Synthesis, characterization, thermodynamics and biological studies of binary and ternary complexes including some divalent metal ions, 2, 3-dihydroxybenzoic acid and N-acetylcysteine - Widya Mandala Catholic University Surabaya Repository

Contents lists available at ScienceDirect

Journal

of

the

Taiwan

Institute

of

Chemical

Engineers

journal homepage: www.elsevier.com/locate/jtice

Synthesis,

characterization,

thermodynamics

and

biological

studies

of

binary

and

ternary

complexes

including

some

divalent

metal

ions,

2,

3-dihydroxybenzoic

acid

and

N

-acetylcysteine

Shella

Permatasari

Santoso

a

,

Artik

Elisa

Angkawijaya

b

,

Yi-Hsu

Ju

a,∗

,

Felycia

Edi

Soetaredjo

c

,

Suryadi

Ismadji

c

,

Aning

Ayucitra

c

a_{DepartmentofChemicalEngineering,NationalTaiwanUniversityofScienceandTechnology,Taipei10607,Taiwan} b_{InstituteofPlantandMicrobialBiology,AcademiaSinica,Taipei11529,Taiwan}

c_{DepartmentofChemicalEngineering,WidyaMandalaSurabayaCatholicUniversityKalijudan37,Surabaya60114,Indonesia}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9April2016 Revised1July2016 Accepted1August2016 Availableonline9September2016

Keywords:

2,3-Dihydroxybenzoicacid Acetylcysteine

Divalentmetal Complexsynthesis Metal–ligand Thermodynamic

a

b

s

t

r

a

c

t

The binary complexes with the [M(Dhba)] −_{core and ternary complexes with [M(Dhba)(Nac)]} 3−_{core have}

been synthesized and characterized by physical and spectral analyses, where M is divalent metal (Mn 2+_,

Co 2+_{, Ni} 2+_{, Cu} 2+_{or Zn} 2+_{) and Dhba and Nac are 2, 3-dihydroxybenzoic acid and N-acetylcysteine, respec-}

tively. The synthesized complexes are practical in biological applications since they are soluble in wa- ter. The synthesized complexes were found to possess enhanced antimicrobial activity, especially against Staphylococcusaureus, but reduced DPPH scavenging activity of Dhba. The binary and ternary complexes of Zn 2+_{were shown to possess the most remarkable properties thus their structures were further ex-}

amined. Thermodynamic properties of the Zn 2+_{complexes in aqueous solution at various temperatures}

were also determined with an ionic strength of 0.15 mol/dm 3_NaCl.

© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction

Synthesis of chelate complexdrugs is one of the solutions to deal with pathogenic microbes which are becoming more and moredrugresistance[1,2].Bioactiveorganicligandssuchasamino acids, phenolicacids andalkaloids coupled withessential metals are frequentlyemployedinchelatecomplexsynthesis[3–6].2, 3-Dihydroxybenzoic acid(Dhba) is an effective phenoliccompound not only with regard to its biological ability such asantioxidant, antibacterial and anti-inflammatory [7,8], but also to its ability to formstable chelatecomplexes withvarious metal ions[9–11]. Therefore, Dhba was chosen as the ligand for the synthesis of metal–ligandbinarycomplexesinthisstudy.Ternarychelate com-plexesinvolving mixedligands (Dhba and N -acetylcysteine(Nac)) werealsosynthesized.Nacwaschosenastheconsortligandsince ithasbeenknowntoplayanimportantroleasaprecursorof glu-tathione, an important antioxidant, and also known as potential

∗

Correspondingauthorat:DepartmentofChemicalEngineering,National Tai-wanUniversityofScienceandTechnology,#43,Sec.4,KeelungRoad,Taipei106-07, Taiwan.Fax:+886227376644.

E-mailaddress:yhju@mail.ntust.edu.tw(Y.-H.Ju).

sulfur replenishment for living organisms [12,13]. Moreover, Nac canformcoordinationcompoundswithmetalions[11,14,15].

Inthisstudy,binaryandternarycomplexesincludingDhba,Nac andsomedivalentmetalionssuchasMn2+_,Co2+_,Ni2+_,Cu2+_and Zn2+ _{were synthesizedand characterized. The chelatecomplexes} were initially characterized by FTIR and UV–vis spectroscopy analyses.Scavengingactivityandantibacterialactivityofthe com-plexeswereexamined.Subsequently,binaryandternarycomplexes withhighbiologicalactivitywere furtherstudiedusingelemental analysis,1_{HNMRandthermogravimetricanalysis;their} thermody-namicpropertieswerealsodetermined.

2. Experimental

2.1. Materials and instruments

Copper chloride dihydrate (CuCl2·2H2O, 99% purity), cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 98% purity), zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 98% purity), Dhba (C7H6O4, 99% purity) and Nac (C5H9NO3S, 99% purity) were purchased from Sigma–Aldrich (St. Louis, MO); nickel chloride hexahy-drate (NiCl2·6H2O, 98% purity) was obtained from Alfa Aesar (Lancashire, UK); manganese chloride tetrahydrate (MnCl2·4H2O, 99.8% purity) was suppliedby Fisher Scientific (Bridgewater, NJ).

http://dx.doi.org/10.1016/j.jtice.2016.08.003

Sodium hydroxide (NaOH, 96% purity) was supplied by Yakuri Chemical(Kyoto,Japan).Ethanol(C2H6O,95%purity)wasprovided by Echo Chemical (Miaoli, Taiwan). All chemicals were analytical gradeandwereusedwithoutfurtherpurification.

N,C,SandHanalysiswasperformedusinganElementarVario ELCube analyzer(Hanau, Germany).Metal contents (Mn, Co,Ni, Cu,ZnandNa)ofthe complexeswere determinedby anICP-AES JY2000-2.ThecommercialICPmetalstandardsolution(1000mg/l ofmetal in 0.5mol/l HNO3) was used asthe calibrant. Chloride content was determined by using an ion chromatograph Dionex ICS-1000.FTIRspectrawere recordedwithaBio-RadFTS-3500on KBrdiscwithspectrarangeof400–4000cm−1_{.UV–visspectraof} thecomplexes in H2Osolvent were measured in the wavelength rangeof 200–500nm by using a Jasco V-550spectrophotometer. Conductivities of the complexes in water were measured using a Knick Konduktometer 703. Magnetic susceptibilities of powder samples at 300K were measured using a MPMS7 Quantum De-sign SQUID Magnetometer. 1_{H NMR spectra were measured on} a Bruker AVIII-600MHz FT-NMR in D2O solution. Thermogravi-metric analyses in the range of 30 to 900°C were examined by using a Perkin Elmer Diamond TG/DTA. Thermodynamic proper-tiesin0.15 mol/dm3 _{NaCl ionicmedium at varioustemperatures} weremeasuredpotentiometricallyunderN2atmospherebyusinga Metrohm888TitrandopotentiometerwithEcotrodePluspHglass electrode.

2.2. Synthesis of binary and ternary complexes

Binarycomplexes weresynthesizedby dissolvingDhba(0.16g, 1mmol)in14mlH2Owiththeaddition of1ml95% ethanol.The ligand wasallowed to dissolve by slowly adding a few drops of ±5MNaOHsolutionuntilpH11.0wasreached,NaOHsolutionwas directly used without standardization. Five milliliters of 1mmol metal salt solution (0.17g CuCl2·2H2O; 0.30g Zn(NO3)2·6H2O; 0.24g NiCl2·6H2O; 0.30g Co(NO3)2·6H2O; or 0.20g MnCl2·4H2O) wasadded into the solution. The pH againwas adjusted to 11.0 by using ±5M NaOH. The mixture wasallowed to react for 6h withconstant stirring.Any solid in thesolution wasremovedby filtration.The solution wasfreezed in−40°C and then subjected tolyophilization.Thedriedcomplexwaswashedwithethanoland driedina50°Covenfor4h,thenstoredinadesiccator.

Ternarycomplexesweresynthesizedsimilarlytothatofbinary complexeswiththeadditionofNac(0.16g,1mmol).

2.3. Scavenging activity

Scavengingactivityof complexeswastestedagainstthe stable radical2,2-diphenyl-1-picrylhydrazyl(DPPH)[16].Thetested com-plexwaspreparedatconcentrationsof1and2

μ

g/ml. The com-plexwasfirstlydissolvedinwater,thenmethanolwasaddedata watertomethanolratioof1:19(v/v).DPPHsolutionwasprepared ataconcentration of5×10−4 _{mol/linaqueoussolution withthe} samewaterto methanolratio.DPPH solution(0.2ml)was added tothepreparedcomplex(0.8ml).Thetestedcomplexesincubated at37°C for 30min andits absorbance was measured at 517nm against methanol blank. DPPH (0.2ml) in methanol (0.8ml) was usedasthecontrol.PercentDPPHinhibitionwascalculatedas:

%DPPHinhibition=

(

[A c−A s]/A c

)

×100 (1)

where A cand A saretheabsorbanceofcontrolandtheabsorbance of sample, respectively. Ascorbic acid was used as the positive controls.

2.4. Antimicrobial activity

The antimicrobialpotencyofthecomplexeswastestedagainst grampositive Staphylococcus aureus andgram negative Escherichia

coli .Brothmacrodilutionmethodwasusedfortheinhibitory activ-itydetermination[17].Ampicilin(895.5

μ

g/mlpotency)wasused asthereference.Thecomplexwaspreparedatfourconcentrations (100,600,1000and2000

μ

g/ml).Theassaywasperformedintest tubes eachcontainingthetestedcompound dissolvedin Lysogeny Broth media. Fifteenmicroliters of the preparedbacteria suspen-sion(1×108_{cfu/ml)wereinjectedintoeachtube.Afterincubated} for24hat37°C,opticaldensityat600nm wavelength(OD600nm) ofeachsamplewasmeasured.Asthegrowthcontrol,15

μ

lof bac-teriasuspensionwasinjectedintotesttubecontainingnosample. Theantimicrobialactivityisexpressedas%inhibition,calculatedas:

%inhibition=

(

[I c−I s]/I c

)

×100 (2)

where I c is theabsorbanceofcontrol and I s istheabsorbanceof sample.

2.5. Thermodynamic properties

Thermodynamicpropertiesweredeterminedpotentiometrically by titrating50 cm−3 _{mixtureofmetal salts (0.001mol/dm}3_)and 0.001–0.003mol/dm3 _{ofDhbaand/or Nac.The mixturewas} acid-ifiedto pH2.5 byadding 0.003mol/dm3 _{HCl.Theionic strength} maintainedbyadding0.15mol/dm3_{NaCl.Themixturewastitrated} by 0.1 mol/dm3 _{carbonatefree-NaOH underN}

2 atmosphereuntil pH11.0wasreached.Stabilityconstantofbinarycomplexwas de-terminedatmetaltoligandratiosof1:1,1:2and1:3whileternary complexwasdeterminedfrommetal toligandratioof1:1:1. Sta-bilityconstantsweredetermined at25,45and55°C.Analysesof thetitrationdataweredonebyusingHyperquad2008[18].

3. Resultsanddiscussion

Metal–ligand complexes with deprotonated species as core (specifically [M(Dhba)]− _{and [M(Dhba)(Nac)]}3−_{, for binary and} ternaryspeciesrespectively)weresynthesizedatpH11.0.ThispH waschosen forthesynthesis reactionsinceatthispH valuecore specieswerefoundtoexistinthehighestconcentration,asshown inthedistributiondiagraminSupplementaryDataFig.S1forZn2+ systems. The synthesized complexes were then characterized by FTIR and UV–vis spectra analyses. DPPH scavenging and antimi-crobialactivities ofcomplexes wereevaluated. Theresults of ele-mentalanalysis,conductivityandmagneticmeasurements ofZn2+ binaryandternary complexesare presentedinthissection while theresults ofother metal ionscomplexes aresummarized inthe supplementarydata(TableS1).

3.1. FTIR spectra

3.1.1. Binary complexes

Thecharacteristic mainbandsinIR spectraofthe ligandsand binary complexes are listed in Table 1, while the IR spectra are presentedinthesupplementary data(Figs.S2 andS3). Inthe ab-senceofmetalions,Dhbapossessedbandsat3240cm−1_and3048 cm−1 _{corresponding to v (OH) ofhydroxylandcarboxylgroup,} re-spectively.TheIRspectrumofthefreeDhbarevealsabandat1680 cm−1 _{due to v (C}=_{O) and v (C–OH) of protonated carboxyl group,} respectively.Thebandat1566cm−1 _{isattributedtothe v (C=C)of} Dhbaaromaticring,inthecomplexesthisband isfoundat1530– 1548cm−1_{.Severalnewbandscanbefoundinthemetalcomplex} system. Thefirstis the v (OH) bandat3478–3471cm−1 _and837– 811 cm−1 _{regions are due to coordinated water molecule [6,19].} Twonewbandsat1618–1617cm−1 _{and1473–1431cm}−1_are con-tributedto v (COO−₎

Table1

SelectedIRspectraforthesynthesizedbinaryandternarycomplexes.

Complex IRspectra(cm−1₎

Binary Ternary

v(OH) v(C=_{O) v(COO}−

) v(C=_{C) v(C}–OH) v(MO) other v(NH) v(OH) v(SH) v(C=_{O) v(COO}−

) v(C=_{C) v(C}–OH) v(MO) Other

Ligand

Dhba 3240 1680 – 1566 1259 – – – 3240 – 1680 – 1566 1259 – –

3048 3048

Nac – – – – – – – 3376 2810 2548 1718 – – 1229 – –

796

Complexof

Copper 3478 – 1618 1548 1216 477 620 755 3449 – – 1548 1548 1217 516 588

811 1451 806 1440

Zinc 3471 – 1618 1537 1192 476 1384 746 3385 – – 1627 1573 1214 457 1384

820 1445 1222 810 1501 1254

Nickel 3477 – 1617 1533 1196 476 621 745 3373 – – 1628 1565 1228 458 599

837 1445 834 1502

Cobalt 3478 – 1618 1530 1191 476 1384 758 3352 – – 1615 1580 1218 457 1384

834 1473 1251 826 1471 1258

Manganese 3478 – 1618 1530 1218 478 621 746 3371 – – 1659 1566 1220 457 597

813 1431 813 1492

The v (C–OH)bandat1191–1218cm−1_{isduetoethanol.The} com-plexofCu2+_,Ni2+_andMn2+_{possessedbandat621–620cm}−1 in-dicatingtheionicCl−_;whileforZn2+_andCo2+_{thebandsat1384} and1251–1222cm−1_{indicatethecoordinatedNO}

3− in monoden-tatemode[20].TheionicgroupsofCl−_andNO

3−wereoriginated fromthetype ofmetalsaltsused.Thenewbandappearedat477, 476,476,476and478cm−1_{areattributedto v (MO)forCu}2+_,Zn2+_, Ni2+_,Co2+_andMn2+_{,respectively.}

3.1.2. Ternary complexes

TheIRabsorption bandsoftheternary complexesarelistedin

Table1aswellasbinarycomplexes(thefullspectraarepresented in supplementary data Fig. S4). The ligand Nac v (NH) stretching andbendingvibrationwasobservedat3376and796cm−1_, respec-tively. The stretch of v (OH), v (C=_{O) and v (CO) bond from} proto-nated carboxylategroupproduced bandat2810cm−1_,1718cm−1 and1229cm−1 _{respectively.Thethiol v (SH)frequencyofNacwas} characterized at2548 cm−1_{,while inthe complexthisband} dis-appeared,evidencingthatthisgroupwasinvolvedinchelation af-terdeprotonation.Thesynthesizedternarycomplexesinheritedthe main spectra fromboth Dhba and Nac. The v (OH) at 3449–3352 cm−1_{andbandat834–806cm}−1 _{inallmetalsystemsindicatethe} coordinated watermolecule [6,19].The bendingof v (NH)at758– 745 cm−1 _{was still visible in the complexes, while the} stretch-ing of v (NH)at higherregioncannot be observed sincethe band is probably stacked with the v (OH) band. Two bands at 1659– 1548 cm−1 _{and 1502–1440 cm}−1 _{are contributed to v (COO}−₎

asy and v (COO−₎

sym of the deprotonated carboxylic group,

v =108– 167cm−1 _{indicating the monodentate binding of the carboxylic} group [6,19]. The v (C–OH) band at 1214–1228 cm−1 _{is due to} ethanol. Theionicgroup Cl− _{originatedfrommetalsaltsappeared} at 599–588 cm−1 _{for Cu}2+_{, Ni}2+ _{and Mn}2+_{, while coordinated} NO3− in monodentated mode appeared at 1384 and 1258–1254 cm−1 _forZn2+ _andCo2+ _{[20].The band which characterized the} bondofligandtometal, v (MO)wasobservedat516,457,458,457 and457cm−1_forCu2+_,Zn2+_,Ni2+_,Co2+ _andMn2+_{,respectively.}

3.2. UV–vis spectra, conductivity and magnetic properties

Absorptionspectraofthesynthesizedbinaryandternary com-plexes are shown in Fig. 1. The complexes show two absorp-tion bands at 214–230nm and314–360nm which correspond to

π

–

π

∗ _andn–

π

∗ _{bonding ofthe benzenering [21].In binaryand} ternary cobalt complexes, n–

π

∗ _{bonding was observed at longer}

wavelength(370–395nm),whileformanganesecomplexesthe ab-sorbance of this bonding was low and cannot be observed. The absorption bands ford-orbital of the metals cannot be observed since usually these spectra have low absorbance. In particular for ternary complex of Co2+_{, the spectrum corresponding to d-} orbitalisobservedat499nm whichisattributedtothetransition 4_A

2g(F)→4T1g(P),indicatingitsoctahedralgeometry[22].

Magneticsusceptibilities (µ_eff) ofthe binaryandternary com-plexeswere measured at 300K andare summarized inTable S1. Low µeff values (0.69–1.00BM for binary and 0.62–1.75BM for ternarycomplexes)suggestthatthecomplexesonlyhaveone un-pairedelectron,andthecomplexesexhibitweakparamagnetic be-havior. In Zn2+ _{binaryand ternary complexes, the results of µ}

eff indicatediamagneticbehaviorofthecomplexes.Meanwhile, Co2+ ternary complexexhibitshigher µeff (2.38BM) thanother ternary metal complexes, this value is often observed forCo2+ _complex withoctahedralgeometryandisalsosupportedbytheoccurrence ofthebandat499nminUV–visspectra.

The molar conductivity (

M) measurements gave values of 153–179Scm2_{/molforbinarycomplexes,whileforternary} com-plexes the values were 217–242 S cm2_{/mol (Table S1).}

M of a complexindicatestheelectrolytenatureofthecomplex.The elec-trolytebehaviorofacomplexmightbeduetothepresenceofNa+ ionswhichwereinvolvedintheformationofthecomplex.Higher

MofaternarycomplexisbecauseitcontainsmoreNa+ionsthan abinarycomplex.

3.3. Biological activities

3.3.1. DPPH scavenging activity

Scavengingactivityofthecomplexesatconcentrationsof1and 2mg/l were evaluated against DPPH. The testsin scavenging ac-tivitywere done intriplicate.The results are summarizedas the averagevalue,andthedifferencebetweenthethreetestswas indi-catedbytheerrorbars,asshowninFig.2.Thesynthesizedbinary andternarycomplexesdidnotshowanyincreaseinDPPH inhibi-tioncomparedtoDhba.ThepossiblereasonisthatoriginallyDhba inhibits DPPH by transferring H atom from —COO and meta —O group [23] whileNacinhibits DPPHby transferring Hatom from —COOgroup[24];intheformedcomplexHatomsofligandwhich were originallyinhibiting free radicalformation disappearedthus inhibitionactivityactuallydecreased.

Fig.1. Spectraofthebinaryandternarycomplexesatconcentrationof187.5mg/l.

Fig.2. DPPHscavengingactivityofthecomplexes,∗_{AA-ascorbicacidasthestandardreference.}

Nac,where aminegroup ofNacalsocontributes tobonding with DPPHthroughweakhydrogenbond.Howeverforternarycomplex ofCu2+_{, theinhibition activity islower than that ofbinaryCu}2+ perhapsdueto the reactionof NacandCu2+ _{whichis known to} producefreeradical[25].

3.3.2. Antimicrobial activity

Antimicrobialactivitiesofthesynthesizedcomplexeswere eval-uated against E. coli and S. aureus . The inhibition values are re-ported as Supplementary Data (Tables S2–S4). Dhba was shown tohavebetterantimicrobialactivitythanNac.Forthesynthesized complexes,some exhibited lower antimicrobialactivity than that ofDhba.Thecomplexeswerefoundtobemoreeffectivein inhibit-ing S. aureus .It wasfoundthat in binaryandternary complexes, thecomplexofZnpossessedthehighestinhibitionactivityagainst

thegrowth ofbacteria.Thus it isworthwhile to diagnosethe ef-ficacyof zinccomplexes bydetermining the minimuminhibitory concentration (MIC) as shown inTable 2. The complexes of zinc gavehigherinhibitionactivitythanligandalone(especiallyagainst S. aureus )asindicatedbythesmallerMICvalues.

Fig.3. StructureofligandDhba(left)andNac(right).

Table2

MICa_{(μg/ml)ofthetestedcompounds.}

Compound E.coli S.aureus

MIC50 MIC90 MIC50 MIC90

Ampicilin 4.1 49.1 0.3 2.6 Dhba 589.8 1085.7 702.0 932.6 Nac 1293.9 >2000 >2000 >2000 Binaryzinc 603.5 867.0 149.3 383.8 Ternaryzinc 773.9 971.6 347.1 554.8

MIC50andMIC90aretheminimumconcentration

require-menttoinhibit50%and90%growthofmicroorganism, re-spectively.

containinglayer lowersthe permeabilityof thecomplex intothe bacteriainnermembraneandcausedecreaseininhibitionactivity.

3.4. Complexes of zinc

SincethecomplexesofZnshowhigherbiologicalactivitythan othermetalcomplexes,onlyZncomplexeswerechosenforfurther characterizationincludingelementalanalysis,1_{HNMRspectra,and} thermogravimetricanalysis.

3.4.1. Binary complex of zinc

ThebinarycomplexofZnhasthechemicalformulaC9H11NNa2 O9Zn or Na2[Zn(NO3)(C7H3O4)(H2O)]·C2H6O, mw =388.54. It is darkgreen-brownincolor.Elementalanalysisonthecomplex in-dicated its composition asfollows:C,27.89; H,2.84;N,3.69; Na, 11.80;Zn,16.52%whilethecalculated(theoretical)compositionis: C,27.82; H,2.85;N,3.61;Na,11.83;Zn,16.83%.The 1_HNMR sig-nalsofDhba(Fig.3)werefoundat

δ

H7.47(d,1H,H-1, J =6.5Hz), 7.16 (d, 1H, H-2, J =6.4Hz), 6.88 (t, 1H, H-3, J =8.0). The binary complexsignalswereshiftedtolowervaluesspecifically

δ

H7.12(s, 1H,H-1),6.82(s,1H,H-2),6.61(s,1H,H-3),followedbynew sig-nals at

δ

H 3.69(q, 2H,CH2, J =7.1Hz),1.22(t,3H,CH3, J =7.1Hz). The1_{HNMRsignalsarepresentedinTable3,whilethespectrums} are presentedinFigs.S5 andS6. Thenew1_{HNMRsignalsinthe} complex were observed which corresponds to ethanol molecule. The attachment of ethanolmolecule may also causethe shiftin v (OH)IRspectraat3471cm−1_.

Asindicatedintheelementalanalysis,Na+ _{moleculeswere} in-volved intheformation ofcomplex.Na+ _{moleculesseem tohelp} neutralizingthechargeofthecomplex,whichoriginallyhasa neg-ativecharge of2,thus isolationof thecomplex insolid phaseis possible.Resultofelementalanalysissuggeststhattherewasone NatomcorrespondingtoNO3involvedincomplexformation.Thus theproposedstructureofzincbinarycomplexisshowninFig.4.

Fig.4. Proposedstructureofzincbinarycomplex.

3.4.2. Ternary complex of zinc

Ternary complex was found with chemical formula C14H18N2 Na4O12SZn or Na4[Zn(NO3)(C7H3O4)(C5H7NO3S)(H2O)]·C2H6O, mw =595.70.Thecomplexhasadarkpale-browncolor.Elemental analysisfound the composition as: C, 28.46; H, 3.06;N, 4.72; S, 5.22;Na, 15.31; Zn, 11.01% while thecalculated (theoretical) one is:C,28.23;H,3.05;N,4.70;S,5.38;Na,15.44;Zn,10.98%.More complicated 1_{H NMR signals were observed in ternary complex.} 1_{H NMR signals forDhba were the same asthose in the binary}

complex,while for Nac(Fig. 3) the signals observed are

δ

H 4.66 (q, 1H, CH, J =4.9Hz), 3.02 (sep, 2H, CH2, J =9.4Hz), 2.11 (s,3H, CH3). 1H NMR signals for zincternary complex (Table 5) are

δ

H 7.12–7.03(m,1H,H-1),6.78(br,1H,H-2),6.69–6.59(m,1H,H-3), 4.53(q,1H,CHNac, J =4.1Hz),3.69(q,2H,CH2ethanol, J =7.1Hz), 3.02–2.99(dd,2H,CH2 Nac, J =8.9Hz),1.60(s,3H,CH3 Nac),1.22 (t,3H,CH3 ethanol, J =7.1Hz).The1HNMRspectrumsofNacand ternarycomplexarepresentedinFigs.S5andS6.

Similarly in Zn ternary complex, Na+ _{molecules also helped} neutralizingthechargeofthecomplex,whichoriginallyhasa neg-ativecharge of 4. Elemental analysis resultalso suggests the in-volvementofNO3incomplexformation.Thusthestructureofzinc ternarycomplexcanbeproposedasshowninFig.5.

3.4.3. Thermogravimetric analyses

Table3

Selected1_{HNMRdataofzincbinaryandternarycomplex.}

Protonchemicalshifta_(assignment)b

Ligand Znbinarycomplex Znternarycomplex

7.47 (d,1H,H-1Dhba) 7.12 (s,1H,H-1) 7.12–7.03 (m,1H,H-1Dhba) 7.16 (d,1H,H-2Dhba) 6.82 (s,1H,H-2) 6.78 (br,1H,H-2Dhba) 6.88 (t,1H,H-3Dhba) 6.61 (s,1H,H-3) 6.69–6.59 (m,1H,H-3Dhba)

3.69 (q,2H,CH2) 4.53 (q,1H,CHNac)

4.66 (q,1H,CHNac) 1.22 (t,3H,CH3) 3.69 (q,2H,CH2ethanol)

3.02 (sep,2H,CH2Nac) 3.02–2.99 (dd,2H,CH2Nac)

2.11 (s,3H,CH3Nac) 1.60 (s,3H,CH3Nac)

1.22 (t,3H,CH3ethanol)

a _{Protonchemicalshift(δ)inppmunit.}

b_{Signalassignmentinparenthesisrepresentsthesplittingpattern,numberofproton,}

pro-tonlocationinthe compoundgeometryandsplittingdistance.Splittingpattern abbrevia-tions:s=singlet,d=doublet,t=triplet,q=quartet,sep=septet,dd=doubletofdoublets, br=broad,m=multiplet(complexpattern).

Table4

EquilibriumconstantsatvarioustemperaturesandIof0.15mol/lNaCla_.

Parameters Equilibriumconstantsatdifferenttemperature

25°C 37°Cb _{45°C 55°C}

Dhba Nac Dhba Nac Dhba Nac Dhba Nac

Dissociationconstant

pKa1 2.68(2) 3.17(3) 2.63(4) 3.18(2) 2.60(3) 3.12(2) 2.54(1) 3.12(1)

pKa2 10.11(2) 9.60(2) 9.98(2) 9.48(5) 9.84(4) 9.42(2) 9.81(4) 9.33(2)

pKa3 ndc – 13.00(2) – 12.72(1) – 12.60(2) –

σd _{1.13 1.06 1.02 1.02 1.05 1.26 1.23 1.09}

Binary–stabilityconstant

log10β1 10.55(3) 6.53(7) 10.48(4) 6.23(8) 10.25(2) 6.19(2) 10.02(5) 6.05(5)

log10β2 16.53(4) 12.11(2) 16.52(9) 11.97(8) 16.51(7) 11.98(4) 16.11(3) 11.91(4)

σd _{1.08 1.34 1.48 1.41 1.40 1.21 1.11 1.19}

Ternary–stabilityconstant

log10βT 16.59(8) 16.38(7) 16.02(5) 15.51(6)

σd _{1.56 1.55 1.61 1.69}

log10K −0.49 −0.33 −0.42 −0.56

log10X 4.54 4.27 3.55 3.00

a _{Standarddeviationsinparenthesesatlastdecimalplacerepresentthestandarduncertaintiesoftemperature}

u(T)=0.1°C.

b_{ValuescitedfromRefs.[11]and[15},potentiometrymethodatI}=0.15mol/lNaCl.

c_Notdefined.

d_{Sigma(σ),thegoodnessoffittinginasystemwithexpectationvalueof1.00.}

Fig.5. Proposedstructureofzincternarycomplex.

3.4.4. Effect of temperature on thermodynamic parameters

Thedissociationconstant(pK a)ofDhbaandNacaswellasthe stabilityconstant (log10

β

) oftheir binaryandternary complexes withZn2+ _{in aqueous solutionwith I =0.15 mol/l NaClwere} de-terminedat differenttemperatures (25°C, 37°C, 45°C and55°C) andthe results are presented inTable 4. Particularly for pK a3 of Dhbaat25°C,abigerroroccurredduringrefinementthus result-inginundefined value.The errormaybecaused bythe decrease

ofthe acidityof theligandDhba atlow temperatureresultingin delayin dissociation.The change ofacidity is well demonstrated inTable 4 since pK a value decreased asthe temperaturewas in-creased. In order to observe the capability of the ligand in pre-venting the formationof metal hydrolyticspecies, the hydrolysis constantswereincludedinthedeterminationoflog10

β

.No metal hydrolytic species were observed in the systems, indicating that the ligands were capable in preventing their formation (Fig. S1). The log10

β

of binary andternary complexes also decrease with increasing temperature indicating that complex is more likely to disassociateathighertemperature.

Additional parameters

log10K and log10X were determined with respect to the ternary species, where their values are calculatedas:

log10K = log10

β

Zn(Dhba)(Nac)−[log10

β

Zn(Dhba)+ log10

β

Zn(Nac)] (3)

log10X = 2log10

β

Zn(Dhba)(Nac)−[log10

β

Zn(Dhba)2+ log10

β

Zn(Nac)2] (4)

Table5

ThermodynamicspropertiesofDhba,Nac,binaryandternarycomplexesofZn.

Species −G(kJ/mol) −H(kJ/mol) S(J/K·mol)

25°C 37°C 45°C 55°C 25°C 37°C 45°C 55°C

H3Dhba 15.30 15.68 15.84 15.96 8.49 22.85 23.19 23.11 22.78

H2Dhba 57.72 59.27 59.95 61.64 19.45 128.35 128.39 127.29 128.57

HDhba – 77.20 77.49 79.17 41.80 – 114.14 112.17 113.88 H2Nac 18.10 18.89 19.01 19.60 11.24 22.99 24.64 22.68 23.38

HNac 54.81 56.30 57.39 58.62 16.33 129.07 128.89 129.06 128.90 Zn(Dhba) 60.23 62.24 62.44 62.96 33.04 91.19 94.14 92.41 91.18 Zn(Dhba)2 94.37 98.11 100.58 101.23 22.77 240.15 242.91 244.57 239.09

Zn(Nac) 37.28 37.00 37.71 38.02 28.36 29.93 27.86 29.40 29.43 Zn(Nac)2 69.14 71.09 72.98 74.84 11.43 193.56 192.37 193.48 193.24

Zn(Dhba)(Nac) 94.71 97.28 97.59 97.46 65.78 97.04 101.56 100.00 97.64

indicating thatthe formationofternarycomplex islesspreferred than binary complex. Such phenomenon probably occurred be-cause of electrostatic effect between the ligands thus lowering thestabilityofthecomplex.Meanwhile,forlog10X parameter,the value higherthan statistical value (+0.6 forall geometries) indi-cates remarkable stability of ternary complex. All of the ternary complexesexhibitpositivelog10X greaterthanthestatisticalvalue. Thispointoutthatthesecondaryligand(Dhba)preferstoattachat thebinarycomplexZn(Nac)ratherthanattachtometalion.Nacis designatedastheprimaryligandandDhbaasthesecondaryligand since theformation ofNacbinarycomplexes took place atlower pHthanthatofDhbabinarycomplexes.Thelog10X decreaseswith increasingtemperature,indicatingthatternarycomplexisless sta-bleat highertemperature. Thistrendissupported by thelog10

β

valueswhichalsodecreasedwithincreasingtemperature.

Thermodynamicpropertiessuchas

G (Gibbsfreeenergy),

H (enthalpy) and

S (entropy) provides significant information re-latedtothecomplex.

G canbecalculatedas:

G = −2.303RT log10

β

(5)

H canbedeterminedastheslopeoftheVan’tHoff plotwhich isdefinedas:

log10

β

=

−

H

2.303R

1/T +

−

S

2.303R

(6)

Thermodynamic propertiesof Dhba,Nacaswell astheZn bi-naryandternarycomplexesaregiveninTable5.

Negative values of

G and

H in the dissociationof the lig-ands and chelation of binary and ternary Zn complexes indicate that thereactions arespontaneous andexothermic, sothat lower temperatureismorefavorableforcomplexsynthesis.All

S values arepositiveindicatingthatcomplexformationisfavorable.

4. Conclusion

BinaryandternarycomplexesofM/Dhba/Nacweresynthesized. FTIR spectraandUV–vis spectraindicate that all complexeshave similar structure. The complexes did not increase the scavenging activityofDhbaagainstDPPH.Howeverintheantimicrobial evalu-ationsagainst S. aureus and E. coli ,thecomplexeswerefoundtobe moreeffectivethanthatofDhbawhichiscausedbythe enhance-mentofhydrophobicity ofthecomplexes.Thebinaryandternary complexes of Zngave the bestenhancement in antimicrobial ac-tivityofDhbaandwerecharacterizedfurther.Thebinarycomplex was found to bind metal ionthrough ortho and meta —O group, which has the molecular structure C9H11NNa2O9Zn. The ternary complexwasfoundtobindmetalionthrough ortho and meta —O groupofDhba,and—COOand—SofNac,whichhasthemolecular structureC14H18N2Na4O12SZn.Theformationofbinaryandternary complexwasfoundtobespontaneousandexothermic.

Acknowledgments

Thisstudywassupported byaproject(MOST 103-2221-E-011-148)fromtheMinistryofScienceandTechnology,Taiwan.The au-thorsthankShang-MingTsengofInstrumentationCenter,National Taiwan University for magnetic (MPMS7 Quantum Design SQUID Magnetometer)experiments.

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.jtice.2016.08.003.

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