Polyaromatic label-assisted laser desorption ionization mass spectrometry (LA-LDI MS): a new analytical technique for selective detection of zinc ion

Abstract

An external matrix-free LDI technique for selective detection zinc ion is described. An internal polyaromatic label helped desorption cum ionization processes. The method is free of cross-interference which as often faced in fluorescence based methods.

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Selective and sensitive methods for the detection of metal ions have created a substantial impact in various research areas especially in molecular biology,¹ and environmental monitoring.² Metal ions, in particular the transition metal ions like Fe³⁺, Cu²⁺, Zn²⁺ play key roles in regulating important biological processes; for example, as cofactors for a host of enzymes (like carbonic anhydrase,³ cytochrome P450,⁴ amine oxidase,⁵ β-lactamase),⁶ maintenance of protein structural motif (Zn-finger),⁷ DNA-synthesis⁸ etc. Although these processes are essential for normal cellular function, presence of these ions in excess can have profound toxic effects on health and environment, even at extremely low concentrations. Thus it is important to be able to detect transition metals in low concentrations to assess health risks and for environmental monitoring. Various analytical techniques have been reported in the literature for detection/estimation of transition metal ions, which include UV-vis spectroscopy,⁹ atomic absorption spectrometry,¹⁰ fluorescence spectroscopy,¹¹ plasmon resonance energy¹² transfer etc. However, these have their own limitations especially the interference by other contaminants (inorganic/organic). A method which can detect specific metal ions in actual biological or environmental samples will be extremely useful. Our attention was drawn to a recent paper¹³ where a large number of potential chemical reactions were screened by labelling one of the reactant with a polyaromatic tag and recording matrix-free LDI mass spectra. This intrinsic matrix assisted desorption cum ionization mass spectrometry is now popularly known as LA-LDI MS.¹⁵ We reasoned that such a technique can be used to design polyaromatic tagged ligands for detection of metal ions in solution. If the ligand is tailor made for a specific metal ion, it will be possible to detect the presence of that particular metal ion in a mixture of other metal ions as well as other organic contaminants. The principle is based upon the fact that in absence of matrix, only those peaks will be observed where the polyaromatic tagged ligand is present, free or bound to metal ions. The situation is depicted in Fig. 1. In this communication, we describe a novel polyaromatic based oxinyloxy acetic acid 1 (oxine = 8-hydroxyquinoline) for selective detection of Zn²⁺ by LA-LDI MS.

Fig. 1 Principle behind metal ion detection.

Regarding the basis of ligand design, the alkynyl anthracene moiety¹⁴ was chosen as the polyaromatic tag because of its expected ability to assist the ionization process in LDI measurement acting as an internal matrix. Secondly, it is strongly chromophoric with a wide absorption window. Also, the tag can act as a fluorophore where emission intensity or change in wavelength can be monitored during metal ion chelation thus adding an additional handle for detection. Oxinyloxy acetic acid moiety was chosen for its well-known¹⁵ behaviour as a tridentate ligand towards transition metal ions, especially towards zinc.

The ligand was synthesized starting from 9,10-dibromo anthracene as shown in Scheme 1. Three sequential Sonogashira coupling¹⁶ followed by hydrolysis completed the synthesis of 1. The compound was fully characterized by NMR and mass spectral analysis.¹⁷ As expected, 1 showed a strong MH⁺ peak at m/z 503.27 in the LDI spectrum, recorded without adding any matrix, thus confirming the ability of the alkynyl anthracene moiety to act as an internal matrix (Fig. 2). The spectrum also showed peaks at m/z 548.42, 458.33, 445.32 assignable to structures A–C (Fig. 3).

Scheme 1 Synthesis of compound 1. (a) K₂CO₃, bromoethylacetate, acetone, rt, 6 h; (b) PdCl₂(PPh₃), ethynylethylsilane, Et₃N, toluene, reflux, 18 h; (c) NaOH, MeOH : water (10 : 1), 15 min, rt; (d) PdCl₂(PPh₃)₂, Et₃N, rt, 7 h; (e) NaOH, acetone : MeOH (1 : 2), 8 h.

Fig. 2 LA-LDI MS of ligand.

Fig. 3 Ligand and its different fragments produced in MS.

Because of the reported selective complexation of the in situ generated 8-hydroxyquinoline-based ligand 2 with zinc by Chen et al. as shown in Scheme 2, the free acid 1 was first treated with Zn(II) perchlorate, in acetonitrile–water (final concentration ligand 100 μM, salt 60 μM) for 1 h at room temperature to study its complexation behaviour.¹⁸ An aliquot (2 μL) from the reaction mixture was subjected to LDI MS (Fig. 4M) which showed the appearance of new peaks at m/z 543.5 and 607.38 generated due to 2 : 1 and 1 : 1-complex formation respectively (X and Y in Fig. 4M) between the tridentate ligand and with one molecule of solvent (H₂O or CH₃CN) as additional ligand (Fig. 5). The typical isotopic distribution expected for Zn²⁺ was also observed (see inset). The conventional MALDI using sinapinic acid showed the peaks for ligand–Zn complexes (1 : 1 and 1 : 2) with a lower sensitivity (Fig. 4N).

Scheme 2 Reported work by Chen et al.¹⁵

Fig. 4 (M) LA-LDI MS of ligand (100 μM) + Zn(II) perchlorate (60 μM); (N) MALDI spectrum of ligand (100 μM) + Zn(II) perchlorate (60 μM) in presence of sinapinic acid matrix.

Fig. 5 Proposed structures of complexes between ligand and Zn²⁺.

The complexation experiment was then repeated with several other metal ions (Cr³⁺, Fe³⁺, Co³⁺, Ni²⁺, Cu²⁺, Cd²⁺ and Hg²⁺) as perchlorates keeping the same concentrations of ligand and salt. Except for Ni²⁺ which showed a small peak (<10% intensity) at m/z 602 corresponding to a 1 : 1 complex (Fig. 6), for all other metal ions, no peak corresponding to metal ion–ligand complex appeared. Thus except for a small interference from Ni²⁺, our ligand is capable of selectively forming complex with Zn²⁺ as revealed by the LA-LDI MS.

Fig. 6 LA-LDI MS of ligand (100 μM) + Ni(II) perchlorate (60 μM).

One important attribute of this LA-LDI based detection method is that the peak for the complex with Zn²⁺ is not affected by the presence of other metal ions or organic molecules. Thus, the peak for the ligand–Zn²⁺ complex at m/z 607 could be seen in the presence of metal ions like Hg²⁺/Fe³⁺/Cd²⁺/Cu²⁺ (only the spectrum for Zn²⁺/Hg²⁺ is shown in Fig. 7, for others see ESI†) and organic molecules like amino acids (Fig. 8). A spectrum taken in presence of an external matrix sinapinic acid with ligand, Zn²⁺ and amino acids are also shown for comparison. The spectrum without matrix is much cleaner and mostly showed the peaks of species containing the ligand.

Fig. 7 LA-LDI MS of ligand (100 μM) + Zn(II) (60 μM) and Hg(II) perchlorate (60 μM).

Fig. 8 LA-LDI MS of ligand (100 μM) + leucine (50 μM) + phenylalanine (50 μM) + Zn(II) perchlorate (100 μM) (a) without sinapinic acid matrix; (b) with sinapinic acid matrix.

Having been successful in using the LA-LDI MS technique for detection of Zn²⁺ with our ligand 1, the fluorescence behaviour of the latter in isolation and in presence of metal ions was then studied. For the ligand, excitation at 380 nm showed two emission maxima at 490 and 518 nm (fluorescence quantum yield, Φ = 0.32). While metal ions like Cu²⁺, Co³⁺, Ni²⁺ and Mn²⁺ showed only quenching of fluorescence, other ions like Fe³⁺, Cr³⁺ and Hg²⁺ did not cause any appreciable change. The situation was interesting with Zn²⁺. In this case, a new broad hump centering at 570 nm causing an orange fluorescence (fluorescence quantum yield, Φ = 0.09) started to appear along with quenching of the original emission peak (Fig. 9P). Thus the ligand acted as a fluorescence sensor for Zn²⁺. However, the sensitivity of sensing is seriously affected as the orange fluorescence observed for Zn²⁺ is quenched in presence of those ions (the situation is shown for presence of Ni²⁺) (Fig. 9Q). However, the LA-LDI MS of a mixture of Zn²⁺ and Ni²⁺ clearly showed the presence of Zn²⁺ along with a small peak for Ni-complex (Fig. 9R). This clearly demonstrated the matrix-free LA-LDI MS method may be a suitable alternative to the fluorescence based techniques for sensing of metal ions.

Fig. 9 (P) Fluorescence spectrum of ligand 1 (10 μM) on addition of Zn²⁺ (0–2.5 eq.) in CH₃CN–HEPES buffer (2 : 1), λ_ex = 380 nm; (Q) fluorescence behavior of the 1 in presence of Zn²⁺, Ni²⁺ and mixture of Zn²⁺ and Ni²⁺; (R) LA-LDI MS of 1 (100 μM) + Zn²⁺ + Ni²⁺ (60 μM each).

As already pointed out that Chen et al.^15a has reported on the basis of ESI MS and NMR the formation of a 2 : 1 (ligand : zinc) complex formation with in situ generated oxinyloxy acetic acid derivative. However, in our case, both 2 : 1 and 1 : 1 complex were seen in the LDI MS. To explore the possibility that the stoichiometry may be concentration dependent, we carried out the complex formation at different concentrations of ligand and zinc and recorded the LDI MS which is shown in Fig. 10. The spectra clearly supports the existence of equilibrium between 2 : 1 and 1 : 1 complex (ligand : metal). At a concentration of 10 μM, the peak at 543.5 is the only prominent peak indicating the formation of mostly the 2 : 1 complex. With increment of metal ion concentration, the equilibrium shifts from 2 : 1 complex to 1 : 1 complex which is clearly reflected in the spectra. The study showed that upto a concentration of 30 μM of Zn²⁺ (with ligand concentration kept at 100 μM), 2 : 1 complex exists in major amount; concentration beyond 50 μM shifts the equilibrium in favour of the 1 : 1 complex. Job's plot (based on UV titrations) (Fig. 11) also suggests the coexistence of these two complexes.

Fig. 10 LA-LDI MS of ligand + Zn(II) perchlorate at different concentrations (ligand concentration kept at 100 μM).

Fig. 11 Job's plot diagram of the ligand for Zn²⁺ (where X_h is the mole fraction of the host and Δh indicates the change of the absorbance). Job plot shows a maximum at 0.4 mole fraction of the receptor. This value indicates compound 1 and Zn²⁺ exists in a equilibrium between a 1 : 1 and 1 : 2 complex.

In conclusion, we have shown the application of label assisted LDI technique in detecting metal ions. The method has been validated by the ability of the alkynyl anthracene–oxinyloxy acetic acid hybrid 1 to capture metal ions like Zn²⁺ and Ni²⁺, as confirmed from their LDI MS with a strong preference for Zn²⁺. Although the detection limit is ∼10 μM with the present label–ligand hybrid 1, we believe the sensitivity may be improved by modifying the label or the ligand. Current research activities are aimed towards that aspect.

Acknowledgements

We thank DST, Government of India (GoI) for a research grant and for J C Bose fellowship to AB. PSA is grateful to CSIR, GoI, for a research fellowship. We like to thank Ms M. Ganguly for her help during fluorescence data collection and Professor N. Sarkar for his valuable suggestions.

Notes and references

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17. Spectroscopic data: For 1: ¹H-NMR (400 Mz, d₆-DMSO): 9.09 (1H, d, J = 4 Hz), 9.01 (1H, d, J = 8Hz), 8.76–8.69 (4H, m), 8.24 (1H, d, J = 8.1 Hz), 7.95 (1H, bs), 7.89–7.82 (5H, m), 7.53 (3H, s), 7.39 (1H, d, J = 8.1 Hz), 5.1 (2H, s). ¹³C-NMR (100 MHz, d₆-DMSO): 170.2, 154.4, 149.7, 133.5, 132.2, 132.0, 131.8, 129.9, 129.5, 129.3, 129.2, 128.5, 128.3, 127.4, 125.4, 124.1, 123.6, 123.2, 122.7, 118.2, 112.8, 111.3, 103.4, 100.0, 90.6, 86.3, 66.0. HRMS: calcd for C₃₅H₂₂NO₃ + H⁺ 504.1594 found 504.1608.
18. Metal capture protocol: compound 1 (2.5 μL, 4 mM in DMSO) was added to an acetonitrile solution of metal perchlorates (100 μL, 9.6–80 μM). The final concentration of the ligand was thus maintained at 100 μM. The solution was incubated for 1 h at 30 °C. 2 μL from this solution was spotted into the well of the plate for LA-LDI (label assisted laser desorption ionization) and the MS spectrum was recorded.

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedure, compound characterization, copies of NMR, various MS spectra and fluorescence pictures. See DOI: 10.1039/c4ra02250e

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