JFluoresc(2017)27:1201–1212 DOI10.1007/s10895-017-2053-y
FLUORESCENCENEWSARTICLE
SpectroscopicStudiesofFluorescenceEffects
inB ioactive4-(5-Heptyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3- Dioland4 -(5-Methyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3-Diol MoleculesInducedbypHChangesinAqueousSolutions
ArkadiuszM atwijczuk1&D ariuszKluczyk2&A ndrzejGórecki3&
AndrzejNiewiadomy4,5&MariuszGagoś2
Received:7November2016/Accepted:19February2017/Publishedonline:1March2017
#TheAuthor(s)2017.ThisarticleispublishedwithopenaccessatSpringerlink.com
AbstractT hispaperpresentstheresultsofstationaryfluores- cencespectroscopyandtime-
resolvedspectroscopyanalysesoftwo1,3,4-
thiadiazoleanalogues,i.e.4-(5-methyl-1,3,4-thiadiazol-2- yl)benzene-1,3-diol(C1)and4-(5-heptyl-1,3,4-thiadiazol-2- yl)benzene-1,3-
diol(C7)inanaqueousmediumcontainingdifferentconcentrati onsofhydrogenions.Anin-
terestingdualflorescenceeffectwasobservedwhenbothcomp oundsweredissolvedinaqueoussolutionsatpHbelow7forC1a nd7.5forC7.Inturn,forC1andC7dissolvedinwateratpHhighe rthanthephysiologicalvalue(mentionedabove),singlefluores cencewasonlynoted.Basedonprevi-
ousresultsofinvestigationsoftheselected1,3,4-
thiadiazolecompounds,itwasnotedthatthepresentedeffectsw ereasso-
ciatedwithbothconformationalchangesintheanalysed
ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:
10.1007/s10895-017-2053-
y)containssupplementarymaterial,whichisavailabletoauthorizedusers .
* ArkadiuszMatwijczuk
arkadiusz.matwijczuk@up.lublin.pl;a rekmatwijczuk@gmail.com
* MariuszGagośmariusz.gagos@poczta .umcs.lublin.pl
1 DepartmentofBiophysics,UniversityofLifeSciencesinLublin,Ak ademicka13,20-950Lublin,Poland
2 DepartmentofCellBiology,InstituteofBiology,MariaC urie-SkłodowskaUniversity,20-033Lublin,Poland
3 DepartmentofPhysicalBiochemistry,FacultyofBiochemistry, BiophysicsandBiotechnologyoftheJagiellonianUniversity,Gro nostajowa7,30-387Krakow,Poland
4 InstituteofIndustrialOrganicChemistry,Annopol6,0 3-236Warsaw,Poland
5 DepartmentofChemistry,UniversityofLifeSciencesinLublin,20- 950Lublin,Poland
2 JFluoresc(2017)27:1201–1212
moleculesandchargetransfer(CT)effects,whichwereinflu- encedbytheaggregationfactor.However,inthecaseofC1and C7,thedualfluorescenceeffectswerevisibleinahigherenerge ticregion(differentthanthatobservedinthe1,3,4- thiadiazolesstudiedpreviously).Measurementsofthefluores -cencelifetimesinamediumcharacterisedbydifferentcon- centrationsofhydrogenionsrevealedclearlengtheningofthee xcited-
statelifetimeinapHrangeatwhichdualfluorescenceeffectscan beobserved.Animportantfindingoftheinvesti-
gationspresentedinthisarticleisthefactthatthespectroscop- iceffectsobservednotonlyareinterestingfromthecognitivep ointofviewbutalsocanhelpindevelopmentofanappro- priatetheoreticalmodelofmolecularinteractionsresponsible forthedualfluorescenceeffectsintheanalysed1,3,4- thiadiazoles.Furthermore,thestudywillclarifyabroadrange ofbiologicalandpharmaceuticalapplicationsofthesecom- pounds,whicharemorefrequentlyusedinclinicaltherapies.
KeywordsD ualfluorescenceeffects.Molecularspectrosc opy.Molecularaggregation.Substituenteffects.1,3,4- thiadiazole
Introductio n
AsreportedbytheWorldHealthOrganisation,oneofthe majorchallengesofmodernmedicineisthefightagainstcan- cerandneurodegenerativediseases.Accordingtoliteratured ata,thesediseasesarecurrentlytheleadingcausesofpa- tients’mortalityworldwide[1,2].Greathopesinthefightag ainstcancerandneurodegenerativediseasesareplacedonsynt heticcompoundsfromthe1,3,4-
thiadiazolegroup,whosedifferentderivativesarealreadykno wnandclinicallyapplied.The1,3,4-
thiadiazoleswithasubstitutedresorcylfragment
-
analysedinthisstudyarehighlypromisingcompoundsi nanticancerandneurodegenerativediseases.Asindicatedin mostresearchpapers,1,3,4-
thiadiazolesarethemostattractiveneuroprotective-
activitymoleculesystemofalltheotherthiadiazolesystems[3 –
5].Manycompoundsofthisgrouphavebeenusedinmedici nease.g.oxidationinhibitors,colourants,andmetalcompl exingcompounds[6].Additionally,theliteratureshowsthat thethiadiazolefamilycomprisescompoundswithantitumour[
7–10],antifungal[11],antibacterial[11],anti- inflammatory[12],anticonvulsant
[13],antiviral[14],antituberculosis[15],antihypertensive [16],andantidepressant[17]activity.
Twohighlypromising1,3,4-
thiadiazoleanalogueswithaconfirmedneuroprotectiveactivit y,i.e.4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3- diol(C1)(C1,Scheme1A–C)and4-(5-heptyl-1,3,4-thiadiazol-2- yl)benzene-1,3-diol(C7)(C7,Scheme1D–
F),wereselectedinthisstudyfortheinves-
tigationsofthemechanismofmolecularinteractions.Note worthy,these1,3,4-
thiadiazolecompoundsexhibitnotonlyremarkableandconfir medpharmacologicalpropertiesbutalsoveryinterestingspectr oscopictraits,whichcontribute
totheirbiologicalactivity.Thespectroscopiceffectsexhibitedb ythe1,3,4-
thiadiazoleanaloguesincludee.g.theeffectsofketo/enoltauto merisminducedbychangesinmediumpolar-izability[18–
21],effectsassociatedwithcrystalpolymor-
phism[22]andsolvatomorphism[23],andinterestinginter- actionsinmodellipidsystems[24,25].Additionally,theanaly sedcompoundgroupexhibitsaninterestingdualfluo-
rescenceeffectoraneffectofseveralfluorescencespectra,dep endingontheconcentration[26],pH,orchangesinthemedium temperature,whichwillbepresentedinthispaper[27].Moreo ver,thesecompoundsaregoodligands,whichcanformcom plexeswithd-
blockmetalions[28],andcanthereforebeusedinnovelmedica lapplications.Thecombi-
nationofthespectroscopicandcrystallographiceffectsreport- edinthepaperscitedabovehasgreatimportanceforelucida- tionofthebroadspectrumofthepharmacologicalactivityofthes ecompounds.
Theaimofthepresentspectroscopicstudywastoinvesti- gateC1andC7inamediumwithdifferentconcentrationsofhydr ogenionsandtodescribethedualfluorescenceeffectobserv edinthesemolecules.Inpreviousstudies,thedual
Scheme1Chemicalstructureof OH
theC1molecule(a–enolform,b
–formionisedwiththe–O− N N
group,c–formionisedwiththe–
a
N+–Hgroup)andC7molecule(d OH
–enolform,e–formionisedwith− S
the–O group,f–formionised O
withthe–N+–Hgroup)
N N OH
b
S OH H
N+N
c
OH
S OH
N N OH
d
S O-
N N OH
e
S OH H
N+N
f
OH
S
1203 JFluoresc(2017)27:1201–1212
fluorescenceeffectwasobservedforonerepresentativeofthean aloguegroup(FABT)[27].However,inthisstudy,theef- fectwasobservedinadifferent(substantiallyhigher)energyran ge,whichcompletelychangedthephotophysicalproper- tiesoftheanalysedmolecules.Usingspectroscopicap- proaches,suchastheelectronabsorptionspectroscopictech- nique,fluorescencemethodscombinedwiththeRLStech- nique,andmainlymeasurementsoffluorescencelifetimes,w ehaveshownthecomplexityofphysicalprocessesthatex- ertanimpactontheobservedeffects.Thedualfluorescenceeffe ctscanbeinducedindifferentmoleculesbychangesinmediu mpolarity,pHofthesolution,temperature,orthecon-
centrationofthesample[29–
33].Accordingtotheoriesattemptingatelucidationoftheobser vedfluorescenceeffects,theycanberelatedtoappearanceofintr amolecularCTstates[34]andtheso-
calledTICTstates(TwistedIntramolecularChargeT ransfer) [35,36].Anotherexplanationoftheanalysedphenomenaist heprocessofExcited-
StateIntramolecularProtonTransferESIPT[37–
42].Othertheoriespostulateformationofcompoundconcentr ation-inducedexcimersystems[43,44],varioustypesofacid- basereactionsintheinvestigatedsystem,orveryfrequentforma tionofexcited-statetautomericsystems[45,46].Theanti- Kashamechanismofthedualfluorescenceeffectpostulatedin 2015intheJournalofPhysicalChemistryBbyBrancato etal.shouldalsobementioned[47].However,assuggestedbyth eresearchresultspresentedinthispaper,noneofthesemechanis msseemstobesuccessfulinelucidationoftheob-
servedeffectsandmolecularmechanismsinvolvedinthespect roscopicchanges.
Thestudiesperformedwiththeuseofstationaryfluores- cencespectroscopyandtime-
resolvedspectroscopyinanaqueousmediumwithdifferentpH valuesrevealedthedualfluorescenceeffectinbothanalogues.
Formulationofaconcisetheoryforthedualfluorescenceeffe ctsobservedinthe1,3,4-
thiadiazoleanaloguesiscrucialforunderstandingtheirbiologic alactivity.Theeffectisattrac-
tivefortheoreticreasons,asitcanbeusedforexaminationoftheex citationstateinvariousmoleculartransformationsandfordesig ningnewmolecularprobesof(dual)fluorescence.
MaterialandMethods
Materials
4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol(C1) (seeScheme1D)and4-(5-heptyl-1,3,4-thiadiazol-2- yl)benzene-1,3-
diol(C7)weresynthesizedintheDepartmentofChemistryo ftheUniversityofLifeSciencesinLublin,detailsoftheprocedur
earedescribedelsewhere[3].ThepurificationprocedureoftheC1 andC7compoundisdescribedindetailin
JFluoresc(2017)27:1201–1212
6
thereferences[3].Theconcentrationofthecompoundswasc=
1.25×10−6MforC1andc=1.19×10−6MforC7.
Metho ds
pHMeasurementandPreparatio n
AllsolutionsweremeasuredwithanElmetronCP-502pH- meteratroomtemperature.InthecaseoftheaqueousC1andC7s olutions,0.1MNaOHwasfirstaddedtowatertoobtainpH12.A fterwards,powderC1andC7wasdissolvedtherein.Next,0.1 MHClacidwasslowlyaddedtoobtainacertainpHvalueinthew aterC1andC7solution.ThepHwascontinu-
allycontrolled.Respectivetitrationcurvesforboth1,3,4- thiadiazoleanaloguesareshowninFig.S1intheSupple mentaryMaterials.TheinsetsinFig.S1presentionisedfunctio nalgroupsandpKpointsforbothcompounds(seebelo w).
ElectronicAbsorptionSpectrosco py
ElectronicabsorptionspectraofC1andC7wererecordedonado uble-beamUV-
VisspectrophotometerCary300Bio(Varian)equippedwit hathermostattedcuvetteholderwitha6×6multicellPeltierblo ck.Temperaturewascontrolledwithathermocoupleprobe (CarySeriesIIfromVarian)placeddirectlyinthesample.
Allexperimentswerecarriedoutat23°C.Thespectralslitwi dthwas1.5nminthemeasurementsoftheelectronabsorptionsp ectra.
ElectronicFluorescenceS pectroscopyw iththeR LST echnique
Fluorescenceexcitationandemissionaswellassynchronouss pectrawererecordedwithaCaryEclipsespectrofluorometer(
Varian)at23°C.Fluorescencespectrawererecordedwith 0.5nmresolutionandcorrectedforthelampandphotom ultiplierspectralcharacteristics.Resonancelightscat- tering(RLS)measurementswereperformedasinPasternacka ndCollings[48,49].Theexcitationandemissionmonochro- matorsofthespectrofluorimeterwerescannedsynchronously (0.0nmintervalbetweenexcitationandemissionwave- lengths),theslitsweresettoobtainspectralresolutionof 1.5nm.ThespectralanalysiswasperformedwiththeuseofGra ms/AI8.0software(ThermoElectronCorporation).
Time-
CorrelatedSinglePhotonCounting(TCSPC)
Time-correlatedsinglephotoncounting(TCSPC)measure- mentswereperformedonaFluoroCubefluorimeter(Horiba,F rance).ThesampleswereexcitedwithapulsedNanoLEDdio
deat372nm(pulsedurationof150ps)operated with1 MHzrepetition.Toavoidpulsepile-up,thepowerofthe
Σατ
τ
pulseswasadjustedtoanappropriatelevelusinganeutralgra dientfilter.Fluorescenceemissionwasrecordedusingapicose conddetectorTBX-
04(IBH,JobinYvon,UK).TheDataStationandtheDAS6softw are(JobinYvon(IBH,UK))wereusedfordataacquisitionandsi gnalanalysis.Allfluores-
cencedecaysweremeasuredina10×10mmquartzcuvette,using anemittercut-offfilterwithtransmittanceforwave- lengthslongerthan408nm.Theexcitationprofilesrequiredfort hedeconvolutionanalysisweremeasuredwithouttheemi tterfiltersonalightscatteringcuvette.Allmeasurementswerep erformedinwaterat20°CandvariouspH.Fluorescencedec aywasanalysedwithamultiexponentialmodelgivenbytheeq uation:
experimentaldata.Theaveragelifetimeoffluorescencedecayw ascalculatedaccordingtothefollowingequation:
Σ iαiτ2
hτi¼ i ð2Þ
iii
ResultsandDiscussion
ByplottingthepHtitrationcurvesforC1andC7,character- isticpKpointsfortheionisation-associatedgroupswerede- terminedinthestudied1,3,4-thiadiazoles(Fig.S1inthe
t SupplementaryMaterials).Forthe–O−g r o u pintheortho
It¼∑αiexp−
i ð1Þ positioni ntheresorcylr ing,pKC7=8.8andpKC1=8.1(Fig.S1inth
eSupplementaryMaterials-figureinsets)and whereαiandτiarethepre-
exponentialfactorsandthedecaytimeofcomponenti,respectiv ely.
Best-
fitparameterswereobtainedbyminimizationofthereducedχ [ 2]valueaswellasresidualdistributionof
forthe–
NH+group,pKC7=4.9andpKC1=4.1(Fig.S1intheSupplementar yMaterials-figureinsets).
PanelsAandCinFig.1presentresultsobtainedwithelectr onabsorptionspectroscopyforC1(PanelA)andC7
Fig.1PanelsAandCpresentelectronabsorptionspectraforC1(PanelA)an dC7(PanelC)generatedintheaqueoussolutionatpH12,10,8,6,4,and2.For
C7inPanelC,theabsorptionspectraatpH2,4,and6weremultipliedby10forbet terpresentationandcomparisonoftheanalysed
effects.PanelsB andD showfluorescenceemissionspectracorres pondingtothespectrafromPanelsAandCforC1(PanelB)andC7(PanelD ).Theexcitationwavelengthcorrespondedtothemaximumoftherespecti veabsorptionband
JFluoresc(2017)27:1201–1212 120 5 (PanelC)overtheentirerangeofhydrogenionconcentrations(fr
ompH1topH12).Thereresultsshowdistinctchangesintheshap eofthespectra,inparticularintheregionthatisrelevan ttothephysiologicalvalues.SpectraforpH2,4,6,8,10,and12f orbothanalysedcompoundsarepresentedforbetterclarity.The spectrainthepHrangefrom1to6weremultipliedby10(specifi cally:forC7atpH2,4,and6)forclearerpresentationoftheanaly sedeffectsforC7(PanelCinFig.1).AsshowninbothpanelsinFig .1,thedissociationofthe–
OHgroupfromtheresorcylringintheorthoposition(Scheme1 BandE)resultsinaclearhypsochromicshiftby301cm−1inthec aseofC7andby303cm−1forC1inthecompoundspectraatp H12.Inaddition,thereisa bathochromicshiftby3845cm−1fo rC7and3864cm−1forC1forspectraofbothcompoundsatpH1 ,comparedwiththeirspectraatpH7.Theseshiftsareusedforcal culationofthedistancesbetweenthemoleculesinthedimerusi ngtheexcitonsplittingtheory.Inboth1,3,4-
thiadiazoleanalogues,theionisationprocessisaccompaniedby compoundaggrega-
tion[27].AtpHca.7–
8,distinctbroadeningoftheabsorptionspectraisevidentforC1a ndC7(dashedgreylineinPanelsAandC,Fig.1),whichindicates aprobabilityofthepresenceofotherthanmonomericspectralfo rmsoftheanalysedstruc-
tures[27].InthecaseoftheC7spectrumatpH2,theabsor- banceisthelowest,whichimpliessubstantialpredominanceofa ggregatedformsinthiscompound(seePanelBinFig.2).Inturn,i nthecaseofC1,thelowestabsorbanceintensityisobservedinth esamerange,butitissubstantiallyhigherthanthatforC7atthecor respondingpH.Thisindicatesanimpactofthestructureofthean alysedcompounds,inparticularthestructureoftheiralkylsubsti tuents,onthemodeofformationofaggregatedformsofthesemol ecules.Thestructureofsub-
stituentgroupscanhaveasignificantinfluence(throughpro- cessesrelatedtostrongeraggregation)onthesolubilityoftheana lysedmoleculesindifferentorganicsolvents.ForC1atpH4,th ecompoundabsorbanceunexpectedlyincreasesslightly,sug gestinganincreaseinthenumberofthemono-
mericformsofthisanalogue.Nosuchphenomenonisob- servedinthecaseofC7inthespecifiedconcentrationrange,whic hevidencesstrongerinteractionsbetweenC7moleculesandfor mationofmoredurableaggregates.InC7,aremark-
abledecreaseintheabsorbancelevelisvisible,whichimpliesver ystrongaggregationata(probably)constantlevelalongthedec reaseinthepHvalue(atlowpH).
Basedontheexcitonsplittingtheoryandthespectralshiftspre sentedinFig.1andabove(andinFig.S2intheSupplementary Materials),itwaspossibletocalculatethedis-
tancebetweenadjacentchromophoresofmoleculesC1and
C7inthedimericstructure[49].Fig.S2(intheSupplementary
Fig.2PanelApresentstheratioofthemaximumelectronabsorptionatca.36 0/313nmforC1(whitecircles)andat361/314nmforC7(blacktriangles),i.
e.theratiobetweenthepredominantmonomericform(ionisedwiththe–
O−g
roup)andthepredominantassociatedformforagivencompound(ionis edwiththe–N-
H+g
roup)dependingonthepHoftheaqueoussolution.PanelBshowsthera tioofthefluorescenceemissionintensityforC1(whitecircles)andC7(bla cktriangles)dependingonchangesinthepHoftheaqueoussolution.T hepointswerereadfromtheabsorptionandfluorescenceemissionspect rapresentedinFig.1.Themeasurementsoftheabsorptionandelectronflu orescencespectra,fromwhichtherespectiveabsorptionandfluoresce ncemaximawereread
maximumatca.319nm(31,348cm−1)atpH7isslightlyshift edt owards317 nm(31,5 46c m−1)anda bandw itha maximu matca.351nm(28,490cm−1)appearsonthelongwaveside.Si milarly,inthecaseofC1,themainbandswithamaximumatca.3 16nm(31,646cm−1)isshiftedatlowpHvaluestowardsca.314n m(31,847cm−1)andabandwithamaximumatca.357nm(28,01 1cm−1)canbeseenonthelongwaveside.
Basedintheexcitonsplittingtheory,thedistancebetweenadjac entchromophoresRβcanbecalculatedusingtheformula:
sffiffiffiffiffiffiffiffi Materials)showselectronabsorptionspectraforC7(PanelA)an
dC1(PanelB)normalisedatthemaximum.Underpinning
Rβ¼ 1:71 3 μ2κ
η2β ð3Þ
ofthebandcanbeobservedforbothC1andC7atpHca.1, comparedwithpH7.ForC7(PanelA),thebandwitha
whereμisthediploemomentoftransitionoftheinteracting molecules,η–refractiveindex,β–dipole-dipoleinteraction
energy(inaclassicalapproach).Intheexcitonicmodel,onecanc onsideranaggregatedstructureformedthroughinterac- tionofidenticalmolecules,inwhichtransitiondipolemo- mentsofadjacentmoleculesareparallel,henceα=0(where,κ=
1 -
3cos[2]θ,whereθistheanglebetweenthetransitiondipolemom ents).Asproposedintheexcitonsplittingtheory[50,51],κ= 1for thecardpackmoleculearrangementintheaggregateandκ=− 2f ort heheadtot ailaggregate.T hetransitiondipolemomentcalcu latedviaintegrationoftheab-
sorptionspectrumisμ=4.16D(inH2O)andμ=4.25DforC7(the valuesoftransitiondipolemomentsinothersolventsandwatera swellasthemolarvalueoftheextinctioncoefficientsareprese ntedinTableS1intheSupplementaryMaterials).Thecalculate ddistancebetweenadjacentchromo-
phoresis4.29Å50inthecaseoftheC1dimersinanaqueoussolutio nand3.87ÅforC7.Theseresultsareconsistentwithcrystallogr aphicdatapresentedinsomeother1,3,4-
thiadiazolecompounds[27].Thedistanceincrystalsislowertha ninsolutions,whichisthecauseoftheobviouslydenserpacking oftheanalysedmoleculesinthecrystallinestructureandstronge rintermolecularinteractions.However,themostimportantfact isthatthedistancebetweenadjacentmoleculesinC7issubstanti allysmallerthaninC1.
PanelAinFig.2showstheratiooftheabsorbancemaxi- mumat360nm(predominantmonomericform,Scheme1B,and E–ionisedwiththe–
O−group)and313nm(predominantassociatedform,Scheme1C ,andF–ionisedwiththe–
NH+group)forC1(opencircles)aswellastheratiooftheabsor- bancemaximumof361nmand314nmforC7(blacktrian- gles),dependingonthepHoftheaqueoussolution.Thepre- dominanceofthenegativelyionisedform(predominanceofthe monomericform)forbothC1andC7compoundsismostevident atthehighpHvalues(pH12forC7andpH10.5forC1).Incontrast ,thepredominanceoftheformsionisedwiththeNH+groupcanbe observedatthelowpHvalues(atpH1forbothC7andC1).Inanaci dicenvironment,thepresenceofapositivelychargedNH+group inthethiadiazoleringshouldalsoleadtomonomerisation.How ever,thepresenceofthe–
OHgroupsintheresorcylringfacilitatesgenerationofhydro- genbondsnotonlywithwaterbutalsowithothermolecules,resu ltingintheiraggregation.Inturn,thegreatestchangesintheprese ntedratioinbothcompoundscanbeobservedforthephysiologic alpHvalues,whichcanbeclearly seenintheabsorptions pectrapresentedinPanelsAandCofFig.1.
Inthenextstepoftheinvestigations,thecompoundswereanal ysedwiththefluorescencespectroscopymethods.Notewor thyaretheeffectspresentedinPanelsBandDofFig.1.Thepa nelsshowfluorescenceemissionspectraforC1(PanelB)a ndC7(PanelD),correspondingtotheabsorp-
tionspectrapresentedinFig.1(PanelsAandC),togetherwiththe changeinthepHvalueoftheaqueoussolution(pH2,4,6,8,and10
arepresentedanalogouslyas fortheabsorptionspectra).Thee xcitationwavelengthforalltheanalysed
samplescorrespondstothemaximumofrespectiveabsorption bands.Adualfluorescenceeffectwithamaximumatca.380 nmand440nminthepHrangefrom1to7.5(PanelsCandD)i se videntint hecaseofbothc ompounds.AbovepH7.5,singleflu orescencewithamaximumat438nmforC7and437nmforC 1i s observed.At p H 8,onlysl ightunderpinningoftherespe ctivefluorescenceemissionspec-
trumisnotedforC1.Additionally,itshouldbeemphasisedtha ttheintensityofthefluorescenceemissionspectraforC7issubst antiallylowerthanthatforC1intherangewheretheaforeme ntionedeffectcanbeobserved(thesameconcentra-
tionofbothanalogues).Thisclearlyindicatesaconsiderablygr eaterdegreeofaggregationinC7thanthatinC1andmoreintens ivefluorescenceemissiondecay.Thiswasalsoobservedinthe calculationsbasedontheexcitonsplittingtheory(seeabove), wherethedistancesbetweenadjacentchromophoresweremar kedlysmallerinC7thaninC1.
PanelBinFig.2showstheratioofshort-
andlongwavefluorescenceemissionintensityforbothcomp ounds(emis-
sionwithamaximumof c a.380nm vs.emissionwithamaxi mumofca.440nm)dependingonthepHchangesintheaque oussolution.Evidently,inthepHrangefrom1to7.5forC7andC 1,thepointsarearrangedinonealmosthorizon-
talline,likewiseforthespectrashowninFig.1(PanelsAandC).
Thisimpliesthattheprocessofnitrogenatomprotonationisine quilibriumanddoesnotchangetheintensityoftheratiointhean alysed1,3,4-
thiadiazoleanalogues(Scheme1CandF).AtapHvaluehigher than7.5(forbothcompounds),theratioincreasessignificantly andthefluorescenceat378/9nmdisappearsforbothcompoun ds.
Noteworthy,a longwavefluorescencebandwitha max imumatca.500nmappearedinthecaseofanother1,3,4- thiadiazoledescribedpreviously,i.e.FABT[27],inwhichthe structuraldifferencesarerelatedtothepresenceoftheN- Hgroupandafluorobenzenering.Inturn,inthecompoundsan alysedinthispaper,thedecreaseinthepHvalueisaccompanie dbyashortwavefluorescenceemis-
sionbandwitha maximumatca.380nm.Therefore,changes intheenergystructureofthe1,3,4-
thiadiazolemoleculesareobserved.Thiseffectcanbeassocia tedwiththepresenceoftheaminoN-
HgroupintheFABTstruc-turelocatedatthe1,3,4- thiadiazolering[27].Inthecaseofa 1,3,4-
thiadiazolethatstructurallyresemblesFABT,althoughwith outthefluorobenzenefragment,anddiffersfromC1onlyinth eN-
Hgroup,abandwithamaximumatca.380nmisobservedathi ghpHvalues(resultssub-
mittedforpublishing).Inturn,acidificationyieldedasep- aratebandatca.440nm(asinthecaseofC1andC7atthehighp Hvalues).Theseconsiderationsindicateanim-
pactofthesubstituentsinthe1,3,4-
thiadiazolestructureonthepositionoffluorescencebandsde
spitetheidenticalchromophoreorganisation(fiveconjugated doublebonds)inthemolecule.
Fig.3ThepHeffectofthefluorescencedecayofC7andC1inwater.Dottedc urvesshowthedecayoffluorescenceemissionobservedusingtheTCSPCt echniqueatagivenpHforC7andC1inPanelsAandB,respectively.Solidli nesarebestexponentialfits.Theexcitationpulse
profile,setupat372,isshownbythedottedblackcurve.Residualsdet erminedforthepresentedfitsareshownbelowthedecaycurvesinPanelsC andD
FluorescenceLifetimeStudy
Figures3and4aswellasTables1and2presenttheresultsofmeas urementsoffluorescencelifetimesforC1andC7intheaqueouss olutionovertheentirepHrange(shownforpH4,7,
9,and12).Lightwithawavelengthof372nmwasusedforexcitat ion.AtpH7,theselectedexcitationwavelengthistyp-
icalforthelongwaveabsorptionedgeofthemonomericformand resonantexcitationoftheaggregatedform(~370nm).Fluore scencedecaywasmonitoredwiththeTCSPCmethod
Fig.4TheeffectofpHonthefluorescencelifetimeforC7(PanelsAand B) andC1(PanelsCandD).Thedependenceofthemeanfluorescencelifeti mesonthebestexponentialfitsatgivenpHareshowninPanelsA
andC,whileintensitiesofthefractionalfluorescencelifetimesarepresent edinPanelsBandD
Table1Fluorescencelifet imesofC7inH2Oinrelatio ntochangesinpH
Table2Fluorescencelifet imesofC1inH2Oinrelatio ntochangesinpH
C7
pH τ ± Δτ
0.8 1.59 ±
0.03
1.0 1.59 ±
0.03
1.6 1.60 ±
0.03
2.0 1.59 ±
0.02
2.5 1.55 ±
0.02
3.1 1.53 ±
0.02
3.6 1.51 ±
0.02
4.1 1.52 ±
0.02
4.5 1.52 ±
0.02
4.9 1.61 ±
0.02
5.5 1.58 ±
0.01
6.1 1.51 ±
0.01
6.3 1.51 ±
0.02
6.6 1.47 ±
0.01
6.8 1.46 ±
0.01
7.1 1.44 ±
0.01
7.3 1.46 ±
0.01
7.5 1.48 ±
0.01
7.8 1.44 ±
0.01
8.0 1.45 ±
0.01
8.6 1.18 ±
0.01
9.1 0.75 ±
0.01
9.4 0.68 ±
0.02
10.0 0.72 ±
0.06
10.6 0.12 ±
0.10
11.1 0.29 ±
0.03
11.6 0.17 ±
0.02
12.3 0.11 ± 0.07
C1
pH τ ± Δτ
atatemperatureof2 2°Cforlightwitha wavelengthover40 8nm.Theresultswe reanalysedbydeco nvolutionoftheflu orescencedecayusi ngEq.
(1)eachtimefori=1, 2,and3.Inamajority oftheanalysedcase s,thedoubleexpon entialmodeloffluo rescencedecaypro vedtobeoptimal.T hemono-
exponentialdecay modelwasinsuffici ent,andadditionofa thirdcomponentdi
dnotimprovethequalityofthefit, whichwasverifiedbythevalueofth efitparameterandanal-
ysisofresiduedistribution(Fig.3,P anelsCandD).Asingleexponential wasonlyobservedforC1atpHlowe rthan5.Inthesecases,anadditional componentdidnotimprovethefit.F orC7,bothcomponentsofthefluore scencelifetimesexhib-
itedverylittlevariabilityatpHbelo w8,5.Inthisrange,thelifetimeofth elongercomponentisbetween2an d2,5nsandisrapidlyreducedtoca.1 nsoverthethresholdpHvalue.Itsco ntributionisca.70%atthelowpHva luesanddeclinestoca.10%atthehi gherpHvalues.Thelifetimeofthes econdcomponentisca.0.5nsatthel owerpHvaluesanddecreasestosev eraltensofpsforthehigherpHvalu
es.Asaresultofthisvariability,theaveragelifetimeisca.1,6nsfo rpHlowerthan8andisreducedtoseveraltensofpsatthehigher pHvalues.InthecaseofC1,thecomponentwiththelongerlifet imehasasimilarvaluetothatobservedforC7andis2–
2,5nsovertheentirepHrange.FrompH5,asecondcompo- nentwithalifetimeof0,5nsisobservedanditsvaluechangesinsig nificantlyovertheanalysedpHrange.Withtheaddition- alcomponent,theaveragelifetimeismarkedlyreducedatthepHi ncreaseuptoca.6andthenremainsrelativelyconstantatca.0,5–
1nsuptothepHvalueof11.
Noteworthy,drasticchangesinthelengthofthelifetimesare observedfromapHvalueofca.8inthecaseofC7(Fig.4,PanelA) and frompHofca.5.5 inC1(Fig.4,PanelC).Similarly,t hepercentageproportionofthecomponentsoftheaveragelifeti mesforC1andC7changesatexactlythesamepHvalues(seeFig.
4,PanelsBandD).Thisclearlyindicatessubstantiallystrongera ggregationoftheC7molecules,com-
paredwithC1,whichhasalreadybeenobservedintheabsorp- tionspectra(Fig.1,PanelsAandC),whereasignificantre- ductionintheabsorbancelevelwasnoted.Thisprovescon- siderablybettersolubilityofC1intheaqueousmediumwith
1.0 2.45 ±
0.06
2.0 2.24 ±
0.03
3.0 2.17 ±
0.01
4.0 2.15 ±
0.01
5.0 1.29 ±
0.01
6.0 0.73 ±
0.01
7.0 0.63 ±
0.01
8.0 0.60 ±
0.01
9.0 0.62 ±
0.01
10.0 0.72 ±
0.01
11.0 0.81 ±
0.04
12.0 0.81 ±
0.04
pHrelevantfromthephysiologicalpointofview.
Thelengtheningofthefluorescencelifetimeinthecaseofmol eculesexhibitingthedualfluorescenceeffectischaracter- isticforcharge-
transfersystemsandexcimers[52],incontrasttoprocessesindu cedbythephenomenonofaggregation(dimerisation),inwhich aclearreductioninthelifetime[27]ordecayisobserved.
ResonanceLightScatteringStudy
Inordertorelatethefluorescenceeffectsobservedwiththemol ecularaggregationeffects,respectiveResonanceLightScatt ering(RLS,Δλ=0)spectrawereobtainedforC1and
C7intheaqueoussolution,dependingonthechangesinthemedi umpH.PanelsAandBinFig.5demonstrateRLSspec-
traforC1(PanelA)andC7(PanelB)obtainedatthedifferentpHva luesofthemedium.Asreportedintheliterature(mainlybyPaster nackandParkash[48,49]),theappearanceofRLSbandsshould primarilybeassociatedwithchromophoreag-
gregationofthesystemspresentinthesolution.Analysisofallthe RLSspectrashowninFig.5revealsthepresenceofRLSspectra(
withhigherorlowerintensity)inthepHrangeswherethedualflu orescenceeffectisobservedforC1andC7,aspresentedabov einFig.1(PanelsBandD).Additionally,itcanbenotedthatthei ncreaseinthepHvalueisaccompaniedbyreductionoftheRLSsi gnalforbothanalysedanalogues.PanelCinFig.5showstherel ationshipbetweentheRLSsignalintensityandthesolutionp HforC1(blackline)andC7(greyline).Ascanbenoted,theR LSsignalintensitysubstantiallydeclineswiththeincreaseinp H,whichclearlysuggestsanimpactofmolecularaggregationo nthespectraleffects.Theintensityofthesignalsisclearlyhighe rforC7,whereasubstantialdeclineintheabsorbancelevelwas re-vealedbythemeasurementsofabsorptionspectra.Thisevi- dencestheimpactofthealkylsubstituentstructureonth e
strengthoftheaggregationinteractionsinC7.TheRLSsignallos esitsintensityatapHvalueofca.7forC7andC1.Thepresenceo ftheRLSspectraandtheirdependenceonthechangesinthepH oftheanalysedsolutionsclearlyprovesarelationshipbetweent hepresentedeffectandthemolecularaggregationoftheinvesti gated1,3,4-
thiadiazoles.Itisalsoworthmentioningthattheoscillatoryst ructureoftheRLSbandsevidencesthepresenceofvariouspo ssiblydifferent-
sizeaggregationstructuresofbothC1andC7,whichalsoco nfirmstheimpactofthestructureoftheanalysedanalogues,inpa rticulartheiralkylsubstituents,ontheobservedeffect(Schem e1).
Theobservationsclearlyindicateagreaterimpactofmo- lecularaggregationeffectsthantheaforementionedTICT,E SIPT,andanti-
Kashaprocessesorexcimerfluorescence.Additionally,aggr egationhasasignificantimpactonchangesintheelectroncharge distributionaroundtheanalysedmole-
cules,whichyieldsfluorescencespectraleffectsinapHrange.Th edifferenceinthestructureofsubstituentgroupsoftheanalys edanalogues(C1,C7)evokesdifferentaggregationef- fects.Therefore,wepostulatethatthedualfluorescenceef- fectsinthe1,3,4-thiadiazolesareinducedbyatleasttwo
Fig.5PanelsA andB:RLSspectra(ResonanceLightScattering,Δλ=0)for C1andC7obtainedintheaqueoussolutionatthedifferentpHvalues,presen tedinthefigureforpH1,3,7,and10,respectively.
PanelCshowstheratiooftheintensityoftheRLSspectraforC1(blackline)a ndC7(dashedgreyline)dependingonthepHoftheaqueoussolution.Al lRLSspectrawereobtainedatT=23°C
JFluoresc(2017)27:1201–1212 121 3 overlappingeffects,i.e.molecularaggregation(dependingont
hesubstituenttypeinthemolecule)andchargetransferCT(indu cedbytheaggregationfactor).Basedonthespectralshift sintheelectronabsorptionspectra,formationofcardpackr atherthanhe a dtot a i laggregatescanbeassumed.Furthermore ,theaggregationcanbestrongeratlow,neutral,orveryhighpHv alues,whereasadditionalintermediateforms,whicharearesul tantofbothionisedforms,canappearatneutralpH.Thiscanexpl aintherapiddeclineintheinten-
sityoftheRLSsignalatneutralandhighpHvalues.Theseconsi derationswillbecontinuedinfurtherinvestigationsofthishigh lyattractivedualfluorescencespectroscopiceffectinthe1,3,4- thiadiazolegroupwitha resorcylringinthestructure.
Conclusions
Theresultspresentedinthispaperindicateappearanceofthedual fluorescenceeffectinthefluorescenceemissionspectrumofthe analysed1,3,4-
thiadiazoleanaloguesC1andC7intheaqueousmedium.Inboth compounds,thiseffectismostev-
identatthephysiologicalpHandvalueslowerthatca.7.Fu rthermore,thecomparisonoftheanalysedanalogueswiththepr eviouslydescribedFABTcompound,differingstructur- allybythepresenceoftheamino–N-Hgroupinthesubstit- uentgroupandthefluorobenzenering,revealedthattherewasno longwavefluorescencebandwitha maximumatca.500nm.I nthecaseconsideredinthispaper,ashortwavefluoresc enceemissionbandwithamaximumatca.380nmwasfoundto accompanythedeclineinthepHvalue.Thiseffectmayberelat edtothestructuraldifferences(absenceoftheN-
HgroupintheC1andC7structure)betweenthecom-
pounds.Inanotherstudyofacompoundthatisstructurallysimi lartoFABTbutdoesnotcomprisethefluorobenze nefra gmentanddiffersfromC1onlyinthepresenceoftheN-
Haminegroup,abandwithamaximumatca.380nmwasobserv edathighpHvalues(resultssubmittedforpublishing).Acidifica tionofthemediumyieldedaseparatebandatca.440nm(asint hecaseofC1andC7atthehighpHvalues).Therefore,thereisane videntimpactofthesubstituentsinthe1,3,4-
thiadiazolestructureonthepositionoffluorescencebandsdesp itethesimilar/identicalchromophoreorganisation(fiveconju gateddoublebonds)intheanalysedmolecules.Thelength enedfluorescencelifetimeandchangeintheinten-
sityoftheRLSspectruminthespecifiedpHrangeforbothmole culesindicateassociationofthiseffectwiththeaggrega- tionphenomenondependentonthealkylchainlength.Theref ore,ithasbeenproposedthatprobablyacombinationoftwoeffe cts,i.e.molecularaggregation(withtheformde-
pendentontheanalogue)andCTchargetransfer,isresponsi- blefortheobservedfluorescencephenomena.Thishypothesisi slargelyconfirmedbythequantum-mechanicalcalculations
(TDDFT)performedforotheranaloguesfromthisgroupofco mpounds.Thenon-
specificinteractions(e.g.formationofintra-
andintermolecularhydrogenbondsorCTstates)intheanalysed 1,3,4-
thiadiazoleanaloguesmodifytheelectronicstructuresurrou ndingthemolecule,leadingtochangesinthedistributionofelect rondensityandtherebyforcingCTchargetransferintheanalyse danalogues.Thiseffectisreversibleafteralkalinisationofthe mediumandathighpHvalues(sin-
glefluorescence)andafteracidificationofthemediumatpHinth erangefrom1toca.7(dualfluorescence).
Thedualfluorescenceeffectoccursinadifferentenergeticra ngethanthatinthe1,3,4-
thiadiazolesanalysedpreviously.Inthetheoreticalaspect,itcan beusedforanalysisofexcita-
tionstatesintheseandotherstructurallysimilarmoleculesandfo rdesigningnewfluorescenceprobes.
Inconclusion,itshouldbeemphasisedthattheanalysed1,3, 4-
thiadiazolescanserveasexcellentfluorescentprobesthatareh ighlysensitivetochangesinthemediumpH.Additionally,the attractivenessoftheinvestigatedsystemsisenhancedbytheirad vantagesinmedicalandpharmacologicalapplications,whichar emoreoftenbasedonnewcompoundswithdesirableproperties.
AcknowledgementsT h e researchwasp artlysupportedby thegrantfro mtheUniversityofLifeScienceinLublin(TKF/MN/5toAM).
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo m mo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecommo ns.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedyougiveappropriatecredittotheori ginalauthor(s)andthesource,providealinktotheCreativeCommonslicens e,andindicateifchangesweremade.
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