Selective Zn²⁺ sensing using a modified bipyridine complex

Abstract

A novel fluorescent Zn²⁺ sensor, 4-(pyridin-2-yl)-3H-pyrrolo[2,3-c]quinoline (PPQ), has been designed, synthesized and characterized by various spectroscopic and analytical techniques. PPQ exhibits superior detection of Zn²⁺ in the presence of various cations tested, including Cd²⁺ and Hg²⁺, via shifting its emission maxima and fluorescence intensity enhancement. An emission wavelength at 500 nm, ensures probable non-interference from cellular components while performing biological applications.

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Zinc is required by living organisms to optimally perform various life functions: cell-division, growth and even vision.¹ Zinc is also known to play a main role in the functioning of the nervous system in the bodies of bilateral animals,² which is an active research area in neurobiology.^3,4 Dynamic pools of loosely bound zinc ions – termed ‘mobile zinc’ – mediate diverse cellular processes in the physiology of living organisms, ranging from signal transduction to proliferation and death due to the immune response.^5–8 In spite of such diverse roles, direct spectroscopic detection of Zn²⁺ is not possible due to its d¹⁰ electronic configuration. The sensitivity of fluorescence spectroscopy when compared to other optical tools renders this technique ideal for Zn²⁺ detection.⁹ Its widespread use has motivated the development and characterization of catalogues of fluorescent probes which produce readouts specific to this cation. Several chemosensors with wide arrays of structural features are known in the literature for the selective detection of Zn²⁺.¹⁰ For example, bipyridine^9b,11 and quinoline based ligands¹² are well known in this respect. In fact, quinoline based ligands like N-(6-methoxy-8-quinolyl)-4-toluenesulfonamide [TSQ] have garnered a lot more attention as fluorescent chemosensors for Zn²⁺.¹² In this regard, one of the major challenges has to do with the selectivity of the available probes. Namely, a stringent test of an ideal probe comprises selective Zn²⁺ detection in the presence of various transition metals.

The generally accepted mechanisms for Zn²⁺ sensing through fluorescence are photoinduced electron transfer (PET) and intramolecular charge transfer (ICT).¹⁰ Most PET sensors detect Zn²⁺ by fluorescence enhancement, whereas ICT sensors provide more selective ratiometric signals with quantifiable shifts in fluorescence emission maxima. Herein, we embark on a quest to develop sensors which take advantage of both mechanisms. Namely, a wavelength shifted fluorescence response as well as fluorescence enhancement are sought after simultaneously, with an ultimate goal of superior recognition of Zn²⁺.

With all the aforementioned in mind, we attempted to develop a hybrid quinoline–bipyridine system, 2-(pyridine-2-yl)quinoline [PQ] (Scheme 1), and test its sensitivity and selectivity towards Zn²⁺. Our initial attempt proved unsuccessful, namely, a complete lack of Zn²⁺ selectivity was observed when PQ was used as a probe. At this juncture we decided to introduce skeletal modification to the existing PQ structure, by fusing heterocycles on the pyridine half of the quinoline moiety by a 2,3-c mode. The premise was that the introduction of an electron rich system in PQ would allow for the supply of electrons to the electron deficient quinoline. It was also anticipated that attaching heterocycles by the 2,3-c mode would provide an additional site for metal binding. The synthesis of the target molecules was carried out by employing two separate synthetic strategies. The fused pyrrole system, 4-(pyridin-2-yl)-3H-pyrrolo[2,3-c]quinoline (PPQ) was synthesized using the van Leusen method with toluenesulfonylmethyl isocyanide (TosMIC) as the key step.¹³ Suzuki coupling¹⁴ was used as a key step for the synthesis of furan [4-(pyridin-2-yl)-3H-furo[2,3-c]quinoline] (FPQ) and thiophene [4-(pyridin-2-yl)-3H-thieno[2,3-c] (TPQ) based systems. All the required compounds were obtained in 45–85% yield and were fully characterized by ¹H NMR, ¹³C NMR and mass spectroscopy before testing the fluorescence activity of the yielded compounds. Initial steady state fluorescence measurements of FPQ, PPQ and TPQ with respect to Zn²⁺ detection revealed that only PPQ can selectively detect Zn²⁺, whereas the other two compounds were optically silent. The most mundane of considerations is the selectivity of PPQ towards Zn²⁺ binding in the presence of other metals and the binding mode, to which we limit this communication.

Scheme 1 Progressive increase in the complexity of the bipyridine structure.

The binding of PPQ towards different metal ions in DMF was investigated with steady state fluorescence spectroscopy (Fig. 1). The PPQ ligand exhibits a fluorescence emission maximum at 400 nm in DMF. As shown in Fig. 1, we observed negligible changes to the fluorescence response of PPQ upon addition of Li⁺, Na⁺, Mg²⁺, K⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Cd²⁺, Hg²⁺ and Pb²⁺. In contrast, PPQ shows high selectivity for Zn²⁺, marked by the emergence of a red shifted fluorescence peak at 500 nm only upon Zn²⁺ addition. Notably, the fluorescence intensity at 500 nm was enhanced six-fold when compared to PPQ after the addition of only 0.15 equivalents of Zn²⁺. Also, PPQ can be used as a ratiometric sensor for Zn²⁺ (Fig. S11†), when the fluorescence intensity ratio of 500/400 nm is used as the detecting signal.

Fig. 1 Fluorescence response (λ_ex = 360 nm) of PPQ in DMF to various metal ions (0.15 equivalents), measured at 500 nm; 1: only PPQ. Inset: the corresponding fluorescence spectra of PPQ alone and in the presence of various metal ions.

To establish the selectivity of the PPQ molecule towards Zn²⁺, we also performed competitive fluorescence sensing experiments in the presence of other metals (Fig. 2). The observations clearly show that PPQ is highly selective towards the detection of Zn²⁺ in the presence of most of the other metals. It is noteworthy to mention here that PPQ shows prominent selectivity towards Zn²⁺, even in presence of Cd²⁺ or Hg²⁺, although these ions have similar chemical properties and are expected to interfere with the Zn²⁺ signals. On the other hand, we observed moderate interference when Co²⁺, Ni²⁺ and Cu²⁺ were present in the solution which led to fluorescence quenching. MALDI spectra (Fig. S12–S14†) revealed that the aforementioned cations form complexes with PPQ, which possibly result in the decrease of fluorescence intensity.

Fig. 2 Competitive selectivity (λ_ex = 360 nm and fluorescence was recorded at 500 nm) of PPQ towards Zn²⁺ in DMF, in the presence of other metal ions (0.15 equivalent). 1: only PPQ.

The binding stoichiometry for the complex formed between PPQ and Zn²⁺, was established by the continuous variation method (Job's plot), which indicates a 2 : 1 ligand to metal ratio (Fig. 3). Benesi–Hildebrand analysis¹⁵ of the fluorescence titration data gave an association constant of 1.5 ± 0.2 × 10² M⁻² for the complex. The detection limit was found to be 1.5 × 10⁻⁶ M in DMF, according to a reported method.¹⁶ The MALDI spectrum of the complex (Fig. S7†) provides further support to the binding pattern. The mass spectrum showed a prominent peak at m/z 553 in the positive ion mode, along with well resolved isotopic peaks at m/z 555 and 557, which confirm the presence of two ligand molecules bound to Zn²⁺. While these results address questions pertaining to the metal to ligand ratio, they cannot discern between the two possible PPQ–Zn²⁺–PPQ binding motifs (Fig. 4). In an effort to further validate the proposed structures, and ascertain the participation of the pyrrole and/or quinoline nitrogens along with the pyridine nitrogen (Scheme 1) to zinc binding, we decided to introduce a minor modification to the structure of PPQ. The –NH of the pyrrole was methyl-substituted. Synthesis of the aforementioned compound, namely 3-methyl-4-(pyridine-2-yl)-3H-pyrrolo[2,3-c]quinolone (N-methyl PPQ), was accomplished by reacting PPQ with methyl iodide in the presence of sodium hydride. The yielded product was again fully characterized before subsequent fluorescence measurements. As expected based on our prior observations, the compound did not exhibit any shift in fluorescence maxima upon addition of Zn²⁺, indicating a loss of selectivity towards Zn²⁺. This stresses the crucial role played by the pyrrole nitrogen in selective Zn²⁺ sensing.

Fig. 3 Job's plot of PPQ (in DMF) and Zn²⁺, where concentrations of PPQ and Zn²⁺ were 10⁻⁴ M. λ_ex = 360 nm and fluorescence was recorded at 500 nm. The inset shows the Benesi–Hildebrand analysis of the fluorescence changes for the complexation between PPQ and Zn²⁺.

Fig. 4 Two possible complex structures with a 2 : 1 ratio of PPQ and Zn²⁺.

As the dephased radiation process does not provide direct structural information about the yielded complex, we opted to employ IR absorption spectroscopy to further validate our assignments, guided by density functional theory (DFT) for spectral assignments. The IR spectra (Fig. 5) of the PPQ ligand, complex (PPQ–Zn²⁺–PPQ) and N-methyl PPQ were recorded as KBr discs. The spectra of PPQ exhibited the characteristic N–H stretching vibration at 3370 cm⁻¹. Upon complexation with Zn²⁺, the N–H peak disappeared, indicating the involvement of the pyrrole nitrogen in complex formation. This is also supported through the absence of a N–H peak in the IR spectrum of N-methyl PPQ. As such, we propose that both the binding motifs (Fig. 4) are possible for the complex. The computed B3LYP¹⁷/6-311++G**¹⁸ anharmonic IR spectra¹⁹ for all the three species corroborate the experimental results, and provide compelling proof for our assignments. On the basis of the above observations, the selective recognition of Zn²⁺ by the PPQ molecule over a number of other cations is indicative of the fact that the bidentate ligand better satisfies the geometrical requirements of a tetrahedral Zn²⁺ complex.²⁰ It is obvious that Zn²⁺ [3d¹⁰] shows a preference for tetrahedral geometry as this geometry minimizes ligand–ligand repulsion by maximizing L–M–L angles to 109°.²¹ This is evident through the inspection of the fully optimized B3LYP/6-311++G** structures of PPQ and PPQ–Zn²⁺–PPQ (binding mode I of Fig. 4), which are illustrated in Fig. 6.

Fig. 5 IR spectra for PPQ, PPQ–Zn²⁺–PPQ and 2-N-Me PPQ.

Fig. 6 The calculated energy minimized structures for PPQ (left) and PPQ–Zn²⁺–PPQ (right).

The reversibility of Zn²⁺ binding to PPQ was also tested. The strong affinity of EDTA towards Zn²⁺ binding, leads to fluorescence quenching, indicative of a decomplexation of the PPQ–Zn²⁺–PPQ complex (Fig. S8†). Subsequent addition of Zn²⁺ revealed that Zn²⁺/PPQ binding can be recovered, as evidenced by a rise in the characteristic fluorescence response of the complex.

In conclusion, we have developed a new molecular sensor for the selective detection of Zn²⁺. The utilized sensor can be easily synthesized by simple chemical transformations. After the addition of Zn²⁺, a red shift in the emission maxima of the sensor with fluorescence enhancement indicates the involvement of both PET and ICT mechanisms. The stoichiometry of the complex was found to be 2 : 1 by Job's plot and MALDI-MS analysis. The molecule senses Zn²⁺ in the presence of Cd²⁺/Hg²⁺, demonstrating its superior ability. Further work is currently underway in our laboratory for the development of water soluble systems of the aforementioned ligand and a crystal structure of the PPQ–Zn²⁺–PPQ complex.

Acknowledgements

M.A. thanks the University Grants Commission (India) for JRF. P.Z.E acknowledges support from the Laboratory Directed Research and Development Program through a Linus Pauling Fellowship at Pacific Northwest National Laboratory (PNNL), and an allocation of computing time from the National Science Foundation (TG-CHE130003). A.N. acknowledges support from BITS Pilani through a research initiation grant. A.B. thanks the Department of Science and Technology, New Delhi, India for a fast-track grant. The authors would like to thank Dr Aasheesh Srivastava (IISER, Bhopal) for useful discussions.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra00922c

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