Carboxylated ‘locking unit’ directed ratiometric probe design, synthesis and application in selective recognition of Fe3+/Cu2+

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http://dx.doi.org/10.1039/c3ra23442h

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1

Carboxylated ‘locking unit’ directed ratiometric probe design, synthesis and application

in selective recognition of Fe3+/Cu

2+

Sougata Sinha,a Sunil Kumar,a Rik Rani Koner,a Jomon Mathew,b Chayan K. Nandi,a,*

Subrata Ghosh a,*

aSchool of Basic Sciences, Indian Institute of Technology Mandi, Mandi-175001, H.P, India

bSchulich Faculty of Chemistry and the Lise Meitner Minerva Center for Computational

Quantum Chemistry, Technion-Israel Institute of Technology, Haifa, Israel

*Corresponding authors: Tel. +91-1905-237917; Fax: +91-1905-237924 (C. K. Nandi)

Tel. +91-1905-237926; Fax: +91-1905-237924 (S. Ghosh)

E-mail address: [email protected] (C. K. Nandi)

[email protected] (S. Ghosh)

†Electronic supplementary information (ESI†) available: Experimental procedures, additional

spectral data and scanned copies of NMR spectra are available.

Abstract: A ratiometric probe has been designed and synthesized through introduction of a

carboxylated functionality. The probe 1, synthesized in two steps, was found to be a

fluorescence chemosensor for selective detection of Fe3+/Cu2+ ratiometrically over a wide

spectrum of metal ions quite efficiently. The carboxylated unit, ‘CH2COOH’, acts as a

‘locking unit’ restricting the possibility of E/Z isomerization and ESPT. This has been

supported by the theoretical studies. The selectivity was established using various

spectroscopic techniques like UV-vis and fluorescence spectroscopy. The probe was found to

have fluorescence property with quantum yield 0.18. The binding of this fluorescent receptor

with metal ions was also proved by NMR and mass spectrometry. The present probe can

efficiently detect the presence of very low concentration of Fe3+/Cu2+ (0.51 and 4.46 µM of

Cu2+ and Fe3+ respectively).

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Due to their several advantages like operational simplicity, high sensitivity etc. over

traditional and expensive tools like atomic absorption spectroscopy, inductively coupled

plasma-mass spectrometry and inductively coupled plasma-atomic emission spectrometry,1,2

fluorescence chemosensors have been the choice of chemical devices in the last few decades

for selective detection of biologically and environmentally important heavy and transition

metal ions.3-10 Among various types, ratiometric fluorescence chemosensors (RFCSs) are

drawing special attention of scientific community due to their interesting unique spectral

behavior. In general, the RFCSs offer a number of advantages such as different types of

signaling behavior at different wavelengths, built-in correction for general environmental

effects etc.11-14

While iron occupies the number one position amongst the most abundant heavy metal ions

present in human body, zinc and copper occupy second and third positions respectively. This

in turn clearly indicates the importance of iron and copper in regulating large number of basic

and essential biologically processes in living systems. While both of them at low

concentration act as catalytic cofactors for a number of metalloenzymes in living

organisms,15-18 high concentration of iron and copper can create disorders like Alzheimer’s,

Parkinson’s, Menkes, and Wilson diseases.19,20-23

Therefore, enough efforts have been directed to develop novel efficient chemosensors for

selective recognition of Fe3+/Cu2+.24-37 As Fe3+ and Cu2+ are well known fluorescence

quencher because of their paramagnetic nature, the development of fluorescent probes which

can selectively detect Fe3+/Cu2+ following ‘turn on’ mechanism particularly ratiometrically

are always desirable. 38-47

Since its discovery in 1864 by Hugo Schiff, 48 the Schiff bases have found wide applications

in the field of synthetic organic chemistry, biochemistry, petrochemical industry and in

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catalysts development.49,50 In recent years, and due to their easy synthesis the Schiff base-

based chemosensors are attracting the eyes of scientific community.51-60 Generally, the E/Z-

isomerisation make them non-fluorescent or weakly fluorescent. The binding of the Schiff

bases with metal ions through imine nitrogen restricts the isomerisation which in turn leads to

the enhancement of the fluorescence property of the Schiff bases.51,52,55,56,58-60 Therefore,

fluorescent imine preparation is still remain challenging. In this article, and in continuation of

our effort towards the development of chemosensors for selective detection of

environmentally and biologically important metal ion(s),34,60 we report a smartly designed

easy synthesizable novel carboxylated carbazole-based fluorescent Schiff base 1 as a

ratiometric chemosensor for Fe3+/Cu2+ through the introduction of carboxylic functionality as

‘locking unit’ which actually locks the E/Z isomerization through hydrogen bonding. To the

best of our knowledge, there is no report on carboxylic functionality directed synthesis of

fluorescent imine. That the fluorescence property generates due to incorporation of

‘CH2COOH’ functionality has been proved by synthesizing analog 5. The chemosensing

ability of this fluorescent receptor was tested over twenty different metal and non-metal ions,

and established using various spectroscopic techniques.

Base mediated reaction between salicylaldehyde 2 and chloroacetic acid yielded (2-formyl-

phenoxy)-acetyl acid 3 as white powder 61 (Scheme 1). Compound 1 was prepared by the

condensation between (2-formyl-phenoxy)-acetyl acid (3) and 3-amino-9-ethyl carbazole (4)

(Scheme 1). The product was isolated by filtration, washed with methanol, and finally

obtained as orange-red solid. The compound 5 was synthesized by the condensation reaction

between salicylaldehyde 2 and 3-amino-9-ethyl carbazole (4) and obtained as yellow powder.

Sodium borohydride was used as reducing agent to reduce the imine 5 to the corresponding


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amine 6. Common spectroscopic techniques such as FT-IR, NMR and mass spectrometry

were used to characterize all the intermediates and final products.

CHO

OH

O

CHO

MeOH, 70 C, 30 min, 70%

O OH

O

N

N

N

NH2

1

O OH

Reflux, 4 h, 60%

ClCH2COOH, NaOH, H2O

°

23

4N

NH2MeOH, rt, 30 min, 80%

OH

N

N5

OH

HN

N

6

NaBH4

MeOH, rt, 75%

4

Scheme 1. Syntheses of compounds 1, 5 and 6

The selectivity and sensitivity of compound 1 against different metal ions were investigated

using various spectroscopic techniques. The absorption spectral features of compound 1 in

acetonitrile/dichloromethane (7:3, 10 µM) showed four peaks (Fig. 1) at 372 nm (ϵ = 7.9 x

103 M-1 Cm-1), 307 nm (ϵ = 2.42 x 104 M-1 Cm-1), 299 nm (ϵ = 2.15 x 104 M-1 Cm-1) and at

279 nm (ϵ = 7.9 x 104 M-1 Cm-1). These bands arose mainly due to π-π* and n-π* transition

of the aromatic rings. Strong and distinct spectral changes were noticed in the presence of

Fe3+ and Cu2+. Addition of Fe3+ induced 12 nm blue shift in 372 nm absorption band (ϵ =

5.69 x 104 M-1 Cm-1), 7 nm red shift in 307 nm absorption band (ϵ = 5.78 x 104 M-1 Cm-1)

and very little blue shift in 299 nm absorption peak (ϵ = 6.08 x 104 M-1 Cm-1) (Fig. S1). In

the presence of Cu2+ a different spectral feature was observed in 372 nm absorption band. A

new broad band appeared at around 420 nm which was possibly a 76 nm red shift of the 372

nm band. A 10 nm red shift was observed for the 307 nm band (Fig. S2). These spectral

changes of compound 1 could easily distinguish Fe3+ over Cu2+. Fluorescence study was

explored extensively to establish the selectivity and sensitivity of compound 1. Compound 1


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itself was fluorescent in acetonitrile/dichloromethane (7:3) with quantum yield 0.18 (0.5 M

H2SO4 solution of quinine was used as standard). Figure 2 shows emission spectra of 1 (10

µM) and 1 in the presence of several metal ions (λex = 297 nm and λem = 426 nm).

Figure 1. UV-vis absorption spectra of compound 1 (10 µM), and 1 in the presence of 20 µM

Fe3+ and Cu2+ in acetonitrile/dichloromethane (7:3) mixture.

Figure 2. Emission spectrum of compound 1 (10 µM), and in the presence of 20 different

metal and non-metal ions (15 µM Fe3+, 6 µM Cu2+, 20 µM other cations) in

acetonitrile/dichloromethane (7:3) mixture, (λex =297 nm)


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The fluorescence property of 1 could be explained from the fact that due to the incorporation

of COOH functionality into the imine scaffold there is a possibility of hydrogen bonding

between the hydrogen of COOH functionality and imine nitrogen restricting the possibility of

E/Z isomerization. This has also been proved by theoretical studies. The optimized structure

(Fig. 3a) of 1 shows that the H-bonding interactions of the hydrogen of COOH group with

imine nitrogen (N-H distance is 1.88 Å) and with phenoxy oxygen (O-H distance is 2.05 Å)

are prominent and stabilizes the conformation. In addition, the incorporation of the

CH2COOH unit into the imine scaffold also restricted the possibility of excited state proton

transfer (ESPT) 62 which ultimately led to the generation of fluorescence property. To support

this conclusion, we synthesized compound where the imine scaffold doesn't contain

CH2COOH (Scheme 1). As expected, compound 5 didn't show any fluorescence property

proving that due to ESPT the compound 5 become non-fluorescent with the opening of

quenching channels (Fig. S3b) Interestingly, and to prove this further, when compound 5 was

converted to compound 6 by sodium borohydride reduction (Scheme 1) to rule out the

possibility of ESPT, the compound 6 was found to be fluorescent (Fig. S4b). These

experiments indicated that, the CH2COOH functionality, as a ‘locking unit’, was the key in

developing fluorescence property of 1 by locking both E/Z isomerization and ESPT.


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1.882.05

(a)

1.862.01

1.82

1.99

1.81

2.11

(b) (c)

Figure 3. Optimized structures of the (a) 1, (b) 1-Cu2+ complex, (c) 1-Fe3+ complex at

B3LYP/6-311G(d,p) level of theory.

The addition of 20 µM of various metal and non-metal ions to the solution of 1 resulted in

almost no changes except for Fe3+ and Cu2+ (Fig. 2). Interestingly, the addition of 15 µM Fe3+

resulted in a ratiometric phenomenon leading to new fluorescence emission bands at 361 nm

and 378 nm along with strong quenching of fluorescence at 426 nm emission (Fig. 2). To

better understand these spectral changes, a ratiometric fluorescence titration was performed

with varied concentration of Fe3+. Fluorescence titration profile clearly showed that

successive addition of Fe3+ to the solution of compound 1 resulted in gradual quenching of

emission at 426 nm with a very little blue shift and strong fluorescence enhancement at 361

nm and 378 nm, and ultimately reached to saturation (Fig. 4a). Further addition of Fe3+


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beyond a certain concentration (15 µM) led to little quenching of fluorescence emission at

361 nm and 378 nm. Figure 4b indicates a good linearity in between 4 µM to 13 µM of Fe3+

for I378 signal.

Figure 4. (a) Fluorescence titration spectra of compound 1 (10 µM) in the presence of 0-15

µM of Fe3+ in a mixture of acetonitrile/dichloromethane (7:3). (b) Fluorescence intensity of

compound 1 as a function of Fe3+ concentration, (λex = 297 nm).

Similar spectral features were observed upon gradual addition of Cu2+ except for the

generation of a new peak at 520 nm with very low intensity (Fig. 2 & Fig. 5a). As this is

reported that the charge transfer (CT) band is affected when the metal ion comes closer to the

fluorophore,63 the appearance of peak at 520 nm possibly was due to ICT process. From

theoretical studies (Fig. 3), it has been observed that the distance between the center of the

five membered ring of carbazole unit and Cu2+ in 1-Cu2+ complex is 5.85 Ǻ, whereas the

distance is 6.15 Ǻ in case of 1-Fe3+ complex. In addition, the distance between carbazole

nitrogen and Cu2+ is 6.33 Ǻ, and between carbazole nitrogen and Fe3+ is 6.64 Ǻ. These

theoretical data indicate that in comparison to Fe3+, Cu2+ is closer to the fluorophore

carbazole. Therefore, upon binding with Cu2+, the CT band was highly affected and as a


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result a weak band at higher wavelength, 520 nm, was observed. However, and in comparison

to Fe3+ only 6 µM of Cu2+ was sufficient enough to bring these spectral changes. The addition

of Cu2+ beyond this concentration led to some extent of quenching of emission at 361 nm and

376 nm. Figure 5b shows good linearity for I376 signal in between 1-6 µM of Cu2+. From

these fluorescence data it could be concluded that compound 1 was more sensitive towards

Cu2+ than Fe3+. The detection limits of 1 for Cu2+ and Fe3+ were calculated following reported

methodology.64 Our data (Fig. S5) suggested that compound 1 could detect as low as 0.51

µM of Cu2+ and 4.46 µM of Fe3+. These data also indicate the high sensitivity and efficiency

of the probe.

Figure 5. (a) Fluorescence titration spectra of compound 1 (10 µM) in the presence of 0-7

µM of Cu2+ in a mixture of acetonitrile/dichloromethane (7:3). (b) Fluorescence intensity of

compound 1 as a function of Cu2+ concentration, (λex = 297 nm).

The binding constants were calculated following well-known Benesi-Hildebrand equation,65

and were found to be 5.05 x 105 M-1 for 1-Cu2+ complex and 1.2 x 105 M-1 for 1-Fe3+

complex (Fig. S6). The 1:1 complex formation between 1 and Fe3+/Cu2+ was established by

Job’s plot66 (Fig. S7). This was further verified by ESI-mass spectra (Fig. S8). While the

addition of Fe3+ to the solution of 1 showed a peak at m/z 608.6 (calculated value 608.1,


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corresponds to [1+Fe+6H2O+2Cl+2H]+) (Fig. S8a), the addition of Cu2+ resulted in a less

intense peak at m/z 507.34 (calculated value 507.03, corresponds to [1+ CuCl2 + 2H]+) and

an intense peak at m/z 416.34 (calculated value 415.99, corresponds to [1+ CuCl2 + H-

OCH2COOH - CH3]+) (Fig. S8b).

Selectivity of compound 1 towards Fe3+/Cu2+ was checked in the presence of excess amount

(100 µM ) of wide range of other metal and non-metal ions including alkali, alkaline earth,

transition and heavy metal ions (Fig. S9a, S9b). These experiments revealed an excellent

selectivity of compound 1 towards Fe3+/Cu2+.

The recognition of Fe3+/Cu2+ by 1 was rechecked by reversible binding experiment (Fig. 6).

In the presence of little excess of strong chelating agent ethylenediamine, the ratiometric

fluorescence signal developed by the addition of Fe3+/Cu2+ reverted back to its original signal

of compound 1.

Figure 6. Fluorescence recognition profile of compound 1 (10 µM) towards Fe3+ and Cu2+ in

the presence of strong chelating agent ethylene diamine.


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Finally, and as another important spectroscopic tool to study the interaction of compound 1

with Fe3+/Cu2+ the NMR titrations were performed. In 1H NMR spectrum of 1 the CH=N

proton showed a sharp peak at 9.12 ppm. The addition of 0.5 equivalent of Cu2+ led to a

broadening with 0.03 ppm downfield shift of the CH=N peak. Further addition of another 0.5

equivalent of Cu2+ led to additional downfield shift of 0.06 ppm of the CH=N peak (Fig. 7).

Similarly, the addition of 0.5 equivalent of Fe3+ led to a 0.19 ppm downfield shift CH=N

peak (Fig. 8) (further addition of Fe3+ created difficulty in recording spectra). All these results

clearly indicated that the interaction of imine nitrogen with Fe3+/Cu2+ resulted in downfield

chemical shift of CH=N proton.

Figure 7. 1H NMR titration of compound 1 in the presence of different equivalents of Cu2+


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Figure 8: 1H NMR titration of compound 1 in the presence of Fe3+

Theoretical studies on 1-Cu2+ and 1-Fe3+ complexes further support the strong binding of

Cu2+ and Fe3+ with imine nitrogen. The optimized the geometries of 1-Cu2+ and 1-Fe3+

complexes at B3LYP/6-311G(d,p) level of density functional theory are depicted in Fig. 3b

and 3c. The anionic form of 1 is acting as tridendate ligand with three binding sites; imine

nitrogen, COO- and phenoxy oxygen. Both Cu2+ and Fe3+ strongly interact with imine

nitrogen as the Cu-N bond length is 1.86 Å and Fe-N bond length is 1.99 Å. The stabilization

of 1-Cu2+ and 1-Fe3+ complexes by the interaction of Cu2+ and Fe3+ with COO- and phenoxy

oxygen are also not trivial since the Cu-O bond lengths are 1.82 Å and 2.01 Å while Fe-O

bond lengths are 1.81 Å and 2.11 Å.

We further investigated the possibility of using the same probe for ratiometric detection of

Fe3+/Cu2+ in aqueous/semi-aqueous solution. Unfortunately, the probe 1 lost its ratiometric


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sensing property in the presence of water. The quenching in the emission of 1 at 426 nm was

observed upon the addition of Fe3+/Cu2+ in a combination of CH3CN/H2O (98:2) and no

ratiometric peak was generated. The quenching effect of Cu2+ was stronger than Fe3+.

Therefore, the use of water, either alone or in combination with organic solvent, was not

encouraging. Though the probe did not show any ratiometric sensing property in the presence

of water, our synthesis strategy may be useful to develop structurally modified ratiometric

molecular probe containing carboxylated locking unit which might work efficiently in

aqueous or semi-aqueous solution.

To conclude, we have designed and synthesized in two steps, with the incorporation of a

carboxylated 'locking unit', a novel carbazole-based chemosensor which can selectively

detect Fe3+ and Cu2+ in the presence of wide spectrum metal ions by ratiometric fluorescence

signalling. As the incorporation of carboxylated unit restricts the possibility of both E/Z

isomerization and ESPT, the imine 1 becomes fluorescent and thus ultimately makes it a

ratiometric probe as the interaction with Fe3+/Cu2+ leads to a different flourescent signaling.

We believe this approach of synthesis of fluorescent imine through introduction of carboxylic

functionality as ‘locking unit’ will be a new addition to the literature. The sensitivity and selectivity

were tested over 20 metal and non-metal ions, and established using various spectroscopic

tools. This novel chemical device could efficiently detect as low as 0.51 and 4.46 µM of Cu2+

and Fe3+ respectively.


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Acknowledgments

The work was funded by IIT Mandi (Grant No. IITM/SG/SUG/010). SS and SK are grateful

to IIT Mandi for their fellowship. RRK is thankful to the Department of Science and

Technology (DST) for her fellowship (Grant No. SR/FT/CS-57/2010(G)). We thank the

Reviewers for their valuable comments. We thankfully acknowledge the Director, IIT Mandi

for research facilities.


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Graphical Abstract:

“Carboxylated ‘locking unit’ directed ratiometric probe design, synthesis and

application in selective recognition of Fe3+/Cu

2+”

Sougata Sinha, Sunil Kumar, Rik Rani Koner, Jomon Mathew, Chayan K. Nandi,

Subrata

Ghosh

A ratiometric probe has been designed and synthesized through introduction of a

carboxylated functionality and ultimately applied for selective recognition of Fe3+/Cu

2+


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Documents

Carboxylated ‘locking unit’ directed ratiometric probe design, synthesis and application in selective recognition of Fe3+/Cu2+