ring opened (Scheme 25) 23-fold enhancement with Fe³⁺ at 586 nm when excited at 530 nm in aqueous buffer solution. No other competing metal ions show any enhancement. The dissociation constant (K_d) of Fe³⁺ complex is found to be 1.87 × 10⁻⁵ M. The fluorescent probe is found to be safe for long hours at low concentration; it is cell permeable and suitable for the real-time imaging of Fe³⁺ in living MGC803 cells.

Scheme 25 Possible metal ion binding mode of 39.

The rhodamine 6G derived chemosensors 40 and 41 are found to show excellent selectivity towards Fe³⁺ with the binding constant values of 6.74 × 10⁴ M⁻¹ and 3.946 × 10⁴ M⁻¹ respectively with 40 and 41 in aqueous DMSO at pH 7.4.⁶⁹ Upon excitation at 480 nm both the probes show substantial spiro-ring opened enhancement at 553 nm in presence of Fe³⁺. None of the other coexisting metal or heavy metal ions shows any enhancement. Live cell imaging experiments with each probe demonstrated its value of practical applications in biological systems.

Although several rhodamine based chemosensors for Fe³⁺ are available as mentioned above, in most cases either a pure organic solvent or an organic co-solvent will be required to guarantee a high affinity of Fe³⁺ towards a chemosensor. However, in some cases, it is required that the probe be highly soluble in water for detecting Fe³⁺ ions in natural water samples. For increased solubility in water the N-stabilized rhodamine hydrazide fluorescent probe 42 has been synthesized. The N-stablization of rhodamine increases its rigidity resulting in larger Stokes shift and higher intensity of fluorescence.⁷⁰ This chemosensor itself does not show any fluorescence but in presence of Fe³⁺ in aqueous medium (pH 5–8) gives about 164-fold enhancement in less than a minute (Scheme 26).⁷¹ Under similar conditions other biologically relevant metal ions do not show any fluorescence except Cr³⁺ ion which also gives substantial enhancement. This probe forms a 1 : 1 complex with Fe³⁺ and although no association constant data are available, this binding is found to be reversible. Upon addition of 20 equiv. of K₃PO₄ to the 42–Fe³⁺ complex in water, the fluorescence is completely quenched. Further addition of 20 equiv. of Fe³⁺ restores the fluorescence to its original value. This chemosensor is found to be useful in imaging MGC803 cells for monitoring Fe³⁺ ion.

Scheme 26 Possible sensing mechanism of 42.

The fluorescent probe 43 has two fluorophores: a dansyl unit as a donor and a rhodamine 101 as an acceptor for FRET based signaling.⁷² In FRET based sensing, the pseudo-Stokes shifts are larger than the Stokes shift of either fluorophore. This avoids self-quenching as well as fluorescence detection errors due to backscattering effects from the excited source.⁷³ The probe forms a complex of 1 : 1 stoichiometry with Fe³⁺ showing a moderately high association constant (K_a) of 1.74 × 10⁴ M⁻¹. The Fe³⁺ induced spirolactam ring-opening of the rhodamine moiety transforms it as a FRET acceptor (Scheme 27). When the dansyl group is excited at 380 nm, the FRET acceptor emits at 605 nm. The probe displayed a linear response to Fe³⁺ in the range of 5.5–25 μM with a detection limit of 0.64 μM. Other competing metal ions do not give any fluorescence and their presence do not change the fluorescence profile of the Fe³⁺ complex. Thus, this novel rhodamine–dansyl conjugate can selectively sense Fe³⁺ ion in presence of a host of other ions.

Scheme 27 Possible sensing mechanism of 43.

Another FRET based chemosensor on the rhodamine platform for Fe³⁺ is provided by the system 44 has been shown below. A quinoline fluorescent group is covalently linked⁷⁴ to a rhodamine moiety. Metal free 44 exhibits a weak emission at 482 nm which changes upon addition of Fe³⁺ ion, due to an efficient FRET from the conjugated quinoline to rhodamine. Thus, the excitation interference of the new rhodamine based probe is eliminated as a result of efficient FRET (Scheme 28). Binding of the metal ion also causes opening of the spiro-bond of the rhodamine moiety resulting in an enhancement by a factor of 186 times indicating that the new sensor can be used as a highly sensitive Fe³⁺ probe especially when other competing metal ions taken in excess, do not interfere with the Fe³⁺ determination. Its aqueous solubility makes it an useful probe for live cell imaging studies.

Scheme 28 Possible mechanism of Fe³⁺ induced FRET.

A rhodamine B and a coumarin are connected to have the ratiometric fluorescent probe 45 for Fe³⁺ with high sensitivity and selectivity.⁷⁵ Upon addition of Fe³⁺ it binds to both the moiety in aqueous medium with the appearance of spiro-ring opened fluorescence peak at 580 nm and PET suppressed fluorescence from the coumarin moiety at 460 nm (Scheme 29). The association constant for Fe³⁺ (K_a) is found to be 1.172 × 10⁴ M⁻¹ which is quite significant. But a host of other competing metal ions also give enhancement at 460 nm belong to the coumarin moiety. Thus, the design principle is not at all clear in this case.

Scheme 29 Possible sensing mechanism of 45.

In a novel approach, NP conjugate of Fe₃O₄–Rh6G has been synthesized as the chemosensor 46 and found to be sensitive and selective in detecting Fe³⁺ ion.⁷⁶ It forms a 1 : 1 complex with Fe³⁺ with high stability constant (5.0 × 10⁶ M⁻¹). A ∼60-fold enhancement due to spirolactam ring opening is observed for Fe³⁺ with the detection limit reaching as low as 2 ppb (Scheme 30). Amongst other metal ions, only Cr³⁺ shows some enhancement. Since the system can work in aqueous medium, confocal microscopic studies have been made on HeLa cells. The chemosensor 46 is more sensitive compared to the system where no NP is used due to its low solubility.

Scheme 30 Binding of Fe³⁺ to the chemosensor 46.

In addition to mono-rhodamine derived systems, a number of bis-rhodamine derivatives have been designed for detection of iron in different oxidation states. One such bis-rhodamine based chemosensor 47 is easily synthesized from rhodamine B and diethylenetriamine.⁷⁷ The free sensor is colourless in absolute ethanol or in water in the pH range, 5.0–9.0 and does not show any significant emission suggesting that the spirolactam bonds are intact. Upon addition of Fe³⁺, colour of the solution changes immediately to purple both in ethanol and water giving an orange fluorescence while other competing metal ions do not show any change. However, in ethanol medium, Cr³⁺, Fe²⁺ and Cu²⁺ show emission to a small extent. Addition of Fe³⁺ causes both the spirocyclic rings to the open form with a ∼110 and ∼40-fold enhancement at 580 nm in ethanol and water respectively when excited at 510 nm (Scheme 31).

Scheme 31 Binding of Fe³⁺ to the chemosensor 47.

The bis-rhodamine-based dual sensor 48 upon complexation with Fe³⁺ triggers the formation of a highly fluorescent ring-opened form.⁷⁸ This sensing mechanism is reversible, as indicated by disappearance of the emission with the addition of excess EDTA. However, Cu²⁺ interferes and Fe³⁺ cannot be detected in presence of Cu²⁺ ion (Scheme 32).

Scheme 32 Reversible binding of Fe³⁺/Cu²⁺ to the chemosensor 48.

Another bis(rhodamine)-based chemosensor 49 exhibits high selectivity for Fe³⁺ over other transition metal ions in 50% aqueous ethanol.⁷⁹ The spirocyclic rings are opened and a 115-fold enhancement of fluorescence at 575 nm is observed. However, Cr³⁺ also gives enhancement of emission albeit to a lesser degree.

When two rhodamine moieties are connected through a disulfide linkage to have chemosensor 50, it gives about 60-fold enhancement in presence of Fe³⁺ in aqueous ethanol within a wide pH range.⁸⁰ However, as is found for most of the rhodamine derivatives, Cr³⁺ ion also affords enhancement to a less amount. The association constant (K_a) with Fe³⁺ is found to be 7.4 × 10⁴ M⁻¹. Since cytotoxic measurements are found to be negative, the probe has been used for live cell imaging studies.

In an elegant approach, a 12-membered dithia-aza-oxa macrocycle is attached to two different fluorophores through para-substituted benzene to have the chemosensors 51 and 52.⁸¹ In this design, the fluorophores are well-separated from the receptor to minimize metal-fluorophore interaction and possible quenching. The response of Fe³⁺ in presence of potentially interfering metal ions are studied in solvents of different polarity. In organic solvents, fluorescence of metal-free sensors are quenched due to ICT/PET when excited. Addition of Fe³⁺ leads to recovery of the fluorescence without pronounced spectral shifts. Also, metal-free 52 shows dual emissions in water and can be employed for the selective ratiometric signaling of Fe³⁺ in buffered aqueous solution.

The fluorophore 53 has two di-(2-picolyl)-amine (DPA) groups as the metal binding sites attached to a perylene tetracarboxylic diimide fluorophore via benzene as the spacer.⁸² Perylene tetracarboxylic diimides have excellent thermal and photo-stability, high luminescence efficiency and novel optoelectronic properties.⁸³ The receptor and the fluorophore moieties being separated through the rigid spacer, gives low M–F interactions when a metal ion is bound at the receptor. Upon addition of Fe³⁺, PET from the dipicolyl moiety to the fluorophore is blocked leading to recovery of fluorescence. An enhancement factor of 138 in presence of Fe³⁺ is observed although metal ions such as Co²⁺, Zn²⁺ and Cu²⁺ also give emission to a small extent. The Zn²⁺ ion although diamagnetic shows low enhancement and hence it appears that binding strength of the metal ion at the receptor decides the extent of enhancement.

The bis(coumarinyl) Schiff base 54 does not show any significant emission in MeOH–H₂O (98 : 2, v/v) medium.⁸⁴ This low-fluorescence character of free 54 is attributed to a photoinduced charge transfer (PCT) process from the electron donating C=N moiety to the coumarin ring leading to quenching of the excited state of coumarin. Upon addition of Fe³⁺ to 54 which catalyzes the hydrolysis of the imine linkage that releases strongly fluorescent coumarin derivative offering about a 140-fold enhancement in fluorescence (Scheme 33). Other first-row transition metal ions do not catalyze the hydrolysis and hence does not interfere.

Scheme 33 Probable Fe³⁺ induced sensing mechanism of 54.

It is known that Fe³⁺ has a strong binding affinity with imidazole N atom.⁸⁵ The association constant K_a of 55 for Fe³⁺ ion is found to be (2.9 ± 0.2) × 10³ M⁻¹ with the metal ion engaging four N donors equatorially while the axial sites are occupied by counter anions as proven by different spectroscopic techniques. When excited at 288 nm in MeCN–H₂O (95 : 5, v/v) the metal-free 55 gives an emission at 412 nm.⁸⁶ Addition of Fe³⁺ to the solution causes the emission at 412 nm to decrease in intensity with the appearance of a new band at 475 nm. Thus, this chemosensor can be used as a ratiometric probe for Fe³⁺. Other competing metal ions do not afford any ratiometric response neither they interfere with the response obtained with Fe³⁺ ion.

The PET chemosensor 56 has a squarate hydroxamate and a coumarin signaling units linked through a spacer. The squarate dianion itself does not bind first- and second-row transition-metal ions in a chelating fashion because the bite angle imposed by the cyclobutene framework is too large to sustain such coordination. That is why, the squarate hydroxamate has been used which is known to bind Fe³⁺ effectively.⁸⁷ The amine linking the squaramide moiety and the fluorophore is likely to cause a PET-type quenching of the chromophore. The linker moiety is designed to be rigid to prevent a bound metal to have any significant communication with the fluorophore and to lessen the effects of the paramagnetic quenching.⁸⁸

The metal-free sensor shows low emission suggestive of an efficient PET quenching of the coumarin chromophore. Upon addition of Fe³⁺ to an aqueous buffered (pH, 4.4) solution of 56 a 9-fold enhancement of fluorescence is observed due to blocking of the PET (Scheme 34). At a 150 μM concentration of 56 in the presence of 1 equiv. of Fe³⁺ at ambient temperature, equilibration of the emission intensity is reached only after 15 min suggesting its slow response. However, the signal does not degrade over the course of 48 h. Although no stability constant value is given, the fluorescence signal does not diminish even in presence of 1000-fold excess of EDTA.

Scheme 34 Possible binding mode of chemosensor 56.

The chemosensor 57 has been built with two fluorophores viz., a coumarin and a naphthalimide for sensing Fe³⁺ (Scheme 35).⁸⁹ The metal free sensor shows very weak emission when excited at 456 nm. In presence of Fe³⁺ ion, however, it gives an enhancement at 504 nm with high selectivity over other biologically relevant metal ions in aqueous THF solution. The association constant K_a value is, however, found to be moderate at 2.589 ± 0.206 × 10³ M⁻¹. Utility of using two fluorophores is not clear from the studies and also possible interference from Cr³⁺ ion has not been probed for this chemosensor.

Scheme 35 Possible sensing mechanism of 57.

Two 1-naphthylamine derived ICT probes 58 and 59 are found to act as fluorescence turn-on sensors selectively for Fe³⁺ ion in methanol.⁹⁰ Other competing metal ions show quenching of fluorescence like the metal-free probes. However, it is necessary to study the efficiency of enhancement when other metal ions are added in presence of Fe³⁺ ion for its effective use.

Two pyrene moieties are present at the two ends of a linear chain incorporating O and N donors to have the fluorescent probe 60.⁹¹ In the metal-free state, the two pyrene groups are far apart for any possible excimer emission. Besides, no monomer emission is observed due to an efficient PET from the imine N to the excited pyrene. Selectively in presence of Fe³⁺ ion, the probe folds around the metal ion that brings the two pyrene moieties close to one another to give an excimer band at 507 nm when excited at 330 nm. However, no monomer emission is mentioned arising out of the blocked of PET upon Fe³⁺ binding. The association constant of the Fe³⁺ complex is found to be moderately high at 1.27 × 10⁴ M⁻¹.

A BODIPY covalently linked with 8-hydroxyquinoline has been attached to a benzoate linked porphyrin to have the dyad 61.⁹² The 8-hydroxyquinoline benzoate moiety not only facilitates the energy transfer process but also provides binding sites for Hg²⁺ and Fe²⁺ ions. On irradiation at 470 nm where the BODIPY unit absorbs most of the light, 61 gives not only the BODIPY emission at 516 nm but also the porphyrin emission at 650 nm. Addition of Hg²⁺ leads to a significant decrease in the BODIPY emission (almost 90% in comparison with that of the metal-free 61) and a slight increase in the porphyrin emission decreasing the F516/F650 ratio from 16.4 to 1.24. In presence of Fe²⁺, however, an opposite change in the corresponding emission intensities are observed. The emission from BODIPY is remarkably enhanced (83.62% relative to that of metal-free 61) and porphyrin emission, simultaneously diminished slightly, with the F516/F650 ratio increasing from 16.4 to 26.67. It is believed that binding of either metal ion at the 8-hydroxyquinoline site leads to twisting of the dyad bringing the two fluorophores closer. This facilitates FRET from BODIPY to porphyrin. Presence of Fe²⁺ induces partial restraint in the FRET due to non-radiative quenching in the excited states associated with the unpaired electrons in Fe²⁺. This system represents a rare example of Fe²⁺ detection.

2.6. Cobalt chemosensors

Cobalt is an essential trace element in human body and found in cobalamin and also in a few metalloproteins.⁹³ Apart from its biological roles, exposure to high levels of cobalt can lead to health hazards like decreased cardiac output, cardiac and thyroid enlargements, heart disease, elevated red blood cells accompanied by increased cells in the bone marrow, increased blood volume, vasodilation, flushing and so on.⁹⁴ Monitoring its concentration in human is, therefore, of crucial importance. However, there is hardly any fluorescence signaling systems available for Co²⁺ that can be utilized for tracking this transition metal in living cells or other biological specimens. Two principal challenges of Co²⁺ detection in biosystems are its paramagnetic nature and its biologically relevant divalent metal ion competitors like Ni²⁺, Cu²⁺ and Zn²⁺ that typically bind more tightly to common ligands compared to Co²⁺ owing to Irving–Williams series considerations.⁹⁵ In spite of these difficulties, there are several chemosensors available for detecting the Co²⁺ ion.

The chemosensor 62 has been synthesized by connecting a Tokyo Green (TG) dye derivative with a tetradentate N₃O chelator ligand through a cleavable ether linkage.⁹⁶ In presence of O₂, the Co²⁺ ion can break the C–O bond releasing TG free which does not contain a bound, quenching paramagnetic metal center (Scheme 36). This transforms a weakly fluorescent probe into a highly fluorescent dye, that gives about 18-fold fluorescence enhancement in aqueous medium. Cellular concentrations of other competing and biologically relevant s- and d-block metal ions do not elicit any significant fluorescence enhancement. This sensor can also detect changes in exchangeable Co²⁺ pools in living cells as found in case of lung carcinoma A549 cell line.

Scheme 36 Possible sensing mechanism of chemosensor 62.

Two 8-sulfonamidoquinoline derivatives 63 and 64 were designed for the determination of Co²⁺ in presence of H₂O₂.⁹⁷ It has been observed that under basic medium it forms a chelate with the sulphonamide group resulting in intense emission in the ultraviolet region where a range of metal ions, including Cr³⁺, Cu²⁺ and Ni²⁺ do not interfere with the exception of Fe³⁺. This makes these probes useful for the determination of trace amount of Co²⁺ in food and hair samples. While speciation of the complex is not clear, it is likely that Co²⁺ is oxidized to the kinetically inert Co³⁺ complex. Interestingly, no emission is observed in absence of H₂O₂.

Cyanide anions are extremely poisonous as they inhibit the process of cellular respiration in mammals by interacting strongly with a heme unit in the active site of cytochrome a₃.⁹⁸ Also, the high level of intracellular Ca²⁺ concentration is caused by the uptake of CN⁻ and triggers a cascade of enzymatic reactions to increase the level of reactive oxygen species that finally inhibits the antioxidant defense system.⁹⁹ It is thus crucially important to have a sensor to monitor the CN⁻ concentration in biosystems. The coumarinylsalen fluorescence chemosensor 65 binds a Co²⁺ ion at the salen moiety as shown in Scheme 37.¹⁰⁰ While the metal-free ligand shows emission, when it is bound to Co²⁺ no emission is observed due to an efficient PET from the coumarin moieties to the metal center. Upon addition of CN⁻ anion which occupies the two axial sites (association constant, K₁ ≥ 10⁷ M⁻¹ and K₂ = 4.0 × 10⁵ M⁻¹) the LUMO of the cobalt complex is raised above the HOMO of the fluorophore. This blocks the donor excited PET to give a strong fluorescence recovery.

Scheme 37 Complexation of the cyanide ion with 65·Co.

An efficient and selective fluorescent optode membrane based on the chemosensor 66 has been fabricated in the plasticized PVC membrane containing sodium tetraphenylborate as a liphophilic anionic additive.¹⁰¹ The metal-free sensor upon excitation at 345 nm gives a strong emission at 520 nm at pH 5.0. Upon addition of Co²⁺ ion, this emission is quenched with the appearance of two new bands in the region, 385–420 nm. Quenching of the 520 nm band and appearance of the 420 nm band ratio can be used for quantitative estimation of Co²⁺ up to the concentration range of 5.0 × 10⁻⁷ to 2.0 × 10⁻² M with a relatively fast response time of less than 5 min. The proposed fluorescence optode has been successfully applied to find out cobalt content of vitamin B12 ampoule, cobalt cake, cobalt alloy and tap water samples.

The chemosensor 67 based on ICT mechanism has been reported.¹⁰² The free probe on excitation at 404 nm gives a weak, structureless emission band at 428 nm in buffered MeOH–H₂O (1 : 1 v/v) at pH 7.4. Upon addition of Co²⁺, the metal ion binds at the site shown that results in the colour modulation as well as increases the ICT transition by a 37-fold enhancement in the fluorescence output. The selectivity of Co²⁺ arises as it fits well in the pseudo-cavity with N₂O donor set (Scheme 38). Other biologically relevant metal ions give negligible enhancement. A slight modification of the receptor can make this system highly sensitive and selective for Co²⁺ ion.

Scheme 38 Possible Co²⁺ binding mode of 67.

Chemosensor 68 based on 9,10-anthraquinone has been introduced to show that binding of Co²⁺ changes the colour from yellow to pale green.¹⁰³ It also affords a 100-fold CHEF with green fluorescence in 20% aqueous methanol at pH 7 with the Co²⁺ ion. However, metal ions like Cu²⁺ and Ni²⁺ interfere by completely quenching the fluorescence (in presence of Cu²⁺) or significantly reduce the emission (in presence of Ni²⁺). This fluorescent probe is, therefore, not suitable for use in biological samples.

The fluorescent probe 69 with a benzyl ether linker on excitation at 350 nm emits at 380 nm in aqueous solution at pH 7.2.¹⁰⁴ Addition of Co²⁺ to the solution leads to quenching of this emission band and appearance of a new band at 460 nm. The rapid fluorescence quenching of the 380 nm band is due to binding of the paramagnetic Co²⁺. However, Co²⁺ mediated cleavage of the benzyl ether bond releases highly fluorescent 2-(2′-hydroxyphenyl)benzothiazole that gives emission at 460 nm without any metal bonded to it.

The coumarin–quinoline hybrid 70 affords turn-on fluorescence in presence of paramagnetic Co²⁺ and Ni²⁺ ions at 392 nm when excited at 338 nm in methanolic water (9 : 1, v/v).¹⁰⁵ Heavy metal ions like Ag⁺ also elicit slight fluorescence showing its poor chemoselective nature. The fluorescence appears to be due to binding a metal ion at the site providing NO₂ coordination that stops the deactivation pathway.

2.7. Nickel chemosensors

Nickel is an essential element in plants and many other biosystems. It is implicated in various enzyme activities¹⁰⁶ that include ureases, hydrogenases, superoxide dismutases, acetyl-coenzyme A synthases, carbon monoxide dehydrogenases, methyl-coenzyme M reductases and frequently used in catalytic processes. However, as an industrial pollutant, nickel is a toxic element that can cause lung injury, allergy and carcinogenesis. It is, therefore, of importance to have sensors for nickel. A major obstacle is to design receptors selective for Ni²⁺ ion over other biologically relevant metal ions.

In chemosensor 71, a BODIPY unit is covalently connected to a mixed N/O/S receptor to coordinate the Ni²⁺ cation. The receptor forms a 1 : 1 complex with Ni²⁺ with an approximate dissociation constant of (195 ± 5) × 10⁻⁶ M.¹⁰⁷ The probe gives a 25-fold fluorescence enhancement upon Ni²⁺ binding. This turn-on response is reversible as treatment of the Ni²⁺-loaded 71 with the metal ion chelator TPEN restores the fluorescence back to the free 71 level. Biologically relevant alkali, alkaline earth and transition metal ions except Cu²⁺ do not show any enhancement or interfere with the Ni²⁺ response. Cu²⁺ quenches the enhancement obtained with Ni²⁺ restricting its usefulness. Confocal microscopic studies show that this chemosensor can monitor changes in Ni²⁺ levels within living mammalian cells.

Rhodamine derived chemosensor¹⁰⁸ 72 show spiro-ring opened 200-fold fluorescence enhancement in presence of Co²⁺ and Ni²⁺ while other transition metal ions do not interfere in aqueous medium containing 2% DMSO. Both Co²⁺ and Ni²⁺ form 1 : 1 complexes with the association constants of 2.1 × 10⁴ and 4.5 × 10³ M⁻¹, respectively. Binding to both ions is reversible, as the metal can be extracted with EDTA with concomitant reduction in the fluorescence to the metal-free state. Since Co²⁺ competes fairly well with Ni²⁺, the detection of Ni²⁺ can be done only if Co²⁺ is not present.

The PET chemosensor 73 designed with coumarin attached to a piperazine moiety works in alkaline pH.¹⁰⁹ It affords a 7-fold enhancement in presence of large excess of Ni²⁺ ion. However, Zn²⁺ ion also gives a 10-fold enhancement and therefore, limits its utility.

The novel Zn²⁺ Schiff base complex 74 bearing acacen²⁻ (dianion of N,N′-ethylenebis(acetylacetonylideneaminato)) moieties have been constructed.¹¹⁰ The crystal structure of its dinickel complex shows that the two Ni²⁺ ions occupy the two metallacycles. In DMF, 74 gives an emission at 361 upon excitation at 293 nm. When Ni²⁺ is added, the structure becomes more rigid blocking its deactivation pathway and hence, a significant increase in the emission quantum yield is observed.

The malonohydrazide based PET fluorescent probe 75 forms a 1 : 1 complex in DMSO–water (1 : 1, v/v) with Ni²⁺ ion with the association constant of K_a = 3.3 × 10⁴ M⁻¹.¹¹¹ The free probe gives an emission peak at 629 nm on excitation at 317 nm which increases significantly and selectively in presence of Ni²⁺ ion (Scheme 39). The Ni²⁺ binding at the site is quite selective and strong due to good fitting of the metal ion in the binding pocket. The cause of fluorescence enhancement is not mentioned by the authors but it is more likely to be due to increased rigidity of the system that blocks the deactivation pathway.

Scheme 39 Possible metal ion binding mode of 75.

2.8. Copper chemosensors

Copper is the third-most abundant transition metal in the human body. Copper containing proteins are involved in a wide variety of biochemical functions¹¹² including copper transport (ceruloplasmin), copper storage (metallothionein), protective roles (Cu–Zn superoxide dismutase), terminal oxidase for O₂ respiration (cytochrome c oxidase) and so on. Copper ions are mainly metabolized by the liver. Copper in the cytoplasm predominantly binds to metallothionein, and the excess copper is excreted into the bile mainly through a lysosome-to-bile pathway.¹¹³ The level of copper in lysosomes is closely related to a number of neurodegenerative diseases, that include Wilson's disease,¹¹⁴ Menkes disease¹¹⁵ and Alzheimer's disease.¹¹⁶ Copper commonly accumulates in the lysosomes of patients suffering from these diseases. It is therefore, interesting to know the spatial distribution of copper in the biosystems. It follows, therefore, those sensors that can signal the presence of copper ion selectively is of high demand. However, some of the shortcomings for practical applications such as cross-sensitivities toward other metal cations and low fluorescence quantum yields are also to be kept in mind. Also, aqueous medium should be preferred for biological studies. Definitely, development of new Cu²⁺-selective turn-on fluorescent probes is of utmost importance and necessity.

In the PET chemosensor 76 ¹¹⁷ a N₂O donor receptor has been connected via Schiff base condensation to an electron-withdrawing naphthalimide derivative. It forms a highly stable 1 : 1 complex with Cu²⁺ (K_a = 1.1 × 10¹⁰ M⁻¹) in acetonitrile–water (70 : 30, v/v). Free 75 shows a weak emission at 519 nm due to an efficient PET from the receptor to the fluorophore (Scheme 40). Binding of Cu²⁺ stops the PET giving a 5-fold fluorescence enhancement. The detection limit is calculated to be 1.5 × 10⁻⁷ M while changes in fluorescence is observed within 10 s. Other biologically relevant metal ions show negligible enhancement or do not interfere with the Cu²⁺-induced emission.

Scheme 40 Proposed binding mode of 76 with Cu²⁺.

The PET chemosensor 77 incorporated with two thioether and three amine units has been designed to detect Cu²⁺ ion in CH₃CN–H₂O (4 : 1) at pH 7.¹¹⁸ Besides, the larger cavity size of the receptor has lowered the association constant for the 1 : 1 complex with Cu²⁺ to log K= 4.1 ± 0.1. Ni(II), Cd(II), Zn(II), Ag(I), and Hg(II) (1000 μM) do not interfere in the fluorescence studies. Significant participation of amine nitrogen in binding with Cu²⁺ ion is responsible for the enhancement of fluorescence due to suppression of the PET.

In addition to the PET chemosensors mentioned above, intramolecular charge transfer emission has also been used as a mechanism for Cu²⁺ detection. The chemosensor 78 has been synthesized by connecting 2-methoxybenzaldehyde hydrazone with 4-(N,N-dimethylamino)benzamide.¹¹⁹ The methoxy group rather than a phenolic group has been used to nullify excited-state intramolecular proton transfer (ESIPT) that would have opened another channel of deactivation. The metal ions Cu²⁺, Zn²⁺, Hg²⁺ and Pb²⁺ bind to the chemosensor with 1 : 1 stoichiometry to similar extent in the ground state as the binding constant of comparable value (order, 10⁴ M⁻¹) is obtained with each metal ion. In MeCN, 77 emits dual fluorescence, an excited state CT pertaining to the 4-(N,N-dimethylamino)benzamide group at 525 nm and a LE emission at 350 nm. A host of biologically relevant as well as heavy metal ions are used for probing the response of the CT band. These metal ions either quench the fluorescence or have no effect. Only Cu²⁺ exhibits a prominent 30-fold enhancement of the CT band with a substantial blue-shift from 525 to 460 nm. The fluorescence enhancement factor decreased with increasing water content in MeCN, but can still be significant even when the water content is 90% by volume. The low fluorescence quantum yield of 77 in absence of Cu²⁺ can be attributed to radiationless transitions from the nπ* state. Upon addition of Cu²⁺ the metal ion coordinates with the lone pair of the carbonyl O raising the energy level of the nπ* state such that the ππ* state becomes the lowest excited state, leading to a substantial increase in the fluorescence quantum yield. Besides, Cu²⁺ binding can also lead to a conformation restriction which can block a channel of the deactivation pathway.

The PET chemosensor 79 has been fabricated on a calix[4]arene platform by modifying its upper rim with four iminoquinoline subunits.¹²⁰ Metal free 79 shows a very low fluorescence quantum yield in MeCN due to an efficient PET from the imine N atoms. A Cu²⁺ ion coordinates the four imine N atoms with high association constant (K_a) of 3.67 × 10⁷ M⁻¹ to block the PET accompanied by about 1200-fold enhancement of fluorescence. While other transition metal ions do not bind at this site, alkali/alkaline metal ions are expected to bind at the lower rim to the ethereal O atoms without affecting fluorescence. While this is a simple design, its lack of solubility in water restricts its usefulness.

Like in the cases of other transition metal sensors, a number of turn-on sensors for Cu²⁺ has been designed based on rhodamine derivatives. The probe 80 has been synthesized by condensing two rhodamine B hydrazides with terephthalaldehyde. In aqueous medium, free 80 shows very weak emission in aqueous medium. Addition of Cu²⁺ ion gives a remarkably high fluorescence at 575 nm due to opening of spirolactam bond while other metal ions do not exhibit any significant emission (Scheme 41).¹²¹ Spectroscopic data suggest a 1 : 1 stoichiometry although no association constant value is provided.

Scheme 41 Proposed binding mode of 80 with Cu²⁺.

In chemosensor 81, a Cu²⁺ ion is anchored at the site shown in Scheme 42 leading to opening of the spirolactam as well as hydrolytic cleavage of the imine bonds giving high fluorescence.¹²² Since it works in aqueous medium, it affords imaging studies of different cell lines with confocal microscopy. However, the hydrolysis step is sluggish and takes at least 1 h and so this probe cannot be used for measuring temporal distribution of Cu²⁺ ion in biological systems.

Scheme 42 Proposed binding mode of 81 with Cu²⁺.

The chemosensor 82 is possibly the first example of a boronic acid-linked chemosensor for Cu²⁺ ion (Scheme 43).¹²³ The metal ion is believed to occupy the site as shown giving an association constant value of 2.8 × 10³ M⁻¹. When the boronic acid group is replaced by hydrogen, the Cu²⁺ ion does not elicit spiro-ring opened fluorescence enhancement. Thus, boronic acid moiety is important in binding a Cu²⁺ ion at the site. Free 81 forms a colourless solution in 50% aqueous MeCN showing very low emission. Upon addition of Cu²⁺ the colour changes to pink accompanied by spirolactam ring-opened fluorescence enhancement at 585 nm. Other biologically relevant metal ions do not give any fluorescence. Also, metal ion competition experiments show that binding of Cu²⁺ to the chemosensor is unaffected by other metal ions even when used in large excess. When an excess of cyclen is added to the solution containing the Cu²⁺ complex of 82, the fluorescence disappear and the original structure of the chemosensor is regenerated. Thus, in this case, Cu²⁺-induced spirolactam ring opening is a reversible process. Confocal microscopic experiments show that 81 can pass through cell membranes and displays a strong fluorescence response to intracellular copper ions in different mammalian cell lines.

Scheme 43 Proposed binding mode of 82 with Cu²⁺.

The rhodamine B based chemosensor 83 with a six-membered reactive ring was developed by inserting a nitrogen atom in the known fluorescent probe rhodamine B spiro thiohydrazide.¹²⁴ This sensor is shown to be an efficient “turn-on” fluorescent chemodosimeter for Cu²⁺ in a neutral aqueous medium due to opening of the spiro ring (Scheme 44). Other biologically relevant or heavy metal ions do not induce ring-opened fluorescence enhancement. Titration experiments reveal that the fluorescence intensity at 597 nm increases linearly with increasing concentrations of Cu²⁺ between 0.10 and 10.0 μM and further smoothly increase until a maximum is reached up to 20.0 μM Cu²⁺, where an approximately 400-fold enhancement is observed.

Scheme 44 Proposed sensing mechanism of Cu²⁺ by 83.

The chemosensor 84 has also been found to selectively bind Cu²⁺ ion with the association constant estimated to be 8.9 × 10³ M⁻¹ in aqueous MeCN (1 : 1, v/v). Upon binding of Cu²⁺, strong spiro-ring opened fluorescence enhancement is observed (Scheme 45)¹²⁵ while other competing metal ions do not bring any discernible fluorescence changes. Also, Cu²⁺ induced fluorescence of 84 is reversible as addition of excess EDTA completely quenches the fluorescence. Experiments with living cells indicate that the chemosensor is cell viable and can detect intracellular Cu²⁺ ion.

Scheme 45 Proposed binding mode of 84 with Cu²⁺.

Chemosensors 85 and 86 can be easily synthesized using click chemistry.¹²⁶ These compounds are non-fluorescent but with increase in pH in presence of Cu²⁺ lead to Fenton reaction promoting ring-opening of triazole linked fluorescein lactone (Scheme 46) that leads to strong emission with the detection limit down to 200 nM. Selectivity of this protocol has been evaluated by screening various biologically relevant metal ions that shows none of these metal ions can elicit fluorescence enhancement.

Scheme 46 Proposed mechanism for detection of Cu²⁺ by 85 and 86 based on the Fenton reaction.

The indole derivative of rhodamine 87 specifically binds to Cu²⁺ ion with an association constant of 1.71 × 10⁴ M⁻¹ in presence of large excess of other competing ions.¹²⁷ The metal-free dye emits at 490 nm when excited at 340 nm in aqueous MeCN due to the indole moiety. Binding of the Cu²⁺ ion breaks the spiro-bond of the rhodamine moiety (Scheme 47). At this stage, excitation at 340 nm results in FRET from the donor indole to the Cu²⁺ bound xanthene acceptor accompanied by a strong emission at 587 nm with concomitant reduction of the emission due to the indole moiety. The detection limit of 87 for Cu²⁺ ion is found to be 3.6 ppb, which is much lower than the U.S. EPA maximum allowable limit for Cu²⁺ ion (1.3 ppm) in drinking water. Besides, fluorescence microscopic studies confirmed that the chemosensor can be used as an imaging probe to study the uptake of Cu²⁺ in HeLa cells.

Scheme 47 Proposed binding mode of 87 with Cu²⁺.

The rhodamine B semicarbazide 88 show high spiro-bond opened fluorescence (Scheme 48) in aqueous methanol (1 : 1, v/v), exhibiting a fast response time (in seconds) and a detection limit of 1.6 × 10⁻⁷ mol L⁻¹ at neutral pH.¹²⁸ The quick response time allows determination of Cu²⁺ in waste water samples and also monitoring of intracellular Cu²⁺ levels in living cells using confocal fluorescence spectroscopy. The high selectivity of the chemosensor for Cu²⁺ ion might be due to close matching between the Cu²⁺ radius and the size of the binding pocket. However, no association constant value is available.

Scheme 48 Possible sensing mechanism of 88.

The strategy of metal induced bond breaking has been adopted for the coumarin-334 derived chemosensor 89.¹²⁹ This compound is non-emissive due to an efficient PET from the imine N atom. Addition of Cu²⁺ catalyzes breaking of the imine linkage to free the coumarin-334. Since the paramagnetic Cu²⁺ is not bound to the coumarin, it offers a strong fluorescence (Scheme 49). The detection limit for Cu²⁺ is found to be 8.7 × 10⁻⁸ M which is within the EPA (US) acceptable limit in drinking water. Another important property of the chemodosimeter is its high selectivity for Cu²⁺ over other metal ions. The competing metal ions do not catalyze hydrolysis of the imine bond.

Scheme 49 Cu²⁺-induced catalytic sensing cycle of 89.

BODIPY is an important fluorophore and has been used for several metal ions because of its visible excitation and emission characteristics. The chemosensor 90 based on BODIPY has been designed to have a receptor specific for Cu²⁺ ion.¹³⁰ To one arm of the fluorophore is also attached a p-diacetylene benzene moiety to extend conjugation of the system for emission at longer wavelength. Excitation at 620 nm in CH₃CN in presence of Cu²⁺ ion affords an emission with maximum at 651 nm.

Two more BODIPY derived chemosensors 91 and 92 are reported.¹³¹ Compound 91 gives large CHEF enhancement in presence of Pb²⁺ or Cu²⁺ among the metal ions examined. The association constants of 91 with Pb²⁺ and Cu²⁺ are found to be 8.8 × 10³ and 5.5 × 10⁴ M⁻¹ respectively. On the other hand, 92 exhibits absolute chemoselectivity for Cu²⁺ ion over other competing ions including Pb²⁺ and gives high fluorescence in presence of Cu²⁺ ion through hydrolysis of the acetyl groups (Scheme 50).

Scheme 50 Proposed sensing mechanism of chemosensor 92.

When the receptor contains several soft thioether donors as in 93, the BODIPY based system exhibits excellent selectivity towards the soft Cu⁺ ion¹³² giving about 10-fold turn-on response and shows excellent selectivity for Cu⁺ over biologically relevant metal ions, including Cu²⁺. Confocal microscopic studies indicate that this sensor can be used for detecting Cu⁺ levels within living cells.

A slightly different derivative of a BODIPY reporter with the same thioether-based receptor as above has been constructed to obtain 94 for selective detection of Cu⁺ ion over other biologically relevant metal ions, including Cu²⁺ and Zn²⁺.¹³³ Live-cell confocal microscopy experiments show that 94 is membrane-permeable and can sense changes in the levels of labile Cu⁺ pools within living cells by ratiometric imaging, including expansion of endogenous stores of exchangeable intracellular Cu⁺ triggered by ascorbate stimulation in kidney and brain cells. Such knowledge of spatial distribution of Cu⁺ is important to monitor physiology of this important metal.

The fluorescent probe 95 has been designed as a new type of targetable fluorescent chemosensor for imaging exchangeable mitochondrial copper pools in living cells.¹³⁴ The sensor has two distinctive sites: a Cu⁺-responsive fluorophore and a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. The fluorescence intensity of 95 increases 10-fold with a slight blue shift of the emission maximum to 558 nm with the addition of 1 equiv. of Cu⁺. The observed dissociation constant (K_d) value for Cu⁺ complex is found to be 7.2 × 10⁻¹² M. The fluorescence response of the Cu⁺ bound 95 is not affected by the presence of physiologically relevant concentrations of Ca²⁺, Mg²⁺, and Zn²⁺. Besides, other bioavailable divalent transition metal ions do not induce a change in the emission intensity of 95 or do not interfere with the Cu⁺ response. This PET sensor is found to be very useful in detecting labile Cu⁺ in the mitochondria of living cells.

Two-photon fluorescence is a preferable technique for imaging live cells. Two-photon microscopy has a number of advantages over one photon microscopy including increased penetration depth (4500 mm), localized excitation and reduced photodamage and photobleaching effects, thereby allowing tissue imaging for a longer period of time. Besides, no interference from natural amino acid fluorophores occurs which allows for sharp images. The chemosensor 96 has been designed with the same receptor as above for binding a Cu⁺ ion inside. The Cu⁺ complex of 96 affords a TPA (two-photon absorption) value of 67 GM at 750 nm.¹³⁵

The chemosensor 97 is based on coumarin fluorophore that displays high selectivity for Cu²⁺ in aqueous medium¹³⁶ with high emission quantum yield due to blocking of PET from the imine N atom to the fluorophore with association constant K_a = 3.34 × 10⁴ M⁻¹. Its operation in aqueous medium allows for microscopic imaging in LLC-MK2 cells (in vitro) and several living organs (in vivo).

The weakly fluorescent thiosemicarbazone derivative 98 was found to be a selective optical and “turn-on” fluorescent chemodosimeter for Cu²⁺ ion in aqueous ethanolic medium.¹³⁷ It is argued that Cu²⁺ selectivity results from an oxidative cyclization of 98 into the highly fluorescent rigid 4,5-dihydro-5,5-dimethyl-4-(naphthalen-5-yl)-1,2,4-triazole-3-thione (Scheme 51). The chemodosimetric reaction, in combination with selective metal ion-induced catalysis, can be a promising approach to develop selective detection methods for different metal ions.

Scheme 51 Proposed oxidative cyclization mechanism of 98 by Cu²⁺.

A coumarin moiety is covalently attached on either side of benzyl dihydrazone to have the sensor 99.¹³⁸ Upon excitation at 468 nm, the aqueous solution of the metal-free sensor gives an emission at 534 nm with very low quantum yield due to efficient PET from the hydrazone moiety to the coumarin fluorophore association constant (K_a) 3.67 × 10⁶ M⁻¹. Upon addition of Cu²⁺ it undergoes conformational change as shown (Scheme 52) and at the same time, blocks both the PET pathways to afford a strong fluorescence. This system functions in aqueous medium and therefore, is suitable for use in live cell imaging studies.

Scheme 52 Proposed binding mode of 99 with Cu²⁺.

In designing the PET sensor 100, a tricarbocyanine dye is used as the NIR fluorophore and 2,2′-azanediyl bis(N-hydroxyacetamide) as the Cu²⁺ receptor.¹³⁹ The specificity of Cu²⁺ binding at the receptor is accomplished due to matching of the cavity of the binding site with radius of Cu²⁺ ion (Scheme 53). The metal-free system shows very weak fluorescence, however, in presence of Cu²⁺ ion, the PET is blocked giving high fluorescence at 800 nm in aqueous medium with a dissociation constant of 2.7 ± 0.3 μM in buffered aqueous medium. The emission maximum lies within the biological window and more importantly, aqueous medium can be used both of which make this chemosensor quite important.

Scheme 53 Proposed binding mode of 100 with Cu²⁺.

The chemosensor 101 obtained by derivatizing the upper rim of a thiacalix[4]arene by a crown ether and the lower rim by two pyrene moieties.¹⁴⁰ Upon addition of Cu²⁺ in aqueous organic medium, two Cu²⁺ ions are believed to occupy the sites as shown (Scheme 54) with the binding constant >10¹¹. This makes the two pyrene moieties well-separated and offering only monomer emission due to blocking of PET. However, addition of Li⁺ ion which occupies the crown ether cavity leads to ejection of the Cu²⁺ ions. This brings the two pyrene moieties closer for π-stacking giving an excimer emission.

Scheme 54 Proposed binding mode of 101.

The electron-withdrawing fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) derived chemosensor 102 binds a Cu²⁺ ion at the pocket shown (Scheme 55).¹⁴¹ This mode of binding by the Cu²⁺ ion blocks the PET recovering fluorescence in aqueous MeCN.

Scheme 55 Proposed binding mode of 102.

In an elegant approach, a sensor system that separates the sensing and the signaling events have been designed. Two different fluorogenic ligands 103 and 104 are capable of metal chelation.¹⁴² The system functions as illustrated below (Scheme 56).

Scheme 56 Schematic representation of a ratiometric Cu²⁺ sensor system.

In presence of 1 equivalent of Cd²⁺, it preferably chelates with 103 giving strong emission centering at 435 nm due to stoppage of PET. When an equivalent amount of Cu²⁺ is added it displaces Cd²⁺ from 103 leading to fluorescence quenching from the paramagnetic ion. However, Cd²⁺ is now bonded to 104 giving strong emission at 518 nm again due to blocking of PET. The entire operation can be performed in aqueous media in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer at pH 7.2. This way, detection of Cu²⁺ is indirect.

The chemosensor 105 acts as an ICT based ratiometric and colourimetric fluorescent chemosensor selective for Cu²⁺ ion (Scheme 57).¹⁴³ A Cu²⁺ ion binds fairly strongly at the site shown with an association constant estimated to be 2.58 × 10⁴ M⁻¹. Upon addition of Cu²⁺, the metal free absorption band shifts from 328 nm to about 540 nm due to deprotonation as shown with the disappearance of the ICT band centering at 496 nm and the appearance of a new band at 620 nm.

Scheme 57 Proposed binding mode of 105 with Cu²⁺.

The quinoline-indene derived PET chemosensor 106 was found to be sensitive towards Cu²⁺ ion in 1 : 1 aqueous MeCN giving a 5-fold enhancement of fluorescence (Scheme 58).¹⁴⁴ The receptor cavity binds a Cu²⁺ ion with an association constant estimated to be (log K) 6.48 ± 0.48.

Scheme 58 Proposed binding mode of 106 with Cu²⁺.

Two 4,5-disubstituted-1,8-naphthalimide derivatives 107 and 108 have been synthesized as Cu²⁺ ion sensors.¹⁴⁵ The free sensor 107 displays a broad emission centering at 534 nm in aqueous medium. Upon addition of Cu²⁺ this band reduces in intensity with the appearance of a new band centered at 478 nm due to complex formation with the probe. Compound 108 on the other hand, quenches emission of the free probe although colour of the solution changes sharply from yellow to pink.

The compound 109 is non-emissive although in presence of Cu²⁺ ion,¹⁴⁶ it undergoes oxidative cyclization (Scheme 59) to form a highly fluorescent benzotriazole. This cyclization reaction can be triggered by μM level concentration of Cu²⁺ in water at pH = 6–8, at room temperature. The cyclic product exhibits a green emission at 530 nm.

Scheme 59 Cu²⁺ ion induced oxidative cyclization of 109.

The receptor in the fluorescent probe 110 can accommodate a Cu²⁺ ion with a high association constant of 2.95 × 10⁵ M⁻¹. Once the metal ion occupies the site, the two naphthalimide moieties come closer to each other. Upon addition of 1 equiv. of Cu²⁺ ion, the two naphthalimide moieties come closer giving an excimer emission at 558 nm. Further addition of another equiv. of Cu²⁺, triggers a change in the conformation of the chemosensors where the two naphthalimide groups are move far apart from each other giving a naphthalimide monomer emission at 461 nm (Scheme 60).¹⁴⁷

Scheme 60 Proposed binding mode of 110 with Cu²⁺.

The chemosensor 111 has been designed based on the strong tendency of the pyrene moiety to form intermolecular dimers.¹⁴⁸ The free sensor exhibits pyrene monomer emission at 388 nm upon excitation in acetonitrile solution. Upon addition of Cu²⁺ ion, the monomer emission is partially quenched but a strong excimer emission at 460 nm is observed. It is argued that interaction of the quinolinylamide group with the Cu²⁺ ion induces an intermolecular π–π* interaction which is responsible for the fluorescence changes. With increasing the length between the pyrene and quinolinylamide moieties, excimer formation is hindered.

The thiophene based Schiff base 112 was found to be highly selective fluorescence turn-on PET chemosensor for Cu²⁺ in aqueous medium.¹⁴⁹ It affords a high binding constant of 4.23 × 10⁵ M⁻¹ in aqueous medium with the detection limit of 0.418 nM. The sensor gives a 35-fold enhancement at 430 nm in presence of Cu²⁺ when excited at 370 nm. In presence of Cr³⁺ and Sn²⁺ the enhancement maximum is obtained at 465 nm while other metal ions do not interfere. This probe can be used for studying Cu²⁺ ion in the living cell by confocal microscopy.

The fluorescent probe 113 was easily synthesized via Schiff base condensation as a chemosensor for Cu²⁺.¹⁵⁰ The free probe does not give any emission when excited at 400 nm in acetonitrile due to an efficient PET. Upon addition of Cu²⁺ it affords 120-fold fluorescence enhancement at 468 nm as the PET process is blocked. The N and S atoms provide a binding scaffold for recognition of Cu²⁺ (Scheme 61) with fairly high affinity (K_a = 4.35 × 10⁴ M⁻¹). Other competing metal ions do not interfere. The simplicity and elegance of the probe design should spur further research for water soluble signaling systems for biological applications.

Scheme 61 Possible sensing mechanism of 113.

The coumarin derived fluorescent probe 114 is found to be highly selective for Cu⁺ ion.¹⁵¹ The benzylic ether bond of the probe can be cleaved selectively in presence of Cu⁺ under a physiological condition to release the coumarin derivative that is highly fluorescent (Scheme 62). Other competing metal ions do not give any fluorescence as they cannot cleave the benzylic ether bond.

Scheme 62 Possible sensing mechanism of 114.

For monitoring real-time copper fluxes in living animals and thus offers an opportunity to examine copper physiology, the chemosensor 115 is designed.¹⁵² It consists of a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to Cu⁺. This probe with negligible cytotoxicity is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. This new generation sensor is definitely expected to spur research on sensors for monitoring fluctuations in exchangeable metal ions in biosystems.

3. Conclusions

It is now clear that several design principles have been adopted for signalling the presence of first-row transition metal ions through turn-on fluorescence. The nature of the fluorophore assumes great significance. It is often found that PET/PCT based sensors often lead to quenching of fluorescence. Of all the designs, rhodamine derived sensors are particularly attractive as they are capable of overcoming fluorescence quenching by paramagnetic transition metal ions. Several key issues still remain in the fluorescence detection of biologically relevant first-row transition metal ions. Most important being aqueous solubility of the probe and a very quick response with high selectivity and sensitivity for temporal distribution with high fidelity of the metals in live cells. Of course, cell viability of the sensor is an important parameter that should always be kept in mind. For live cell imaging studies, systems that show two-photon fluorescence are of crucial importance as they can nullify the background effect of natural fluorophores. Finally, availability of variable oxidations states of transition metals have not been exploited in designing chemosensors. Thus, despite considerable advances, this subject still requires a lot of attention from chemists to purposely build fluorescence signalling probes.

Acknowledgements

Financial support for this work is provided by the DST, New Delhi, India and DRDO, New Delhi, India. SP and NC wish to thank CSIR for the senior research fellowship. PKB wishes to thank all his students who made significant contributions in the area of fluorescence signalling and fluorescence based logic gates over the years.

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