Synthesis, spectroscopic characterization, insulin-enhancment, and competitive DNA binding activity...

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Cite this: DOI: 10.1039/c2dt12298g

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Synthesis, spectroscopic characterization, insulin-enhancment, andcompetitive DNA binding activity of a new Zn(II) complex with a vitamin B6

derivative—a new fluorescence probe for Zn(II)†

Tirtha Mukherjee,a João Costa Pessoa,b Amit Kumarb and Asit R. Sarkar*a

Received 30th November 2011, Accepted 9th February 2012DOI: 10.1039/c2dt12298g

This paper describes the activity of a Schiff base ligand, derived from pyridoxal, as a promisingfluorescence probe for biologically important Zn(II) ion sensing. This is the first report of a vitamin basedligand as a fluorescent probe for sensing Zn(II) ions. The Schiff base H2pydmedpt, derived from thecondensation of pyridoxal (pyd) and N,N-bis[3-aminopropyl]methylamine (medpt), exhibits around a325-fold increase in fluorescence quantum yield due to zinc triggered fluorescence switching. Theresponse is specific for Zn(II) ions, and remains unaffected by the presence of alkali and alkaline earthmetals but is suppressed to varying degrees by transition metal ions. The corresponding Zn(II)-complex,[Zn(pydmedpt], is isolated. The DFT optimized structure of the complex is compatible with elementalanalysis, mass spectrometry, FT-IR, electronic and NMR spectra. The isolated complex, having pKa

values of ∼5.3 and ∼5, is a moderate intercalator for DNAwith an apparent binding constant of 2.3 × 106

M−1. The complex also shows insulin-enhancing activity at par with other reported complexes, with anIC50 value of 0.65 with respect to ZnSO4.

Introduction

Zinc plays a critical role in numerous cellular functions,1 such asgene expression,2 apoptosis3 and neurotransmission4 and is alsoinvolved with many diseases such as Alzheimer’s disease,5 epi-lepsy,6 cerebral ischemia,7 infantile diarrhea8 and cancer.9 It is aconstituent of many metalloproteins and metalloenzymes10 andis also present as free or loosely bound chelatable Zn(II),especially in the brain,11 pancreas,12 and spermatozoa.13 Moni-toring this “free zinc” (often referred to as non-ligand bound orweakly bound zinc) is of great physiological importance.14 Forexample mobile zinc was used to detect and monitor the devel-opment of prostate cancer in a transgenic mouse model.15 Thus,it is vital to monitor this free Zn(II). However, the low naturalabundance of the NMR active 67Zn isotope, and the silent natureof d10 Zn(II) in EPR and visible spectra are major constraints forits monitoring. Therefore, a fluorescence assay of zinc with thehelp of a zinc specific fluorescence detector is the method ofchoice for detection and estimation of zinc in biologicalsystems.16 Thus, the development of useful fluorescence probesfor Zn(II) has been attracting much interest.17–20 Most of thechemo-sensors reported as fluorescent probes for Zn(II) sensing

are based on quinoline, bis(2-pyridylmethyl)amine or acyclicand cyclic polyamine derivatives.21 Though pyridoxal structu-rally resembles pyridyl amine, it was never used for the develop-ment of Zn(II) fluorescent probes.

Coulston and Dandona demonstrated that the Zn(II) ion stimu-lates lipogenesis in rat adipocytes similar to the action ofinsulin.22 Following these findings, several researchersconfirmed the insulin-enhancing activity of the Zn(II) ion,23–25

indicating that suitable zinc complexes are showing superioractivity compared to ZnSO4. Thus, complexation of Zn(II) by asuitable ligand, besides enabling its use as a fluorescence probe,can also have an insulin-enhancing effect. The mechanism ofaction of zinc as an anti-diabetic agent has not yet been comple-tely established. The published reports indicate several pathwayswhich lead to insulin-enhancing activity of the zinc compounds.Among them are (i) action on the insulin receptor and phospha-tidylinositol 3-kinases (PI3-k), (ii) effect on glucose transporter4 (GLUT 4), (iii) effect on the activation of thephosphodiesterases.26

Owing to the strong Lewis acidity, rapid ligand exchangeproperties and lower toxicity of zinc complexes, another poten-tial biological application of Zn(II) is in the field of artificialnucleases. The number of papers reporting the interactionsbetween DNA and zinc complexes has been increasing rapidly.27

There are three types of binding modes possible for non-covalentinteractions: intercalation, groove binding and external binding.

We, thus strive to develop a Zn(II) complex which can be usedfor the above mentioned bio-applications. Since our aim is to usethe complex in bio-applications, we chose pyridoxal as the

†Electronic supplementary information (ESI) available. See DOI:10.1039/c2dt12298g

aDepartment of Chemistry, University of Kalyani, Kalyani-741235 WestBengal, India. E-mail: ars@klyuniv.ac.inbCentro de Química Estrutural, Instituto Superior Técnico, TechnicalUniversity of Lisbon, Av. Rovisco Pais 1049-001, Lisboa, Portugal.E-mail: joao.pessoa@ist.utl.pt

This journal is © The Royal Society of Chemistry 2012 Dalton Trans.

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aldehyde component of the Schiff base ligand. Pyridoxal is avitamer of vitamin B6, which plays an important role as a co-factor in many biosynthetic processes such as transamination,28

decarboxylation,29 racemization,30 dehydration31 and desulphy-dration32 of amino acids. Pyridoxal and its derivatives have alsobeen implicated in the generation, prevention and treatment ofcancer,33–35 e.g. the pyridoxal isonicotinoyl hydrazone class ofiron chelators shows promising activity as antiproliferativeagents.36,37 Several metal complexes with ligands derived frompyridoxal were reported recently38–41 and some of the complexeswith this type of ligand have already been investigated for bio-applications.42

The amine component of the ligand N,N-bis[3-aminopropyl]methylamine (medpt) is a derivative of the naturally occurringpolyamine, norspermidine, found in some species of plants, bac-teria and algae.43,44 It is known to exhibit significant antitumoractivity against L1210 leukemia, 3LL carcinoma and EL4 lym-phoma in mice.45,46

The compound [H4pydmedpt]2+·2Cl− (Fig. 1) and itsvanadium complex was first reported by our group.47 Thisvanadium complex shows moderate insulin-enhancing activity.47

Keeping these characteristics and potentialities of the ligandin view, we have undertaken the synthesis and characterizationof its Zn(II)-complex. The molecular structure and spectroscopicproperties of [Zn(pydmedpt)], as well as its insulin-enhancingproperties and DNA binding ability are described. Theoreticalcalculations were carried out to support and substantiate themeasured structural and spectroscopic properties. Moreover, thepotential of the ligand as a fluorescence probe for Zn(II) ions isinvestigated, namely the specificity of the fluorescence signal forZn(II).

Experimental

General procedures

Materials: N,N-bis[3-aminopropyl]methylamine (medpt), zincacetate dihydrate [(CH3CO2)2Zn·2H2O], pyridoxal hydrochlo-ride, calf thymus DNA (from calf thymus, Type XV, preparedfrom highly polymerized calf thymus DNA and nicked withDNase using the method of Aposhian and Kornberg48) and ethi-dium bromide (EB) were purchased from Aldrich, Milwaukee.All the other chemicals used were of analytical grade. Com-pound medpt was distilled in the presence of activated charcoalprior to use, solvents were dried by standard procedures, andother chemicals were used as received.

Characterization procedures

Elemental analysis was obtained with a Perkin-Elmer 2400CHN/O Analyzer. Zinc was determined by EDTA complexo-metric titration using xylenol orange indicator.49 FT-IR spectrawere recorded using KBr pellets in a Perkin-Elmer FT-IR spec-trometer (Model: L 120-000A) with resolution of 0.5 cm−1. Theelectronic spectra were recorded in ethanol solvent on a Shi-madzu UV-2401 spectrophotometer, and the mass spectrum wasrecorded in a 4000 Q TRAP MS System in the positive modeusing a methanolic solution of the complex.

The 1H NMR spectra were obtained on a Bruker Avance III400 MHz spectrometer with the usual parameter settings. TheNMR spectra of the complex were recorded in fully deuteratedmethanol solution at 298 K. The protonation/deprotonation of[Zn(pydmedpt)] with change of pD was evaluated using the 1HNMR spectra of the complex in 98% deuterated methanol and2% D2O (v : v) mixed solvent solutions at different pD values(9.1 to ca. 4.6). pD values were measured at approximately298 K, before and after recording each 1H NMR spectra, with apH meter calibrated with Aldrich standard buffers (pH 4 and 7).

The fluorescence spectra were recorded using a Perkin-ElmerLS 55 Fluorescence Spectrometer. The solutions were preparedin a 98% methanol and 2% H2O (v : v) mixed solvent buffered atpH 7.5 by the triethylamine-HCl buffer, to achieve the physio-logical pH. A solution of 0.10 mmol L−1 solution of the ligandwas titrated with a 1.0 mmol L−1 solution of zinc acetate. Theligand solution was titrated with the Zn(II) solution within therange for a zinc : ligand ratio of 0 to 2. At each stage of Zn(II)addition the fluorescence spectrum of the solution was recorded.The reversibility of the fluorescence signal of the solution con-taining 0.100 mmol L−1 ligand and 0.100 mmol L−1 of zincacetate was investigated by gradual addition of 1 mmol L−1

EDTA solution under identical conditions. For investigation ofthe effects of the other cations 3.0 mmol L−1 solutions of NaCl,KCl, CaCl2·2H2O, MgSO4·7H2O, CuCl2·2H2O, NiCl2·6H2O,CoCl2·6H2O, FeSO4·7H2O, Mn(OOCCH3)2·4H2O, Cr2(SO4)3·6H2O, and VOSO4·5H2O were prepared separately in the samesolvent. Fluorescence spectra were recorded with a metal : ligandratio of 3 : 1 for each of the above metal ions separately, and alsofor solution having ligand, zinc ion and one of the above metalions in the ratio of 1 : 1 : 3. All fluorescence measurements wererecorded at an excitation wavelength of 275 nm with emissionand excitation slit width of 15 nm. The fluorescence quantumyield was determined by standard procedures50 using thequantum yield of quinine sulfate in 0.5 mol L−1 H2SO4 sol-ution51 as a standard reference.

Insulin-enhancing studies

This study was performed in compliance with relevant laws andcleared by the Ethical Committee of the Universtiy of Kalyani.Epididymal fat pads were excised from anesthetized (by ether)male Wistar rat (7–8 weeks old, weighing 80–160 g), cut intoappropriate pieces and incubated in type IV collagenase in KrebsRinger Bicarbonate (KRB) buffer (120.0 mmol L−1 NaCl,1.27 mmol L−1 CaCl2, 1.2 mmol L−1 MgSO4, 4.75 mmol L−1

KCl, 1.2 mmol L−1 KH2PO4 and 24.0 mmol L−1 NaHCO3, pH7.4) containing 2% BSA at 37 °C with gentle shaking for ca.

Fig. 1 Structural formula of the Schiff base compound[H4pydmedpt]2+·2Cl−.

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1.5 h. On filtration through sterilized cotton gauze and washingwith KRB buffer, the incubated tissue gave a cell count of1.5–2.5 × 106 cells per mL on hemacytometer after trypan bluestaining. Isolated cells were pre-incubated at 37 °C for30 minutes with various concentrations of the complex orZnSO4 in KRB buffer. The resulting solution was then incubatedat 37 °C for 3.0 hours with added adrenaline, 10 μmol L−1. Thereactions were frozen by soaking in ice water, and the mixturewas centrifuged at 3000 rpm for 10 min at 4 °C. Outer solutionsof the cells are then separated from the precipitates. FFA levelsin this solution were determined spectrophotometrically by themethod described by K. Itaya and M. Ui.52

DNA binding studies

To study the interaction of [Zn(pydmedpt)] with DNA, a stocksolution of DNAwas prepared by dissolving the nucleic acid inTris-HCl buffer [5 mmol L−1 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), 50 mmol L−1 NaCl] at pH 7.5, stored at4 °C, more than 24 hours with gentle shaking occasionally to gethomogeneity, and used within 2 days. Solutions of DNA gaveratios of UV absorbance at 260 and 280 nm, A260/A280 of 1.84,indicating that the DNAwas sufficiently protein free.53 The con-centration of the prepared CT-DNA stock solution per nucleotidewas calculated based on its absorbance at 260 nm by using theextinction coefficient54 ε260 = 6600 M−1 cm−1. Prior to titrationwith [Zn(pydmedpt)], DNA was allowed to be saturated withethidium bromide (EB) by mixing equal volume of solutions ofEB (0.32 μmol L−1) and CT-DNA (10 μmol L−1) and storing itfor ∼24 h.55 Portions of 6 mL of this solution were taken anddifferent volumes of 1 mmol L−1 solutions of [Zn(pydmedpt)]were added to prepare a set of solutions with the final concen-tration of [Zn(pydmedpt)] varying from 0 to 250 μmol L−1. Thesolutions were allowed to stand for 2 hours to reach equilibrium.Fluorescence of the solutions was then measured using an exci-tation wavelength of 545 nm resulting in emission at 589 nm atroom temperature.

DFT calculations

The Gaussian 03 software56 was used for all theoretical calcu-lations. The structure of [Zn(pydmedpt)] was optimized usingthe Perdew–Wang 1991 exchange functional as modified byAdamo and Barone57 and Perdew and Wang’s 1991 gradient-cor-rected correlation functional58 and LanL2DZ59–61 basis set, thefigure was prepared by Chemcraft software.62 Calculation wasrepeated in solution phase with PCM model63 using methanol asthe solvent for the calculation of Mulliken atomic charges in sol-ution. The electronic spectrum of [Zn(pydmedpt)] was computed

theoretically by the TD-DFT method using the B3LYP64,65

method and the 6-31G(d)66 basis set. The 1H NMR spectrum ofthe complex was computed by the GAIO method67 usingB3LYP64,65 level of theory with 6-311+g(d,p)68,69 basis set. Thecalculation was repeated in solution phase in methanol solventusing the PCM model.63

Synthesis of [Zn(pydmedpt)]

[Zn(pydmedpt)] was synthesized by refluxing a mixture of0.50 mmol (0.10 g) pyridoxal hydrochloride, 0.25 mmol(0.04 mL, d = 0.90 g mL−1) of N,N-bis[3-aminopropyl]-methyl-amine and 0.25 mmol (0.04 g) zinc acetate in dry methanol(Scheme 1). A light yellow compound was isolated by additionof acetonitrile, which was collected and recrystallized frommethanol acetonitrile mixture (1 : 2). Its purity was verified byTLC. Yield = 59%. 1H NMR(MeOD-d4): 8.80 (s, 2H, –NvCH),7.41 (s, 2H, pyridoxal H atom (Haromatic)), 4.70 (s, 4H–CH2

attached to –OH), (4.60 (br) 2H 3.58(br) 2H–CH2 attached toNimine), 2.34 (s, 6H, CH3 attached to pyridine ring), 2.33 (s, 3H,CH3 attached Namine). The

13C NMR spectrum is discussed inthe ESI.† Important mass spectral peaks—506.21, 357.16,152.09, 146.18. Selected infrared peaks with possible assign-ments (in cm−1): 3401 ν(O–H); 1639 ν(CvN); 1424 ν(C–N ter-tiary amine); 1021 ν(C–O phenol); For, C23H31N5O4Zn Found(FW 507), (Calcd)%: C 54.63 (54.49) H 5.89 (6.16) N 13.78(13.82) Zn 12.87 (12.90).

Results and discussion

DFToptimized structure of [Zn(pydmedpt)]

Since attempts to isolate single crystals of [Zn(pydmedpt)] suit-able for X-ray diffraction analysis were unsuccessful, the opti-mized structure of the complex was computed by theoreticalmethods. Full optimization was carried out for all possibleisomers of the complex without any symmetry restrictions. Thelowest energy isomer is reported here. The vibrational frequen-cies were analyzed to confirm the identity of the stationary pointand it was found to be a minimum (with no negative frequency).Selected bond lengths and bond angles are given in Table 1. Theoptimized structure is depicted in Fig. 2.

The Zn(II) atom is penta-coordinated with two Ophenolato (O1,O21), two Nimine (N5, N13) and one Namine (N9) donors accord-ing to the calculated optimized structure. The coordinationenvironment around the zinc centre is distorted trigonal bipyra-mid. The structural distortion index parameter70 is τ = 0.69; τ =0 for ideal square pyramid and τ = 1 for ideal trigonal bipyramid.Both Zn–Ophenolato bond distances are almost equal, as well as

Scheme 1 Outline of the synthesis of [Zn(pydmedpt)].

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the Zn–Nimine bond distances. The Zn–Ophenolato bonds areslightly longer than the zinc–imine bonds (average distance of2.00 Å vs. 2.09 Å, respectively). The Zn–Namine bond distance islarger than the Zn–Nimine bonds, as expected from the reportedFukui function value for the atoms in this ligand.47 The Zn–Ophenolato and Zn–Nimine bond lengths obtained from the DFToptimization of the complex are very similar to those reportedfor the structurally characterized Zn-N,N′-bis(pyridoxylidene)ethylenediamine and Zn-N,N′-bis(pyridoxylidene)1,3-propane-diamine complexes reported by Naskar et al.71 The average ofzinc–phenolato and zinc–imine bond lengths reported by Naskaret al. are 2.01 and 2.10 Å whereas the values in our complex are2.00 and 2.09 Å, respectively. The Zn–Ophenolato bonds are alsosimilar to those found in [ZnCl(H2Rpyr2en)]

+Cl− (H2Rpyr2en =N,N′-ethylenebis(pyridoxylaminato)),72 1.976(3) and 2.026(3)Å, which is also a 5-coordinate Zn(II) complex. The Zn–Namine

bond length may be considered as an apical bond; it is alsowithin the range reported for related complexes by X-ray singlecrystal diffraction,73 but it is longer by ∼0.06 Å than those ine.g. [ZnCl(H2Rpyr2en)]

+Cl−, where the two Zn–Namine bondsmay be considered as equatorial.72 The two pyridine rings areoriented at an angle of 66.16°.

Electronic absorption spectrum of [ZnII(pydmedpt)]

The electronic absorption bands of the complex in methanolwere recorded and compared with those predicted by time-dependent DFT (TD-DFT) calculations. The coordinates of the

optimized structure were used for the calculation and the resultsare summarized in Table 2. The major contributions of theatomic orbitals in MOs involved in the transitions, as constitutedby the LCAO-MO method, are given in Table 3. The pictorialrepresentations of the MOs are given in Fig. 3.

Tables 2 and 3 and Fig. 3 indicate that the TD-DFT peak at378 nm (corresponding to the experimental peak at 376 nm) ispartially due to charge transfer from the dxy orbital of zinc to thedelocalized π* orbitals of the pyridine rings and CvN bond,that is, it has a partial MLCT character. Since this transition doesnot involve a HOMO to LUMO transition the MLCT nature ofthe band does not contradict the generally observed redox inert-ness of the ZnII center in zinc complexes. Since the lone pairs onpy and pz orbital of the phenoxo atoms have a major contributionon the HOMO-2 orbitals, the transition at 378 nm also possessessome n → π* character. The band at 310 nm (321 nm experimen-tal) involves the transition of electron from the π orbitals cen-tered at the pyridine ring to the π* orbitals centered at the iminegroups; therefore it can be considered as a π → π* band. TheTD-DFT peak at 284 nm (276 nm experimental) is a σ → π*band which involves transfer of electron from σ orbitals centeredmainly at the amine nitrogen to the π* orbitals centered aroundthe pyridine ring and the imine bonds. Though normal σ → π*transitions are symmetry forbidden, the mixed nature of the orbi-tals (Table 3) involved in the transition evade this restriction.The peak at 239 nm (227 nm experimental) has major contri-butions from two transitions: (i) The transition from (HOMO-2)to (LUMO+2) is associated with the MLCT component, wherethe electron is transferred from the dxy orbital to the π* orbitalscentered around the pyridine ring. Due to contributions of the py

Fig. 2 Optimized structure and atom numbering scheme of [Zn-(pydmedpt)].

Table 2 Observed and computed electronic spectral data of thecomplex and their assignments

Observed peak (nm)(ε/dm−3 mol−1 cm−1)

TD-DFT peak(nm) (osc. str.)

Assignment of majorcontributors

376 (6250) 378 (0.0062) (HOMO-2) → LUMO321 (5900) 310 (0.0012) (HOMO-3) → (LUMO+1)276 (7350) 284 (0.0385) (HOMO-4) → LUMO227 (28 900) 239 (0.0580) (HOMO-2) → (LUMO+2)

(HOMO-10) → LUMO

Table 3 Major contribution of the atomic orbitals in MOs involved inthe transitions as constituted by LCAO-MO methoda

MO% Contribution of AOs[contribution (atom type number-atomic orbital)]

(HOMO-2) 25 (O 1-py), 25 (O 21-pz), 18 (Zn 64-dxy)(HOMO-3) 26 (O 1-py), 0.24 (N 30-py), 0.21 (O 21-pz), 21 (N 18-pz)(HOMO-4) 24 (N 9-1pz), 20 (N 9-py), 16 (N 9-s)(HOMO-10) 39 (O 28-px), 24 (O 24-px), 17 (O 28-pz)LUMO 22 (N 5-pz), 21 (C 4-pz), 17 (N 30-pz), 16 (N 13-py), 15

(C 14-py)(LUMO+1) 21 (N 13-py), 19 (C 14-py), 18 (N 5-pz), 16 (N 18-py), 16

(C 4-pz), 15 (N 18-py)(LUMO+2) 22 (C 2-pz), 21 (C 26-pz), 19 (C 29-pz), 19 (C 31-pz), 19

(O 28-s), 0.18 (C 17-py)

aContribution below 15% are not shown. The atom numbering schemeare as in Fig. 2.

Table 1 Selected bond lengths (Å) and bond angles (°) for [Zn-(pydmedpt)]

Bond lengths

Zn64–N9 2.221 Zn64–N13 2.093Zn64–N5 2.094 Zn64–O21 2.003Zn64–O1 1.997

Bond angles

N9–Zn64–O1 111.4 N9–Zn64–O21 112.6N9–Zn64–N5 88.4 N9–Zn64–N13 89.1N5–Zn64–N13 177.5 N5–Zn64–O1 87.2N5–Zn64–O21 94.3 N13–Zn64–O1 94.1N13–Zn64–O21 86.2 O1–Zn64–O21 136.0

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and pz AOs of the phenolato group in the (HOMO-2) MO, thistransition also has some n → π* character. (ii) The (HOMO-10)to LUMO transition is n → π* in nature, where the electrondensity is transferred from the lone pairs in the alcohol groups ofthe pyridoxal residue to the π* orbital centered around the pyri-dine ring and CvN bond.

1H NMR spectra of [Zn(pydmedpt)] solution

The signals of the various protons observed were tentativelyassigned, with the help of the computed 1H NMR chemicalshifts, by DFT procedures. The experimental and theoreticalvalues are presented in Table 4.

The theoretical 1H NMR chemical shifts were computed forthe optimized structure, which is the lowest energy conformer.However, under the experimental conditions used to record theNMR spectra, the complex will not be present solely in itslowest energy conformer, but as an ensemble of conformers dis-tributed according to the Boltzmann distribution law. Theobserved chemical shifts for a particular type of proton will beaveraged out and will produce a single band. Therefore, for thesame group of protons, there may be more than 1 ppm differenceof shielding constants in the calculated spectrum, while underthe actual experimental conditions they may merge to generate asingle signal. Similar results are obtained using the solvationmodel (Polarization Continuum Model).

The experimental and the calculated 1H NMR chemical shiftsare in good agreement, confirming that the structure of thecomplex in solution closely resembles that obtained by DFToptimization.

Changes of the 1H NMR spectra with pD

The magnitude of the observed chemical shifts of the 1H NMRfor each H atom upon protonation of the Npyridine will changedepending on the nature of the site and its distance to theNpyridine group being protonated. The 1H NMR chemical shiftsof the complex at 298 K in 98% MeOD-d4/2% D2O wererecorded with changes in pD over the range 9.1–5.2. The chemi-cal shift of the pyridoxal aromatic H(a) (Scheme 2) atoms [H(47)

and H(57) (Fig. 2)] should show significant shifts during protona-tion of Npyridine atoms, and indeed significant pD dependence of

the shifts in (δ/ppm) values were observed for protons H(a). The1H NMR spectra recorded are shown in Fig. 4 and the chemicalshifts of the Haromatic are plotted against pD in Fig. 5. The pDtitration curves were fitted to the Henderson–Hasselbalchequation with the assumption that the observed chemical shiftsare weighted averages according to the populations of the proto-nated and deprotonated species. The pKa* (Ka* is the acid

Fig. 3 Pictorial representation of the MOs involved in electronic transitions.

Table 4 Experimental and calculated 1H NMR chemical shifts (δ/ppm)of the [Zn(pydmedpt)] complexa,b

Atomnumber

Type ofhydrogen

Experimentalpeak(multiplicity,number ofprotons)

Theoreticalpeak in gasphase

Theoreticalpeak inmethanolphase

53 CH3 attached toNamine

2.32 (s, 3H) 1.35 1.6362 2.76 2.6054 2.80 2.6537 CH2 of the

aliphatic chain2.00 (br, 4H) 1.83 1.95

43 1.83 2.0136 2.08 2.2642 2.21 2.3758 CH3 attached to

the pyridine ring2.34 (s, 6H) 2.21 2.06

59 2.47 2.6660 2.56 2.6148 2.62 2.6449 2.53 2.6850 2.27 2.0940 CH2 attached to

Namine

2.00 (br, 4H) 2.05 2.4441 3.05 3.0438 2.38 2.7239 3.45 3.3645 CH2 attached to

Nimine

3.70 (br, 4H) 3.49 3.7444 5.15 4.8835 5.15 4.8934 3.50 3.7855 CH2 attached to

OH4.70 (br, 4H) 4.51 4.76

56 4.91 5.1051 4.89 5.1152 4.53 4.7657 pyridoxal H atom

(Haromatic)7.41 (s, 2H) 7.68 7.72

47 7.76 7.7633 HCvN– 8.80 (s, 2H) 9.45 9.4746 9.33 9.46

aAtom numbering as given in Fig. 2. b The experimental 1H NMRspectrum was measured in 98% MeOD-d4/2% D2O (spectrum at “pD9.1” in Fig. 4).

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dissociation constant in the deuterated solvent) values were con-verted to pKa values by the use of the equation pKa = 0.929pKa* + 0.42, suggested by Krężel and Bal74 for comparison with

related values in the literature.75,76 The pKa2 and pKa1 valuesdetermined are ∼5.9 and ∼5.3, and the best fit to the experimen-tal data is shown in Fig. 5.

The 1H NMR spectrum of the [Zn(pydmedpt)] complex at pD9.1 shows 1H NMR signals at (δ/ppm) 7.42 and 8.80 (Fig. 4)due to H(a) and H(b) (Scheme 2), respectively.72,77–80 However,addition of the dilute aqueous acid solution also promotes partialhydrolysis of the [Zn(pydmedpt)] complex (Scheme 2). Thepyridoxal aromatic H(a) shows a signal at 7.61 ppm (H(a1)),which corresponds to the protonated species (CIa and CIb), anda signal at 7.70 ppm (H(a2)) (at pD = 5.6) which probably corre-sponds to species CIIa and/or CIIb, that results from the partialhydrolysis of the pydmedpt ligand. We interpret the signal at7.87 ppm, H(a3), as being due to free pyridoxal (CIII), formedupon total hydrolysis of the complex, the signal observed at∼8.98 ppm (H(b2)) corresponds to the aldehyde proton of thepyridoxal. The hydrolysis of the complex is further confirmed bythe presence of another signal at 6.65 ppm H(c) corresponding tothe –NH2 proton.

The equilibria in solution are therefore complex and upon pro-tonation of [Zn(pydmedpt)] forming [Zn(Hpydmedpt)]+ (CIa)and [Zn(H2pydmedpt)]2+ (CIb), weak peaks at ∼5.58 ppm arealso detected, which probably correspond to the Haromatic ofisomers of these protonated complexes. Isomeric complexesyielding distinct chemical shifts of the Haromatic atom have beenpreviously reported for e.g. [VV(Rpyr2en)] and [Zn(Rpyr2en)](H2Rpyr2en = N,N′-ethylenebis-(pyridoxylaminato)).72,79,80

Fluorescence properties of [Zn(pydmedpt)]

The ligand pydmedpt is fluorescence silent at 469 nm whenexcited at 275 nm (quantum yield 0.1%). The addition of aZn(II) salt results in a prominent emission peak at 469 nm(quantum yield 32.5%). Thus the presences of zinc ions cause analmost 325-fold increase in the quantum yield. The fluorescencespectra of the solutions at different zinc : ligand ratios are givenin Fig. 6.

From the inset graph of Fig. 6, the value of the lower detectionlimit is about 400 μmol L−1 and the fluorescence intensityreaches saturation above 1.7 mmol L−1. The excitation wave-length of 275 nm corresponds to a σ → π* transition where anelectron is transferred from the σ orbital on the Nimine group tothe π* of the pyridine ring system in the complex, as obtainedfrom the TD-DFT calculation (Tables 2 and 3). Since the twopyridoxal units are attached by an aliphatic chain, the structure istoo flexible in the free ligand to give a fluorescence peak, andnon-radiative decay processes prevail. Coordination with zincresults in a rigid structure which gives intense fluorescenceemission.

Reversibility of the fluorescence signal

To establish the mechanism of fluorescence switching, the fluor-escence active solution containing the metal and Zn(II) ion in1 : 1 mole ratio was titrated with an EDTA solution, and fluor-escence spectra were recorded at each stage of EDTA addition. Itis expected that EDTA should sequester and bind zinc from thecomplex, releasing the free ligand. If the fluorescence signal isonly triggered by coordination of the Zn(II) ion to the ligand,

Fig. 4 The 1H NMR chemical shifts of [Zn(pydmedpt)] at 298 K in98% MeOD-d4/2% D2O recorded upon addition of dilute aqueous DClover the pD range 9.1–5.2.

Fig. 5 Dependence on pD of the 1H NMR chemical shifts of the pyri-doxal aromatic H(a) (Haromatic) atom. The curves represent the fit to theHenderson–Hasselbalch equation for determination of pKa* values.

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then the signal will decrease concurrent to release of the ligandto the solution upon addition of EDTA. The change in fluor-escence intensity with addition of EDTA is given in Fig. 7.

As can be seen from the graph, the fluorescence caused by thebinding of the ligand to zinc disappears with the addition ofEDTA. This proves that the coordination of zinc by the

Scheme 2 Protonation and decomposition of [Zn(pydmedpt)] with addition of dilute DCl.

Fig. 6 Emission spectra of 0.10 mmol L−1 of a solution of [H4pydmedpt]2+·2Cl− upon addition of Zn2+ ions in 98% methanol and 2% H2O (v : v)mixed solvent buffered at pH 7.5 by the triethylamine-HCl buffer at room temperature (excitation 275 nm, emission 469 nm); Zn2+ : ligand ratios: (a)–0.0, (b) –0.1, (c) –0.2, (d) –0.3, (e) –0.4, (f ) 10.5, (g) 20.6, (h) 0.7, (i) 0.8, ( j) 0.9, (k) 1.0, (l) 1.1, (m) 1.2, (n) 1.3, (o) 1.4, (p)1.5, (q) 1.6, (r) 1.7, (s) 1.8, (t) 1.9 and (u) 2.0. Inset: Fluorescence intensity versus Zn2+ : ligand ratio.

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pydmedpt ligand is the main driving force towards fluorescenceenhancement.

Specificity of the fluorescence signal

We examined the effects of various metal cation additives on thefluorescence spectral features of the ligand, to examine the selec-tivity of the fluorescence response. The selectivity assay wasdone with metal ion to ligand ratios of 3 : 1, under the same con-ditions in which the fluorescence spectra of the zinc complexwere measured. The high metal ion to ligand ratio hopefullyensures maximum fluorescence enhancement. The metal ionsstudied were Na+, K+, Ca2+, Mg2+, Cu2+, Ni2+, Co2+, Fe2+,Mn2+, Cr3+, VO2+(V4+), Zn2+ and Cd2+, and among these metalions only Zn2+ give a fluorescence signal at the excitation wave-length of 275 nm (Fig. 8).

As mentioned, the free ligand itself is too flexible to give anyfluorescence signal. Na+, K+, Ca2+ and Mg2+ have d0 configur-ation, and form complexes having very low stability. Therefore,the concentration of the fluorescence active rigid complex forthese ions will be very low in solution. Thus, these metal ionsgive only a very weak fluorescence signal. Transition metals, onthe other hand may form stable complexes with the pydmedptligand. However, they also tend to quench the fluorescence of afluorophore through electron transfer from the ligand to vacant dorbitals of the metal.81 The position of the emission band alsodepends on the distance between the Namine donor and the pyri-dine ring and on their mutual orientation. This distance and/ororientation varies from one transition metal complex to another.In fact, Fe2+ gives an emission peak at 391 nm while Cr3+ and

Fig. 7 Emission spectra of a solution containing 0.10 mmol L−1 of [H4pydmedpt]2+·2Cl− and 0.10 mmol L−1 of Zn2+ ions in the presence of (a)0.00, (b) 0.02, (c) 0.04, (d) 0.06, (e) 0.08 and (f ) 0.10 mmol L−1 concentration of EDTA in triethylamine-HCl buffer, pH 7.5 at room temperature(excitation 275 nm, emission 469 nm). Inset: Fluorescence intensity versus EDTA : zinc ratio.

Fig. 8 Fluorescence intensity of solutions containing[H4pydmedpt]2+·2Cl− at pH = 7.5 in the presence of various metal ions.Note that the signal in the case of Zn(II) is multiplied by 10−1.

Fig. 9 Fluorescence intensities for solutions containing Zn2+, theligand and another metal ion in 1 : 1 : 3 molar ratio.

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Mn2+ give emission peaks at 339 nm with moderate intensity(Fig. S-1, S-2 and S-3, respectively in the ESI†). This type ofmetal ion based selectivity has been reported previously.82 It isto be noted that there is no sudden increase in fluorescence inten-sity due to any precipitation during the experiment.

Thus, the fluorescence emission band at 469 nm is specific forthe zinc ion and can be used as an identification tool for Zn2+

Effect of other ions on the fluorescence signal

The effect of other ions on the intensity of the fluorescencesignal was also investigated. The fluorescence spectra wererecorded for a set of solutions containing 0.10 mmol L−1 of theligand, 0.10 mmol L−1 of zinc acetate and 0.30 mmol L−1 ofother ions, namely Na+, K+, Ca2+, Mg2+, Cu2+, Ni2+, Co2+, Fe2+,Mn2+, Cr3+, VIVO2+ and Cd2+ in methanol solutions buffered atpH 7.5 by triethylamine-HCl buffer. The fluorescence intensitiesare presented in Fig. 9.

As shown in Fig. 9 the Na+, K+, Ca2+ and Mg2+ ions exertalmost no effect on the fluorescence spectra of the system. Thiscan be rationalized by considering their poor coordinationability. Compared to these d0 metal ions, the presence of Mn2+,Cr3+ and Cd2+ decrease the fluorescence intensity slightly, whileNi2+, Co2+ and Fe2+ decrease the fluorescence signal signifi-cantly, and Cu2+ and VO2+ totally suppress the fluorescence. Thevarying degree of decrease of fluorescence due to the transitionmetal ions may be ascribed to their ability to form complexeswith the ligand, where the complexes have different stabilities.

Investigation of in vitro insulin-enhancing activity of[Zn(pydmedpt)]

The inhibitory action of [Zn(pydmedpt)] on the FFA-releasefrom isolated rat adipocyte cells treated with epinephrine (adre-naline) was evaluated as percentage inhibition with respect to acontrol. Here the control is the concentration of FFA in themedium without pre-incubation with zinc in any form; the blankwas the amount of FFA in the medium in the absence of epi-nephrine and zinc. The insulin-enhancing activity of the

inorganic zinc salt, ZnSO4, was evaluated as a standard. Thedependence of FFA release inhibition on concentration of thecomplex or ZnSO4 is shown in Fig. 10.

The IC50 values (that is the concentration giving 50% inhi-bition of free fatty acid release), were estimated from a 4-par-ameter logistic nonlinear fit of FFA release inhibition versusconcentration.83 The IC50 values were found to be 0.43 ±0.13 mmol L−1 and 0.66 ± 0.11 mmol L−1 for the complex andZnSO4 respectively. The amount of FFA release when cells werepre-incubated at various concentrations of [Zn(pydmedpt)] andZnSO4 is shown in Fig. 11.

The IC50 values of some complexes reported in the literatureare given in Table 5. To allow an adequate comparison, thevalues are presented considering in all cases the values ofZnSO4 expressed as 1.00 mmol L−1.

Table 5 indicates that the insulin-enhancing activity of [Zn-(pydmedpt)] is on a par with that of several other reported com-plexes; e.g. it is equivalent to [Zn(picolinato)2].

Fig. 10 Dependence of inhibition of FFA release on concentration of[Zn(pydmedpt)] (□) or ZnSO4 (■).

Fig. 11 Amount of FFA release at various concentrations of pre-incubation.

Table 5 Relative IC50 values of some selected zinc complexes,considering ZnSO4 expressed as 1.00 mmol L−1

CompoundRelative IC50values/mmol L−1 Reference

[Zn(pydmedpt)] 0.65 Presentwork

BiszincII 0.61 24

Zn(1-benzyl-3-ethoxycarbonyl-2,5-dihydro-5-oxo-1H-pyrrol-4-olato)2

0.36 24

Zn(γ-pga)a 0.36 84

Zn(maltol)2 0.73 85

Zn(1,2-dihydro-2-oxo-1-pyrimidinolato)2

7.79 86

Zn(ema)2b 0.44 87

Zn(alx)2c 0.37 87

Zn(picolinato)2 0.64 88

Zn(6-methylpicolinato)2 0.31 88

a γ-pga = D,L-poly(γ-glutamic acid). b ema = 2-ethyl-3-hydroxy-4-pyrone. c alx = 3-hydroxy-5-methoxy-6-methyl-2-pentyl-4-pyrone.

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Effect of [Zn(pydmedpt)] on the fluorescence spectra of theDNA–EB complex

It is common and advisable, when featuring the therapeutic useof a compound, to evaluate its interaction with DNA. For thispurpose, the ethidium bromide (EB) fluorescence displacementexperiment is a widely used method.89,90 EB shows no apparentemission intensity in buffer solution due to solvent quenching.However, EB emits intense fluorescence in the presence of DNAdue to its strong intercalation between the adjacent DNA basepairs.89 The competitive binding of any compound to the DNA–EB complex reduces the emission intensity by either quenchingthe emission in the bound complex or by displacement of thebound EB to the free state by the added compound. Therefore,EB can be used as a probe for competitive binding of other com-pounds with DNA.91 The result can be expressed in terms of thevalues of the apparent binding constant of the test compound,Kapp, which may be calculated from the equation,

Kapp½test� ¼ Kapp�EB½EB�;

where Kapp-EB is the apparent binding constant of EB assumed tobe 107 M−1, [EB] is the concentration of EB used, and [test] isthe concentration of the test compound causing 50% decrease oforiginal fluorescence.55,89,92

In our experiments, as depicted in Fig. 12, the fluorescenceintensity of the EB–DNA complex decreases with the increasingconcentration of the complex. This indicates interaction of thecomplex with DNA.

The binding constant was calculated from the intensity datausing the above equation, the value determined for Kapp being2.3 × 106 M−1. The observed constant is in the range reported bySheng et al.93 using a pyridine derivative as ligand. The typicalclassical intercalators and metallo-intercalators give Kapp of theorder94 of 107 M−1. This value of Kapp indicates that [Zn(pyd-medpt)] interacts with DNA in a moderate intercalative mode.

It is known that planarity of the ligand has a significant effecton the intercalating ability of a metal complex with DNA.95 Inthe complex each pyridine ring is coplanar with the adjacent sixmember chelate rings constituted by the coordination ofOphenolato and Ninime with the Zn(II) center (Fig. S-4 in the ESI†).These planar rings, together with their conjugated π electronsystem are responsible for the strong intercalation of the complexwith DNA. Another factor relevant for the interaction of thecomplex with DNA is the protonation state of the two pyridinerings and hence the overall charge on the complex. As men-tioned earlier, the pKa values of the protons at the two pyridinerings in the complex were determined to be 5.9 and 5.3. As thepH for the DNA binding experiment is 7.5, it is expected that insolution the major form of the complex will be the neutralspecies [Zn(pydmedpt)].

Conclusions

A new zinc complex, [Zn(pydmedpt)], was synthesized from arecently reported Schiff base derived from vitamin B6. Thecomplex was characterized by elemental analysis, mass spec-trometry, infrared, electronic and NMR spectral methods, andalso by theoretical studies. The structure of [Zn(pydmedpt)] wasdeduced from DFT optimization. The optimized structure showsa distorted trigonal bipyramid coordination environment aroundthe zinc atom. Each of the two pyridine rings are coplanar withthe adjacent chelate rings formed by the coordination ofOphenolato and Nimine to the zinc center. This planar orientation ofthe ligand is suitably disposed for the DNA binding of thecomplex as an intercalator. The bond lengths and bond angles ofthe optimized structure are in good agreement with related crys-tallographically characterized reported compounds. The elec-tronic transitions of the complex were calculated by TD-DFTmethods and compare well with the experimental ones. Thus, thenature of the electronic spectral bands was assigned with the dataobtained from TD-DFT method.

The experimental 1H NMR peaks show reasonable agreementwith those calculated by DFT methods using the optimizedstructure, thus giving further credence to the predicted structure.1H NMR measurements were recorded at variable pD, the pKa

values of the two protons bound to the pyridine rings of [Zn-(H2pydmedpt)]2+ were determined and the pD dependentdecomposition pattern of the complex evaluated.

This is the first time a pyridoxal derivative has been used as afluorescent probe for Zn2+ ion sensing. The non-fluorescentligand responds giving a strong selective and specific fluor-escence signal in presence of Zn2+ ions, i.e. there is a zinc trig-gered fluorescence switching. Na+, K+, Ca2+ and Mg2+ do notinterfere with this fluorescence switching process. Addition ofMn2+, Cr3+ and Cd2+ slightly decreases the signal, Ni2+, Co2+

and Fe2+ significantly suppress it, while Cu2+, VO2+ suppress itcompletely. The chelating agent EDTA can switch off the fluor-escence signal by coordinating with the zinc ion, releasing theligand to the solution. The fluorescence signal is specific for theZn2+ ion. Na+, K+, Ca2+, Mg2+, Mn2+, Cr3+, Cd2+, Ni2+, Co2+,Fe2+, Cu2+ and VO2+ do not exhibit significant fluorescenceintensity at the emission wavelength under identical conditions.The fluorescence quenching assay on the DNA–EB complexindicates that the complex interacts with DNA as an intercalator.

Fig. 12 Change in fluorescence intensity of the EB (0.32 μmol L−1)–DNA (10 μmol L−1) system when titrated with a solution of [Zn(pyd-medpt)]. Complex concentrations (A) 0, (B) 62.5 μmol L−1, (C)117.5 μmol L−1, (D) 166.5 μmol L−1, (E) 210.5 μmol L−1, (F)250.0 μmol L−1.

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Experiments on inhibitory effects of the complex on FFArelease from isolated rat adipocyte cells clearly showed that theIC50 values of the complex are of the same order of magnitudeas those of several other established insulin enhancing Zn(II)-complexes.

Acknowledgements

The authors wish to thank DST-FIST and UGC-SAP Programmefor providing routine instrumental facilities, University ofKalyani for providing a fellowship (T.M.). J.C.P. and A.K. thankFEDER, Fundação para a Ciência e Tecnologia (FCT), SFRH/34835/2007 and PEst/QUI/UI100/2011 for financial support,and the Portuguese NMR Network (IST-UTL Center).

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