CHEF-Affected Fluorogenic Nanomolar Detection of Al3+ by an Anthranilic Acid-Naphthalene Hybrid: Cell Imaging and Crystal Structure.

We report the synthesis of a novel hydrazine-bridged anthranilic acid-naphthalene conjugate (E)-2-(benzamido)-N'-((2-hydroxynaphthalen-1-yl) methylene) benzohydrazide (BBHAN) and its crystal structure. BBHAN can detect Al3+ by a sharp increment in fluorescence intensity (∼40 times) in aqueous methanolic medium. The limit of detection of BBHAN towards Al3+ is 1.68 × 10-9 M, and the former undergoes a 2:1 binding with Al3+ with a high binding constant of ∼1.15 × 1011 M2-. BBHAN detects Al3+ by the well-known mechanism of chelation-enhanced fluorescence (CHEF), established by fluorescence time-resolved measurement. The mode of interaction between BBHAN and Al3+ has been explored by 1H NMR and electrospray ionization mass spectrometry techniques. Paper strips coated with BBHAN can detect Al3+ under UV light observed through the naked eye. Lastly, BBHAN can detect Al3+ in MDA-MB-468 cells.

Introduction

Aluminum is the third most abundant element on earth’s crust following oxygen and silicon.[1,2] Due to high availability of aluminum, it finds widespread use in preparation of cooking/storage utensils, cosmetics as well as pharmaceutical products.[3] Being a good conductor of electricity, it is used for the manufacture of electric wires, and various electrical and electronic appliances.[4−6] This widespread use of aluminum results in its slow consumption in the human body in its tripositive form, resulting in many toxic effects, such as kidney damage,[7] Parkinson’s and Alzheimer’s diseases, and even cancer.[8−14] Aluminum affects the absorption of calcium in bowel causing softening of the bone and can also cause anemia by hindering the absorption of iron in blood.[15−17] At high concentrations, Al3+ is equally detrimental to fish, algae, bacteria, and other species in aquatic ecosystems.[18−20] Due to such toxic effects exerted by Al3+, the World Health Organization (WHO) has set the upper limit for weekly dietary intake of aluminum for human to be 7 mg/kg of body weight.[21] Hence, selective and sensitive detection of Al3+ by cheap and convenient methods is of utmost importance. Detection of Al3+ ion has always been a challenging task due to the poor coordinating aptitude of the cation along with the interference of similar-sized Zn2+ ions during its detection.[22,23] Commonly employed methods for sensitive detection of Al3+ include atomic absorption spectrometry[24] and inductively coupled plasma mass spectroscopy (ICP–MS).[25] As both processes are expensive and time-consuming, Schiff base derivatives rich in oxygen and nitrogen donor sites for selective fluorogenic detection of Al3+ are gaining prominence and reported in large numbers frequently.[26−32] However, most of them suffer from the problems of poor detection limit and multiple complicated synthetic steps. Thus, reports of synthetic chemosensors that are derived from extremely cheap starting materials and also having low detection limits are scarce. Our group has been involved in designing sensitive optical chemosensors for detecting various detrimental analytes. Recently, we have reported competition-free fluorogenic detection of Al3+ by a chromone–naphthalene conjugate up to ∼10–8 M.[33] In the current work, we report yet another selective Al3+ sensor (E)-2-(benzamido)-N′-((2-hydroxynaphthalen-1-yl) methylene) benzohydrazide (BBHAN), which is derived from anthranilic acid (AA) and 2-hydroxy naphthaldehyde (HN12). Aluminum sensors derived from HN12 show considerably low detection limits as well as appreciable turn-on fluorescence in the presence of Al3+.[34,35] The involvement of anthranilic acid in the metabolism of tryptophan as well as nicotinamide and its presence in various alkaloids and plant signaling compounds, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA)[36] and indole-3-acetic acid,[37,38] make it biologically interesting. This study on BBHAN is the first report of an Al3+ sensor constituting anthranilic acid and HN12. In aqueous methanolic medium, it shows a ∼40 fold increment in its fluorescence intensity in the presence of Al3+ and is completely unaffected by similar metal ions, such as Ca2+, Zn2+, Mg2+, Cr3+, etc. The detection limit for Al3+ is 5.49 nM. Such a low detection limit proves the efficacy of BBHAN, which is even 10 times lower than that in our previous report.[33]BBHAN undergoes a 2:1 binding with Al3+ as evidenced from the electrospray ionization mass spectrometry (ESI-MS) study and Job’s plot. Moreover, BBHAN is able to detect intracellular Al3+ in MDA-MB-468 cells. For rapid assessment of Al3+, successful paper strip experiments have been performed. Lastly, the single-crystal structure analysis of BBHAN shows the presence of a dimethyl sulfoxide (DMSO) molecule attached to the former by hydrogen bonding. Several nonbonding interactions along with hydrogen bonding help BBHAN attain a supramolecular sheet. Hence, BBHAN stands out among other Schiff base derivatives owing to the very low detection limit, multipurpose practical utility and its unique supramolecular architecture.

Results and Discussion

The single crystal of BBHAN obtained was solved using SHELXS 97, PLATON 99, ORTEP-32, and WinGX system ver 1.64.[39]BBHAN was synthesized by a simple two-step reaction[40,41] starting from anthranilic acid, as depicted in Scheme . Both BBHAN and its Al3+ complexes were characterized using Fourier transform infrared (FTIR), NMR, and ESI-MS studies (Supporting Information, Figures S1–S6).

Scheme 1

Synthetic Scheme of BBHAN

Single-Crystal X-ray Structure of BBHAN

As mentioned earlier, the novel architecture of BBHAN prompted us to grow single crystals of the same to explore its absolute structure. Single crystals of BBHAN suitable for diffraction were obtained from a supersaturated solution of the same in dimethyl sulfoxide (DMSO) over a period of 1 week. Important parameters as well as bond lengths are provided in the Supporting Information, Tables S1–S4. BBHAN crystallizes in the P212121 space group and its ORTEP diagram (Supporting Information, Figure S7) shows a DMSO molecule trapped within the former by means of H-bonding interactions (Figure ). The unit cell packing of BBHAN shows five BBHAN units and DMSO molecules in it (Supporting Information, Figure S8). Apart from the mentioned H-bonding between DMSO and BBHAN, the latter shows two other hydrogen-bonding interactions, as pointed out in Figure . Furthermore, BBHAN undergoes nonclassical H-bonding interactions with the trapped DMSO molecule in more than one manner and also with itself, as depicted in Figure . It is due to such nonbonding interactions that BBHAN attains a weblike packing (Figure ).

Figure 1

H-bonding interactions in BBHAN.

Figure 2

(a) CH–O-type interaction between BBHAN and O of the DMSO molecule; (b) CH–O (purple line) and CH···π (red line, black line) interactions between methyl protons of DMSO with two BBHAN units, and (c) CH–O and π···π within three units of BBHAN.

Figure 3

Supramolecular packing in BBHAN through nonbonding interactions observed along the b-axis.

Fluorimetric Sensing of Al3+

As BBHAN is equipped with multiple O and N donor sites, we decided to check its response toward different metal ions. For that purpose, 30 μM solutions of BBHAN in aqueous methanol (water/methanol = 2:8, v:v) were separately treated with 1 equiv of aqueous solutions of different metals (nitrate salts were used). When observed through the naked eye under ambient light, no noticeable color change was observed (Figure ). However, under UV light, the vial containing Al3+ showed brilliant blue luminescence when observed through the naked eye (Figure ). This was the first hint that BBHAN could detect Al3+ through an increase in fluorescence.

Figure 4

Color of BBHAN (30 μM) treated with various metal salts (1 equiv) under (top) ambient light and (bottom) UV light.

Following the naked eye observations, we analyzed the absorption profile of BBHAN in the absence and presence of various metal ions. Bare BBHAN showed bands at ∼326, 368, 438, and 470 nm. Upon addition of metal ions, almost all metals perturbed the absorption profile of BBHAN with Al3+ affecting the same to the greatest extent (Supporting Information, Figure S9). Thus, UV–vis titration for Al3+ was carried out. Upon gradual addition of Al3+, the peak at ∼470 nm diminished gradually along with the appearance of a structured band, centered at ∼418 nm (Supporting Information, Figure S9). However, perturbation from different metal ions curtailed the potential of BBHAN to be a selective sensor for Al3+ as far as naked eye color change and UV–vis response were concerned. As BBHAN contains the well-known fluorophore hydroxyl naphthaldehyde, we checked the emission response of BBHAN in the presence and absence of metal ions. When excited at ∼430 nm, bare BBHAN showed a weak emission profile, having an emission maximum at ∼480 nm. Upon addition of Al3+, the emission profile underwent a massive increment in fluorescence intensity by a factor of ∼30 (Figure ), which was not accomplished by addition of any other metal ion. Even in a mixture of metal ions, an increment was observed by 20 times. Individual interference was also checked to ensure the selectivity toward Al3+ (Supporting Information, Figure S10). Hence, BBHAN could detect Al3+ selectively by fluorescence enhancement (Figure ). The limit of detection (Supporting Information, Figure S11) calculated for Al3+ was found to be 1.68 × 10–9 M based on a standard protocol.[42] Such a low detection limit proves the superiority of BBHAN as a sensitive sensor for Al3+ over other available reported sensors (Supporting Information, Table S5).

Figure 5

(a) Emission response (λex = 430 nm) of BBHAN (1 μM) in the presence of various metal ions (4 equiv). (b) Emission titration of BBHAN (0.5 μM) with 0–1 equiv of aqueous solution of Al3+.

Binding Mode of Al3+ with BBHAN

To ascertain the nature of binding of BBHAN with Al3+, 1H NMR titrations were conducted in DMSO-d6 (Figure ). The most noticeable change was observed for the phenolic −OH signal, which underwent a downfield shift from ∼12.67 δ (in ppm) to ∼13.25 δ (in ppm) upon addition of 0.5 equiv of Al3+, and finally disappearing after addition of 1.0 equiv of Al3+. Downfield shifts were also observed for the two amide proton signals and that of the imine, as listed in Table S6. This indicated that the phenolic −OH group, along with the two amide moieties and imine, was involved in the complexation process with the Al3+ ion. The remarkable shift of the −OH signal compared to that of the others could be understood from the strong affinity of Al3+ toward O. The involvement of imine and amide groups in the complexation process was further confirmed through shifts in the FTIR spectrum of BBHAN and its aluminum complex. The FTIR spectrum of BBHAN showed signals at 1662.38 (imine) and 3399.32 (amide) cm–1, respectively, and its complex showed signals shifted to 1605.21 (imine) and 3434.93 (amide) cm–1 (Supporting Information, Figure S5). The ESI-MS profile of BBHAN–Al complex showed a signal at an m/z value of 843.2380 (Figure ). This could only be rationalized by considering a 2:1 complexation pattern between BBHAN and Al3+ (m/z calculated: 843.2512). The simulated isotopic modeling matched well with the experimental results (Supporting Information, Figure S4). To confirm this assumption, Job’s plot was constructed for finding the stoichiometry of interaction between BBHAN and Al3+ (Figure ). The maxima of the plot showed an abscissa value of ∼0.7, corresponding to a 2:1 type of interaction between BBHAN and Al3+. As Al3+ has a tendency to form strong bonds with O donors, phenolic −OH moieties undergo deprotonation to form strong O–Al bonds, whereas the imine and amide moieties undergo coordinate covalent bonding with Al3+ (Scheme ). The involvement of imine and amide moieties in the complexation process could be understood from the shift in IR stretching frequencies. In bare BBHAN, the amide and imine signals appeared at 3399, 3209, and 1650 cm–1 (Supporting Information, Figure S1). In the Al3+ complex, the signals were observed at 3434, 3231, and 1605 cm–1 (Supporting Information, Figure S5). The change in IR stretching frequencies is a result of covalent bonding of the aforesaid moieties with Al3+.

Figure 6

1H NMR titration of BBHAN with Al(NO3)3 in DMSO-d6.

Figure 7

ESI-MS spectrum of the BBHAN–Al complex.

Figure 8

(Top) Job’s plot and (bottom) BH plot for the determination of stoichiometry and binding constant between BBHAN and Al3+, respectively.

Scheme 2

Mode of Complexation of BBHAN with Al3+

To find the binding constant of BBHAN with Al3+, a modified form of Benesi–Hildebrand method[43] was employed using the following equilibrium equationsHence, K = k2 from eqs and I. The BH plot (Figure ) obtained by plotting the reciprocals of fluorescence intensity with 1/[Al3+]1/2 showed excellent linearity (R2 = 0.98455). The binding constant obtained using the above relation turned out to be 1.15 × 1011 M2–, which reflects the high affinity of BBHAN toward Al3+.

Mechanism of Sensing

To gain insight into the sensing mechanism, a time-resolved fluorescence experiment based on the time-correlated single-photon counting (TCSPC) technique was conducted and the results were calculated using a standard protocol[44] (Figure and Table ). Bare BBHAN shows biexponential decay with two differently populated species having lifetime values of 50.6 ps and 2.92 ns, respectively. However, upon addition of Al3+, a mono-exponential decay pattern was observed, having a lifetime value of 4.28 ns. Such a massive increment in excited-state lifetime indicates the freezing of nonradiative channels in BBHAN after complexation with Al3+. To confirm this, the radiative (kr) and nonradiative rate constants (knr) were also calculated using the equations, kr = quantum yield (Φ)/average lifetime (τav) and kr + knr = 1.0.[44] For this purpose, individual quantum yields of BBHAN and the BBHAN–Al ensemble were measured using quinine sulfate dissolved in 0.05 M H2SO4 as the standard.[45]Table enlists the values of the quantum yield of BBHAN and its Al3+ ensemble and also the radiative/nonradiative rate constants. The increment in the value of radiative constant confirms the attainment of structural rigidity in BBHAN after complexation with Al3+ (Scheme ). Hence, the observed turn-on response for Al3+ in BBHAN is due to the chelation-enhanced fluorescence (CHEF) mechanism.[46]

Figure 9

TCSPC profiles of BBHAN and BBHAN + Al3+ (λex = 450 nm, λmon = 470 nm).

Table 1

Excited-State Lifetime Parameters of BBHAN and the BBHAN–Al3+ Ensemble

species	τ1 (ns)	τ2 (ns)	α1 (%)	α2 (%)	τav (ns)	χ2
BBHAN	0.05	2.92	79.25	20.75	0.65	1.06
BBHAN + Al3+	4.28		1.00		4.28	1.13

Table 2

Comparison of radiative and nonradiative rates of BBHAN and the BBHAN–Al3+ ensemble

species	Φf	kr (ns–1)	knr (ns–1)
BBHAN	0.008	4.6 × 10–3	995.4 × 10–3
BBHAN + Al3+	0.48	11.2 × 10–2	88.8 × 10–2

Practical Application of Al3+ Detection

Live-Cell Imaging for Al3+

Because Al3+ sensing was achieved in aqueous methanolic medium, cell imaging for Al3+ was carried out using MDA-MB-468 cells. Cell survivability assay (Supporting Information, Figure S12) showed that the LD50 value for BBHAN was ∼40 μM. So, most of the cell population would survive at a concentration less than 40 μM. Hence, 15 μM BBHAN was used for cell-imaging purpose. After incubating 3 μM Al3+ in cells loaded with 15 μM BBHAN for 30 min, a prominent blue fluorescence was observed under a fluorescence microscope (Figure ). The blue fluorescence thus observed was due to complexation of Al3+ with BBHAN, which corroborated well with the solution-phase studies. The observed fluorescence also proved that BBHAN can well permeate through the cell membrane within 30 min. Hence, apart from a very sensitive probe for Al3+ in solution, BBHAN shows considerable cell membrane permeability to detect intracellular Al3+, which increases the practical utility of BBHAN.

Figure 10

MDA-MB-468 cells observed under bright field in the absence (a) and presence (c) of Al3+, under dark field in the absence (b) and presence (d) of Al3+.

Paper Strip Test for Al3+

As the detection of Al3+ by BBHAN was carried out in aqueous methanolic medium, it somewhat curtailed the potential of the latter in a pure aqueous medium. To circumvent this shortcoming, Whatman-40 filter paper strips were coated with BBHAN (50 μM) and 2.0 μM aqueous solution of Al3+ was dropped onto it. When observed under UV light, a strong blue fluorescence was observed (Figure ). Thus, by successful demonstration of the paper strip test for Al3+, solid-state detection for the same was achieved, which adds to the merit of BBHAN as a sensitive sensor.

Figure 11

Luminescence observed in a paper strip coated with BBHAN treated with Al3+ (2 μM) under UV light.

Conclusions

We reported the synthesis and crystal structure of an anthranilic acid–naphthalene conjugate, BBHAN, and explored its selective fluorimetric detection of Al3+ by enhancement of fluorescence intensity in aqueous methanol. The limit of detection and binding constant values are 1.68 × 10–9 M and 1.15 × 1011 M2–, respectively. BBHAN experiences the fluorescence enhancement by the CHEF mechanism. BBHAN can detect intracellular Al3+ and also in the solid state as demonstrated by the paper strip.

Materials and Methods

Reagents

Anthranilic acid, 2-hydroxy naphthaldehyde, benzoyl chloride, hydrazine hydrate, and nitrate salts of metal ions were purchased from Sigma-Aldrich and used as received. Triple distilled water was used for the preparation of metal solutions and spectroscopic-grade methanol purchased from Spectrochem was used for dissolving BBHAN.

Apparatus

Steady-state electronic absorption and fluorescence spectra were recorded on a Hitachi UV–vis (model U-3501) spectrophotometer and PerkinElmer LS55 fluorimeter, respectively. The time-resolved emission profile was recorded on a Horiba JobinYvon Fluorocube-01-NL fluorimeter. IR spectra (KBr pellet, 4000–400 cm–1) were recorded on a PerkinElmer model 883 infrared spectrophotometer. 1H NMR spectra were recorded on a Bruker Advance 300 spectrometer, where chemical shifts (δ in ppm) were determined with respect to tetramethyl silane (TMS) as the internal standard. The single crystal of BBHAN was mounted on a Bruker-AXS SMART APEX II diffractometer equipped with a graphite monochromator and Mo Kα (λ = 0.71073 Å) radiation. The crystal was placed 60 mm from the charge-coupled device and 360 frames were measured with a counting time of 10 s. The structure was solved using the Patterson method using SHELXS 97. Subsequent difference Fourier synthesis and least-squares refinement revealed the positions of the remaining non-hydrogen atoms. Non-hydrogen atoms were refined with independent anisotropic displacement parameters. Hydrogen atoms were placed in idealized positions and their displacement parameters were fixed to be 1.2 times larger than those of the attached non-hydrogen atoms. Successful convergence was indicated by the maximum shift/error of 0.001 for the last cycle of the least-squares refinement. Absorption corrections were carried out using the SADABS program. All calculations were carried out using SHELXS 97, PLATON 99, ORTEP-32, and WinGX system ver 1.64.[39] Mass spectrum was recorded on a Waters XevoG2-S Q TOF mass spectrometer. For cell-imaging studies, the cell survivability assay was done by MTT assay. A microplate reader from Biotek, and a fluorescence microscope (Leica) were used for MTT assay and cell imaging, respectively.

Synthesis of BBHAN

The synthesis of BBHAN was achieved in three steps (Scheme ):

Step 1: Synthesis of 2-Phenyl-benzo[d][1,3]oxazin-4-one

It was synthesized by a procedure available from the literature.[40]

Step 2: Synthesis of 2-(Benzamido) Benzohydrazide

It was synthesized by a procedure reported in the literature.[41]

Step 3: Synthesis of (E)-2-(Benzamido)-N′-((2-hydroxynaphthalen-1-yl) Methylene) Benzohydrazide (BBHAN)

2-(Benzamido) benzohydrazide (1.0 mmol) and 2-hydroxy naphthaldehyde (1.0 mmol) were suspended in 10 mL of methanol and refluxed overnight to afford a bright yellow powder, which was filtered under suction and dried under vacuum overnight. It was characterized by IR (Supporting Information, Figure S1), 1H NMR (Supporting Information, Figure S2), 13C NMR (Supporting Information, Figure S3), and mass spectrometry (Supporting Information, Figure S4). Yield: 70%. IR (KBr, cm–1): 3399, 3209, 3054, 2926, 1662, 1650, 1604, and 1524. 1H NMR (DMSO-d6, 298 K, TMS, 300 MHz): 12.66 (s, 1H, −OH), 12.40 (s,1H, −NHCO), 11.87 (s, 1H, −NHCO), 9.50 (s, 1H, −CH=N), 8.54 (d, 1H, Ar–H), 8.23 (d, 1H, Ar–H), 7.89 (m, 5H, Ar–H), 7.60 (m, 5H, Ar–H), 7.36 (m, 2H, Ar–H), 7.22 (m, 1H, Ar–H). 13C NMR (DMSO-d6, 298 K, TMS, 75 MHz): 165.0, 158.4, 148.3, 139.6, 134.7, 133.4, 133.1, 132.4, 129.2, 128.8, 128.1, 127.4, 123.9, 123.6, 121.6, 121.0, 119.1, 108.7. ESI-MS: calculated for [M + Na+]: 432.1324, found: 432.1333.

Synthesis of the BBHAN–Al Complex

To a well-stirred solution of BBHAN (0.5 mM) in ∼5 mL of methanol, an aqueous solution of aluminum nitrate (0.25 mM) was added dropwise, followed by overnight stirring. The solvent was evaporated under vacuum to yield a light yellow powder, which was washed with ice-cold methanol, dried under vacuum, and subjected to FTIR (Supporting Information, Figure S5) and 1H NMR analyses. 1H NMR (DMSO-d6, 298 K, TMS, 300 MHz): 12.42 (brs, 2H), 11.86 (s, 1H), 9.49 (s, 1H), 8.54 (d, 1H), 8.23 (d, 1H), 7.89 (d, 5H), 7.63 (s, 5H), 7.34 (d, 2H), 7.22 (d, 1H) (Supporting Information, Figure S6).