Self immolative dioxetane based chemiluminescent probe for H2O2 detection

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ContentslistsavailableatScienceDirect

Sensors

and

Actuators

B:

Chemical

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b

Self

immolative

dioxetane

based

chemiluminescent

probe

for

H

2

O

2

detection

Ozlem

Seven

a

_,

_Fazli

_Sozmen

b

_,

_Ilke

_Simsek

_Turan

a,∗

a_{UNAM-National}_{Nanotechnology}_Research_Center,_Bilkent_University,_Ankara_06800,_Turkey b_Department_of_{Nanotechnology}_Engineering,_Cumhuriyet_University,_Sivas_58140,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30May2016

Receivedinrevisedform7September2016 Accepted20September2016

Availableonline20September2016 Keywords: Hydrogenperoxide Self-immolation 1,2-dioxetanes Chemosensors Chemiluminescence

a

b

s

t

r

a

c

t

ChemiluminescentdetectionofH2O2hasbeenachievedbyusingselfimmolativedioxetanebasedprobe whichenablesthesignalampliﬁcationviadisassemblyoftwochemiluminogenicmodulesatthesame timeinresponsetosingleanalyte.UpontreatmentoftheprobewithH2O2,boronateesterwas depro-tectedsubsequentlytotriggerthedecompositionof1,2-dioxetaneringviaCIEELmechanismwhich resultsinlightemissionasaselectivesignofH2O2.

1. Introduction

Asa relativelymildreactiveoxygenspecies(ROS),hydrogen peroxide(H2O2)isrecognizedas amessengermoleculein var-ioussignalingprocesses therewithalitis alsoincludedin many biologicalprocesseslikebiosynthesis,immuneresponseandcell signaling[1–3].Additionally,inmanyenzymaticreactions,H2O2is alsoproducedasabyproduct.Ontheotherhand,likeotherreactive oxygenspecies,excessiveproductionofH2O2cancauseoxidative stresswhichbringsaboutthedevelopmentofmanydiseasessuch asAlzheimer’sdisease,Parkinson’sdisease,cardiovasculardisease andHuntington’sdisease[4–6].Inotherwords,intermsof bene-ficialsignalingprocessesandharmfuloxidativestressproperties, thereisadelicatebalancefortheamountsofROS.Thisbalance isdisruptedinfavorofoxidativestresswithaging[7,8].So,the determinationofthesespecieswillallowustoidentifythe pro-duction,accumulation,traffickingofROSandalsorelateddiseases. Therefore,developmentofanalyticaltechniquesforselectiveand sensitivedetectionofH2O2isurgentlyneededinahigh-throughput fashion.Intheliterature,therearemanyproposalsforthe detec-tionofH2O2byusingchromogenicandfluorogenicprobes[9–13]. Asnewinsightsintotheopticalsensingsystems, chemilumines-cence(CL)basedoneswouldbepromisingduetotheirsuperior

∗ Correspondingauthor.

E-mailaddress:ilke.simsek@bilkent.edu.tr(I.SimsekTuran).

advantagessuchasoperationalsimplicity,costeffectiveness,rapid andhighsensitivity ofthetarget,beingfree frominterferences causedbylightscatteringandreducedbackgroundnoisedueto theabsenceofphotonicexcitation[14–16].InCLbasedsystems, lightemissionisobtainedasaresultofspeciﬁcchemicalreaction whichis uniquetotheanalyteof interest.Duetotheemission oflight uponinteractionwiththeanalyteofinterest,CLisalso acceptedascoldlight.Asaresult,chemiluminescencebased sen-sorisdesigned totransform chemicalinformation intoauseful signalasalightemission. Althoughchemiluminescencesensors haveshorterlifetimescomparedtoﬂuorescencesensors,they fre-quentlyhavelowerbackgroundemissionthatmakestheCLasa powerfulanalyticaltechniqueofferinghighsensitivity,wide lin-earrangeandsimpleinstrumentation[17,18].Although,thereare afewreportsforthechemiluminescencebaseddetectionofH2O2

[19,20],untilnow,tothebestofourknowledge,noreportsbased onthechemiluminescencebaseddetectionofH2O2byusing 1,2-dioxetanebasedself-immolativesystemshavebeenpublished.

As a chemiluminogenic unit, our choice was a stable 1,2-dioxetanes which is a four-membered cyclic peroxide usually implicated asthe reactiveintermediates in bioluminescenceas wellasoxalateesters,luminolandacridiniumesters[21–23].Use of1,2-dioxetane derivativesaschemiluminogenicunit isa very promisingalternativestrategysinceitoffersdirect chemilumino-genicresponsewhichcanbetriggeredbythecleavageofachemical bondundermildreactionconditionsspeciﬁctotheanalyteof inter-est[24–26].Thedecompositionof1,2-dioxetanesthat produces

http://dx.doi.org/10.1016/j.snb.2016.09.120

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chemiluminescenceisprobablyoneofthemostinterestingtype of chemical reactionsand therefore it hasbeen focus of many chemical and biological research[27,28].The decomposition of 1,2-dioxetanesisinitiatedwiththeremovaloftriggeringmoiety whichresultsintwomainproducts,oneofwhichisformedinthe excitedstateundergoinganelectrontransferaccordingtoCIEEL (Chemically InitiatedElectron ExchangeLuminescence) mecha-nismandeventuallyrelaxesradiatively withapeakemissionat 466nmwhichdependsonthestructureofthe1,2-dioxetanes.The chemicallyinitiateddecompositionmechanismof1,2-dioxetanes isgenerally triggeredwitha catalyst suchas abaseor ametal ion. Until now, designed chemiluminogenic agents rely onthe decompositionofsinglesubstituted1,2-dioxetanesupon interac-tionwiththesingleanalyte[17,29–33].However,self-immolation enablesthefragmentationoflargemoleculesleadingtothe for-mationofmultipleexcitedstateanionsuponinteractionwiththe singleanalytewhichcouldleadtovaluablesignalampliﬁcationin chemosensorstargetingreactiveanalyteswhenusedjudiciously [34–36].

Herein, we design and synthesize 1,2-dioxetane based self immolative chemiluminogenic probe for H2O2. We wanted to incorporatea self immolativelinker totriggertwo chemilumi-nescenceprocessesatthesametime,inresponsetosingleH2O2 mediateddeprotectionevent.Self-immolationoftheprobewas ini-tiatedwiththedeprotectionoftheboronateestergroupleadingto lightemission.Untilnow,tothebestofourknowledge,noreports forchemiluminescentdetectionofH2O2byusingaself immola-tive1,2-dioxetanebasedsensorshavebeenpublished.Thisﬁnding mayserveasaplatformforfutureH2O2sensorapplications.This studyprovidesanewinsightintoapplicationsof chemilumines-cencebasedsensors.

2. Materialsandmethods

2.1. Materials

Spectrophotometricgradesolventswereusedforspectroscopy experiments.Flashcolumnchromatography(FCC)wasperformed byusingaﬂashgradesilicagel(MerckSilicaGel60(40–63␮m)). Reactions were monitoredby thin layerchromatography (TLC) usingprecoatedsilicagelplates(MerckSilicaGelPF-254), visu-alizedbyUV–vislight.Allorganicextractsweredehydratedover anhydrousNa2SO4 andconcentratedbyusingrotaryevaporator beforebeingsubjected toFCC. Allotherchemicalsand solvents weresuppliedfromcommercialsourcesandusedasreceived. 2.2. Intsruments

1_H_NMR_and13_C_NMR_spectra_were_recorded_on_Bruker Spec-trospinAvanceDPX400spectrometerusingCDCl3asthesolvent. Chemicalshiftsvalues arereportedinppm from tetramethylsi-laneasinternalstandard. Spinmultiplicitiesarereportedasthe following:s(singlet),d(doublet),m(multiplet).HRMSdatawere acquiredonanAgilentTechnologies6530Accurate-MassQ-TOF LC/MS.ChemiluminescencemeasurementsweredoneonaVarian Eclipsespectroﬂuorometer.ApHmeter(Oakton,manufacturedby Eutechinstruments)wasusedtodeterminethepH.

2.3. Synthesisofcompounds 2.3.1. Synthesisofcompound(8)

Compound7(0.191g,0.555mmol)andcompound4(0.150g, 0.555mmol) were dissolved in acetone. K2CO3 (0.105g, 0.761mmol) and catalytic amount of 18-crown-6 was added toreactionmixturewhich wasreﬂuxedfor6h.Theprogressof thereactionwasmonitoredbyTLC.Whenstartingmaterialwas

consumed,mixture wasdilutedwithdiethyl etherand washed withsaturatedNH4Clsolution.Afterwashingwithbrine,combined organicphasesweredriedoveranhydrousNa2SO4.Afterremoval ofthesolvent,theresiduewaspuriﬁedbysilicagelﬂashcolumn chromatography using EtOAc/Hexane (1:5, v/v) as the eluent. Compound8wasobtainedascolorlesswaxyoil(0.212g,78%).1_H NMR(400MHz,CDCl3)ı7.85(d,J=7.7Hz,2H),7.47(d,J=7.7Hz, 2H),7.27(t,J=7.7Hz,1H),6.90-6.95(m,3H),5.12(s,2H),3.30(s, 2H),3.27(s,1H),2.63(s,1H),1.74-1.99(s,1H),1.37(s,12H).13_C NMR(100MHz,CDCl3)ı158.5,143.3,140.2,136.8,135.0,131.6, 135.0,131.6,128.9,126.5,122.2,115.6,114.1,83.8,69.8,57.7,39.2, 39.0, 37.2,32.2, 30.2,28.3, 24.8ppm.MS(TOF- ESI):m/z:Calcd for C31H39BO4: 486.30505 [M+H]+, Found: 486.30941 [M+H]+, =−8.97ppm.

2.3.2. Synthesisofprobe1

Compound 8 (0.070g, 0.144mmol) was dissolved in DCM. Methyleneblue(5mg)wasaddedtothereactionmixturewhich was irradiated while oxygen gas was passing through it. The progressofthereactionwasmonitoredbyTLC.WhenTLCshowed nostartingmaterial,themixturewasconcentratedundervacuo andtheresiduewassubjectedtothesilicagelﬂashcolumn chro-matographybyusingDCMastheeluent. Probe1wasobtained as whitesolid (0.061g,81%).1_H _NMR ₍₄₀₀_MHz,_CDCl 3)ı 7.83 (d, J=7.9Hz, 2H),7.45(d, J=7.9Hz, 2H), 7.13–7.39(m, br,3H), 7.02–7.05(m,1H),5.18(s,2H),3.23(s,3H),3.02(s,1H),2.09(s, 1H),1.42-1.89(m,12H),1.36(s,12H).13_C_NMR₍₁₀₀_MHz,_CDCl 3)ı 139.9,136.1,135.0,129.2,126.4,116.4,112.0,95.4,83.8,69.9,49.9, 36.4,34.6,33.0,32.8,32.3,31.6,26.0,25.8,24.8ppm. 2.3.3. Synthesisofcompound(9)

2,6-Bis(hydroxymethyl)-p-cresol (1.0g, 5.95mmol) was dis-solved in DMF and cooledto 0◦C by using ice-bath. Imidazole (0.809g,11.9mmol)wasaddedtoreactionmixtureat0◦C.After 30min,tert-butyldimethylsilylchloride(1.820g,11.9mmol)was added toreactionmixture which waslefttostir atroom tem-peraturefor12h.Whenstartingmaterialwasconsumed,mixture wasdilutedwithdiethyletherandwashedwithsaturatedNH4Cl solution.Afterwashingwithbrine,combinedorganicphaseswere driedover anhydrousNa2SO4.Afterremovalofthesolvent,the residue waspuriﬁed bysilicagelﬂash columnchromatography using EtOAc/Hexane (1:5, v/v) as the eluent. Compound 9 was obtained as colorless waxy oil(2.1g, 89%).1_H _NMR ₍₄₀₀_MHz, CDCl3)ı8.04(s,1H),6.93(s,1H),4.85(s,4H),0.97(s,12H),0.15 (s,6H).13_C _NMR₍₁₀₀_MHz, _CDCl

3)ı150.9,128.2,126.2,125.7, 63.0, 64.8, 25.8,20.6, 18.3, −5.4ppm. MS(TOF–ESI): m/z:Calcd forC21H40O3Si2:419.24082[M+Na]+,Found:419.24523[M+Na]+, =−10.52ppm.

2.3.4. Synthesisofcompound(10)

Compound 9 (0.50g, 1.26mmol) and compound 7 (0.433g, 1.26mmol)weredissolvedinacetone.K2CO3(0.209g,2.52mmol) andcatalyticamountof18-crown-6wasaddedtoreaction mix-turewhichwasrefluxedfor6h.Theprogressofthereactionwas monitoredbyTLC.Whenstartingmaterialwasconsumed,mixture wasdilutedwithdiethyletherandwashedwithsaturatedNH4Cl solution.Afterwashingwithbrine,combinedorganicphaseswere driedover anhydrousNa2SO4.Afterremovalofthesolvent,the residue waspurified bysilicagelflash columnchromatography usingEtOAc/Hexane(1:5, v/v)astheeluent.Compound10 was obtainedascolorlesswaxyoil(0.718g,93%).1_H_NMR₍₄₀₀_MHz, CDCl3)ı7.97(d,J=7.7Hz,2H),7.54(d,J=7.7Hz,2H),7.30(s,2H), 5.02(s,2H),4.82(s,4H),2.45(s,3H),1.45(s,12H),1.04(s,12H), 0.19(s,6H).13_C_NMR₍₁₀₀_MHz,_CDCl

3)ı151.2,140.8,135.1,133.8, 133.6,128.0,127.0,83.8, 76.2, 63.1, 60.4,26.0, 24.9, 21.2, 18.4,

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−5.1ppm.MS(TOF-ESI):m/z:CalcdforC34H57BO5Si2:634.37661 [M+Na]+_,_Found:_635.37999_[M₊_Na]+_,₌_−11.3_ppm.

Compound10(0.287g,0.468mmol)wasdissolvedinmethanol andcatalyticamountofp-toluenesulfonicacidwasaddedto reac-tion mixture which was stirred at room temperature for 2h. TheprogressofthereactionwasmonitoredbyTLC.When start-ingmaterialwasconsumed,mixturewasdilutedwithDCMand washedwithsaturatedNaHCO3solution.Afterwashingwithbrine, combined organic phases were dried over anhydrous Na2SO4. Afterremovalof thesolvent,theresidue waspuriﬁed by silica gelﬂashcolumnchromatographyusingDCMastheeluent. Com-pound11wasobtainedascolorlesswaxyoil(0.176g,98%).1_H_NMR (400MHz,CDCl3)ı7.83(d,J=7.8Hz,2H),7.39(d,J=7.8Hz,2H), 7.30(s,2H),7.12(s,2H),4.85(s,2H),4.60(s,4H),3.12(s,br,2H), 2.28(s,3H),1.37(s,12H).13_C_NMR₍₁₀₀_MHz,_CDCl

3)ı152.2,140.0, 135.1,134.2,133.8,129.3,127.1,83.9,76.6,60.4,24.8,20.8ppm.MS (TOF-ESI):m/z:CalcdforC22H29BO5:406.20366[M+Na]+,Found: 406.20562[M+Na]+_,₌_−4.83_ppm.

Compound11(0.40g,1.04mmol)wasdissolvedindryTHFand mixturewascooledto0◦Cbyusingice-bath.Diisopropylamine (1.21mL,8.0mmol)andpyridine(0.05mL)wereaddedtoreaction mixtureunderAr.p-nitrochloroformate(0.897g,4.16mmol) dis-solvedindryTHFwasaddeddropwisetoreactionmixtureunder Arat 0◦C. The progressof thereaction wasmonitoredbyTLC. Whenstartingmaterialwasconsumed,mixturewasdilutedwith DCMandwashedwithsaturatedNaH4Clsolution.Afterwashing withbrine,combinedorganicphasesweredriedoveranhydrous Na2SO4.Afterremovalofthesolvent,theresiduewaspuriﬁedby silicagelﬂashcolumnchromatographyusingDCM/Hexane(3:1, v/v)astheeluent.Compound12wasobtainedascolorlesswaxy oil(0.580g,78%).1_H_NMR₍₄₀₀_MHz,_CDCl 3)ı8.26(d,J=9.1Hz, 4H),7.86(d,J=7.8Hz,2H),7.50(d,J=7.8Hz,2H),7.36(s,2H),7.32 (d,J=9.1Hz,4H),5.36(s,4H),5.08(s,2H),2.40(s,3H),1.39(s,12H). 13_C_NMR₍₁₀₀_MHz,_CDCl 3)ı155.4,154.5,152.3,145.4,139.6,135.1, 134.8,132.7,128.1,126.7,126.1,125.2,121.7,115.6,84.0,66.2,24.8, 20.7ppm.MS(TOF-ESI):m/z:CalcdforC36H35BN2O13:736.21607 [M+Na]+_,_Found:_736.21755_[M₊_Na]+_,₌_−1.94_ppm.

Compound12(0.03g,0.420mmol)wasdissolvedindryDCM. DMAP(0.123g,1.0mmol)wasaddedtoreactionmixtureunderAr. Compound4(0.272g,1.0mmol)dissolvedindryDCMwasadded dropwisetothereactionmixturewhichwasstirredatroom tem-peratureovernight.Theprogressofthereactionwasmonitored byTLC.Whenthestarting materialwasconsumed,themixture wasconcentratedundervacuoandtheresiduewassubjectedto thesilicagelﬂashcolumnchromatographyusingEtOAc/Hexane (1:10,v/v)astheeluent.Compound13wasobtainedaswhitesolid (0.258g,63%).1_H_NMR₍₄₀₀_MHz,_CDCl 3)ı7.87(d,J=8.0Hz,2H), 7.51(d,J=7.9Hz,2H),7.34-7.38(m,4H),7.22(m,2H),7.36(s,2H), 7.15(m,2H),7.08-7.11(m,2H),5.34(s,4H),5.06(s,2H),3.31(s, 3H),3.26(s,2H),2.67(s,2H),2.39(s,3H),1.80-1.99(m,28H),1.37 (s,12H).13_C_NMR₍₁₀₀_MHz,_CDCl 3)ı154.1,153.5,150.9,142.5, 139.7,137.1,135.1,132.7,132.2,128.9,128.5,127.1,126.9,121.7, 119.9,83.8,57.9,39.2,39.1,37.1,32.1,30.2,28.2,24.8,20.8ppm. MS(TOF-ESI):m/z:CalcdforC60H69BO11:1062.45505[M+Na]+, Found:1062.45455[M+Na]+_,₌_10.12_ppm.

2.3.8. Synthesisofprobe2

Synthesisofprobe2wasachievedbyusingsameprocedureas inprobe1startingwithcompound14(0.128g,0.13mmol).Probe 2wasobtainedaswhitesolid(0.122g,90%).1_H_NMR₍₄₀₀_MHz,

CDCl3)ı8.14(d,J=9.0Hz,1H),7.86(d,J=8.2Hz,2H),7.51-7.45(m, 6H),7.36(s,2H),7.22(d,J=8.2Hz,2H),6.91(d,J=9.0Hz,1H),5.33 (s,4H),5.05(s,2H),3.23(s,3H),3.05(s,2H),2.39(s,3H),2.15(s, 2H),1.90-1.47(m,28H),1.37(s,12H).13_C_NMR₍₁₀₀_MHz,_CDCl 3)ı 154.1,153.4,151.1,139.6,135.1,134.6,132.3,129.3,128.4,127.0, 126.1,122.1,115.6,111.5,95.5,83.9,65.7,50.6, 39.3,36.3,34.7, 33.1,32.8,32.2,31.6,31.5,25.9,25.8,24.8,20.8ppm.

3. Resultsanddiscussions

OurstrategyreliesontheselectiveH2O2mediateddeprotection ofboronateesterswhichleadstotheformationofm-oxybenzoate anionasaresultofthedecompositionoftheprobes1and2.So, we designed and synthesizedtwo different probes1 and 2 for comparison.Inthecaseofprobe1whichisthereferenceprobe, singleanalyteleadstoformationofunamplifiedchemiluminogenic responsewhereas inthecaseof probe2which istheamplifier probe,disassemblyoftheselfimmolativemoleculeupon interac-tionwithsingleH2O2leadstocompletefragmentationandthusin turn,signalamplificationisachieved.

3.1. Synthesisofprobes

Compounds1–4and 5–7forthesynthesis ofprobe 1and 2 wereprepared according totheliterature [37,38].To that end, thesynthesisof triggeringmoietywasstarted withthe protec-tionof4-formylphenylboronicacid.Formylgroupwasreduced toobtaincompound6whichisfurtherconvertedintoiodinated form7.Meanwhile,3-hydroxybenzaldehydewasreactedwith ben-zoyl chloride and resulting phenyl ester 1 was converted into dimethylacetalderivative2subsequenttotheconversionofthis intophosphonatederivative3 viaArbuzovreaction.Compound 3 wasreactedwith2-adamantanone tocomplete thesynthesis of chemiluminescenceprecursor, compound 4which is further reacted with compound 7 to complete the synthesis of probe 1.Theselfimmolativelinkerwasprepared accordingtoShabat andco-workers[39,40].Triggeringmoietywasintroducedtoself immolativecoregroupwhich wasobtainedbythesilyl protec-tionof2,6-bis(hydroxymethyl)-p-cresol.Constructionofthetarget compoundscontinuedwiththeincorporationofcarbonategroups intothesilyldeprotectedcompound11.Chemiluminogenicunits wereassembled bythereaction ofactivatedlinkerwiththe 3-hydroxyphenylmoietyofthereportercompound4.Intheﬁnalstep, theelectron-richenoletherswereefﬁcientlyphotooxygenatedto yield1,2-dioxetanederivativesasprobe1andprobe2(Scheme1). 3.2. Chemiluminescencemeasurements

InordertodeterminetheH2O2sensingcapabilitiesofprobe1 and2,asolutionofH2O2wasaddedinportions(50␮M–2.5mM) andaftereachaddition,theCLspectrawererecorded.Asexpected, both probe 1 and 2 showed increase in CL response (CL on-mode) after each addition depending on the decomposition of boronategroupandformationofthephenolate ion.Verybright bluechemiluminescenceisobtaineduponadditionofH2O2 and largerconcentrationsleadtomoreintenseluminescence(Fig.1).

Dataprovideclearevidencethatprobe2ampliﬁesthe chemi-luminogenicsignalquartertimesmoreaccordingtoprobe1and senses theloweramount ofH2O2 (Fig.2).The detectionlimits werecalculatedas75␮Mforprobe2(␴=0,007887,m=314,36). For probe 1, the detection limit was determined as 240␮M (␴=0,020766,m=258,55)inDMSOand0.67mMinDMSO/NaHCO3 (140mM,90/10,pH8.3).So,probe2hadmorelightemissiondueto thepresenceofthetwo1,2-dioxetanegroups.Inotherwords,the more1,2-dioxetanemodulesmeanslargerproductionofexcited stateintermediate,whichmeansenhancedCLintensity.

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Scheme1.SynthesisofselfimmolativedioxetanebasedCLH2O2probes1and2.(TEA:triethylamine,DMAP:4-dimethylaminopyridine,LDA:Lithiumdiisopropylamide,

TMS-Cl:trimethylsilylchloride,p-TsOH:p-toluene-sulfonicacid,TBDMS-Cl:tertbutyldimethylsilylchloride,DIPEA:N,N-diisopropylethylamine).

Fig.1.Chemiluminescenceresponseofprobes1(A)and2(B)inthepresenceofincreasingH2O2concentrationswherein1–50equiv.in(A)and0,25to50equiv.in(B).Probe

concentrationis50␮M.SpectrawereacquiredinDMSO/NaHCO3(140mM,90/10,pH8.3)forbothprobesandalldatawereobtainedafterincubationwithH2O2at25◦Cfor

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Fig.2.Comparisonofchemiluminescenceresponseofprobe1and2inthe pres-enceofincreasingH2O2concentrationswherein1to50equiv.inprobe1and0,25

to50equiv.inprobe2.Probeconcentrationis50␮M.Spectrawereacquiredin DMSO/NaHCO3(140mM,90/10,pH8.3)forbothprobesandalldatawereobtained

afterincubationwithH2O2at25◦Cfor30s.

WealsostudiedtheoptimalbufferratiosforCLresponse of probe2atdifferentpercentagesofbuffer(NaHCO3,140mM,90/10, pH8.3)inDMSO(Fig.3).TheCLintensitiesdecreasedwith increas-ing bufferpercentages due tothe possibility of protonation of phenoxideionwhichpreventschargetransferfromphenoxideion to1,2-dioxetanegroup.Spectroscopiccharacterizationofprobe1 wasgiveninSupplementarymaterial.

3.3. Selectivityoverotherrelatedspecies

Further,theselectivityof probe 2 for otherreactiveoxygen specieswasalsoinvestigated.Tothatend,selectivityofprobe2 towardvariousreactiveoxygenspeciesliketert-butyl hydroperox-ide(TBHP),hypochlorite(OCl−),superoxide(O2−),hydroxylradical (•OH),tert-butoxyradical(.Ot_Bu)_are_determined_according_to_the literature[41]anddisplayedinFig.4.Thus,treatmentoftheprobe 2witha numberofpotentialcompetitorROSdoesnotresulted inanyluminescence.CLdataprovideclearevidencethat ampli-fier2offerverygoodselectivityforH2O2.Nootherspecieswas abletodecomposetheboronategroups;as aresultnochanges intheCLspectrawereobserved(Fig.4).Digitalphotographsof thesolutionsshowtheselectivityofchemiluminescentamplifier probe2underambientlight(Fig.5).H2O2inDMSO-buffermixture elicitsclearresponsewithluminescenceintensityreflectingH2O2 concentration.

3.4. Proposeddecompositionmechanism

Allexperimentsareincompleteagreementwiththefollowing mechanismsdescribedinScheme2.Wehaveproposedthat chemi-luminescent disassembly of the ampliﬁer probe was preceded viahydroboration-oxidationreactionbetweenH2O2andboronate esterleadingtoformationofphenoxideionandthus,releasesthe

Fig.3.Chemiluminescenceresponseofprobe2atdifferentpercentagesofbuffer (NaHCO3,140mM,90/10,pH8.3)inDMSO.Probeconcentrationis50␮M.Alldata

wereobtainedafterincubationwithH2O2at25◦Cfor30s.

Fig.4. Chemiluminescenceresponsesof50␮Mprobe2tovariousreactiveoxygen species(ROS).SpectrawereacquiredinDMSO/NaHCO3(140mM,90/10,pH8.3)for

bothprobesandalldatawereobtainedafterincubationwiththeappropriateROS at25◦_C_for₃₀_s._Data_shown_are_for₁₀_mM_for_O₂−_,₂₅₀_␮M_for_all_other_ROS.

tert-butylhydroperoxide(TBHP),andhypochlorite(OCl−₎_were_delivered_from_30%,_70%,

and5%aqueoussolutions,respectively.Superoxide(O2−)wasaddedassolidKO2.

Hydroxylradical(._OH)_and_tert-butoxy_radical₍•_Ot_Bu)_were_generated_by_reaction

of2.5mMFe2+_with₂₅₀_␮M_H

2O2or250␮MTBHP,respectively.

activatedcoremoleculeA.Subsequentdissociationofthe inter-mediatecompoundAleadstotheformationofactivatedformof 1,2-dioxetaneBduetothepKaofthemedium.Phenoxideionof 1,2-dioxetaneBtransferselectrontoO–Oofdioxetanetoinitiateits decompositionforthegenerationofexcitedm-oxybenzoateanion Cwhileitrelaxesbacktogroundstate,resultingintheemissionof photonasshowninScheme2.

4. Conclusion

In summary, we demonstrated that multiple chemilumino-genic groups canbetriggeredby usingself-immolativelinkers. Thisapproachoffersachemicalavenueforenhancingthesignal producedinresponsetoagivenanalyte.Wehavedescribedthe synthesis and CL propertiesof self immolativeprobe 2 for the selectivedetectionofH2O2.UpontreatmentofprobewithH2O2, deprotectionofboranatesubsequentlytriggerthedecomposition of1,2-dioxetaneringviaintramolecularchargetransferduetothe negativelychargedphenolategroups.Thisdecompositionresultsin formationofCLasasignalofH2O2.Probe2hasfulﬁlledtheH2O2 detectioninahighlyselectivemanner.Webelievethatthisstudy islikelytoprovidenewinsightsintothedevelopmentof chemi-luminescencebasedH2O2sensorsandbrightchemiluminescence oftheprobe2orstructurallyrelatedderivativescouldprovidea promisingalternative.

Fig.5.Thedigitalphotographshowingtotheselectivechemiluminescentresponse ofprobe2underambientlight.

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Scheme2.ProposedchemiluminescentdecompositionprocesscatalysedbyH2O2forprobe2.

Acknowledgment

TheauthorsthankProfessorDr.EnginU.Akkayaforfruitful dis-cussions.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.snb.2016.09.120.

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Biographies

OzlemSevenPhDisapostdoctoralresearcherinUNAM-NationalNanotechnology

ResearchCenteratBilkentUniversity,Ankara,Turkey.In2010,shecompletedher postdoctoralworksinChemistryDepartmentatMiddleEastTechnicalUniversity (METU),AnkaraTurkey.SheearnedherDoctoraldegree(2007)andM.Sc.degree (2003)fromSolarEnergyInstituteatEgeUniversity,Izmir,Turkey.Sheearned herB.Sc.degree(2001)inchemistryeducationfromDokuzEylulUniversity,Izmir, Turkey.Herresearchinterestsarephotocatalyticreactions,photodynamictherapy, syntheticchemistry,chemiluminescenceandenergytransfercassettes.

Fazli SozmenPhDis an Associate Professor inNanotechnology Eng. Dept. at

CumhuriyetUniversity(Sivas,Turkey).Heearnedhis Doctoral,M.Sc.andB.Sc.

degreesinchemistryfromAkdenizUniversityin2012,2005and2002,respectively. Hiscurrentresearchisfocusedonthedevelopmentofnovelfunctional nanomateri-alsandtheirapplications,lightharvestingsupramolecularsystems,photodynamic therapy,self-assembledfunctionalsystemsandmolecularsensors.HisPh.D. the-siswasonthesynthesisofnewBODIPYderivativesincludingterpyridineand phenantroline,preparationoftheirmetallosupramolecularcomplexesandenergy transfer.

IlkeSimsekTuranPhDisapostdoctoralresearcherinUNAM-National

Nanotech-nologyResearchCenter atBilkentUniversity,Ankara,Turkey.She earnedher DoctoraldegreeinMaterialScienceandNanotechnologyfromBilkentUniversityin 2014.SheearnedherM.Sc.degreeinchemistryfromMETUin2009andB.Sc.degree inchemistryeducationfromMETUin2007.Herresearchinterestandexperience aredevelopmentofnovelchemiluminescentcompounds,photodynamictherapy, molecularsensorsandmolecularlogicgates.