Femtosecond spectroscopic study of carminic acid DNA interactions [Elektronische Ressource] / vorgelegt von Radu Comanici

De
FemtosecondSpectroscopicStudyofCarminicAcidDNAInteractionsDenNaturwissenschaftlichenFakultätenderFriedrichAlexanderUniversitätErlangenNürnbergzurErlangungdesDoktorgradesvorgelegtvonDipl.Phys.RaduComaniciAusPiatraNeamt2007Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten derUniversitätErlangenNürnbergTagdermündlichenPrüfung:29.07.2007VorsitzenderderPromotionskommission: Prof.Dr.BänschEberhardErstberichterstatter: Prof.Dr.CarolaKryschiZweitberichterstatter: Prof.Dr.RainerFinkContents1.Introduction........................................................................................................................ 102.MaterialsandMethods ...................................................................................................... 132.1Materials....................................................................................................................... 132.1.1.1DNA ................................................................................................................... 132.1.1.2BindingMode..................................................................................................... 172.1.1.3Structural,ElectronicandSpectroscopicPropertiesofCarminicAcid............. 192.2.1StationaryOpticalSpectroscopy............
Publié le : lundi 1 janvier 2007
Lecture(s) : 43
Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2007/659/PDF/FEMTOSECOND%20SPECTROSCOPIC%20STUDY%20OF%20CARMINIC%20ACID-DNA%20INTERACTIONS.PDF
Nombre de pages : 91
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Femtosecond
Spectroscopic
Study
of

Carminic
AcidDNA
Interactions

DenNaturwissenschaftlichenFakultäten
derFriedrichAlexanderUniversitätErlangenNürnberg
zur
ErlangungdesDoktorgrades

vorgelegtvon

Dipl.Phys.
Radu
Comanici

Aus
PiatraNeamt

2007


Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
UniversitätErlangenNürnberg

TagdermündlichenPrüfung:29.07.2007

VorsitzenderderPromotionskommission: Prof.Dr.BänschEberhard
Erstberichterstatter: Prof.Dr.CarolaKryschi
Zweitberichterstatter: Prof.Dr.RainerFink


Contents

1.Introduction........................................................................................................................ 10
2.MaterialsandMethods ...................................................................................................... 13
2.1Materials....................................................................................................................... 13
2.1.1.1DNA ................................................................................................................... 13
2.1.1.2BindingMode..................................................................................................... 17
2.1.1.3Structural,ElectronicandSpectroscopicPropertiesofCarminicAcid............. 19
2.2.1StationaryOpticalSpectroscopy............................................................................... 21
2.2.1.1UV/VISAbsorptionSpectroscopy..................................................................... 22
2.2.1.2FluorescenceSpectroscopy ................................................................................ 25
2.2.1.3MeasurementofFluorescenceQuantumYieldandFluorescenceLifetime...... 27
2.2.2TimeResolvedOpticalSpectroscopy....................................................................... 31
2.2.2.1FemtosecondTransientAbsorptionSpectroscopy............................................. 31
2.2.2.2FemtosecondFluorescenceUpConversion....................................................... 41
3.Experimental...................................................................................................................... 48
3.1Materials:ChemicalsandSampleSolutions ............................................................... 48
3.2Methods........................................................................................................................ 49
3.2.1UV/VISAbsorptionandFluorescenceSpectroscopy........................................... 49
3.2.2DeterminationoftheFluorescenceQuantumYield ............................................. 49
3.2.3FluorescenceTitrationExperiments ..................................................................... 50
3.2.4FemtosecondTransientAbsorptionSpectroscopy................................................ 50
3.2.5FemtosecondFluorescenceUpConversionTechnique........................................ 51
3.2.6Computations ........................................................................................................ 53
4.ResultsandDiscussion ...................................................................................................... 54
4.1.StationaryOpticalSpectroscopy................................................................................. 54
4.2.FemtosecondSpectroscopy......................................................................................... 67

4.2.1TransientAbsorptionSpectroscopy ...................................................................... 67
4.2.2FluorescenceUpConversionSpectroscopy ......................................................... 70
5.Conclusions........................................................................................................................ 80
6.References.......................................................................................................................... 82


List
of
Figures

Fig.2.1.1HeterocyclicbasesA:pyrimidines1:uracil,2:thymine,3:cytosine; ................ 14
Fig.2.1.2Structurecomponentsofthecommonnucleotides. .............................................. 14
Fig. 2.1.3Tautomersofuracil. .............................................................................................. 15
Fig2.1.4Bindingofguaninewithcytosine .......................................................................... 16
Table2.1.5RedoxpotentialoftheDNAbasesatapHvalueof7. ...................................... 16
Fig. 2.1.5StructureofasectionofDNA............................................................................... 17
Table 2.1.1.2 Thermodynamic binding parameters for the interaction of doxorubicin,
daunorubicin,hydroxyrubicinandtheβanomerofdoxorubicinwithcalfthymusDNA;K eq
isthebindingconstantandnisthenumberofbasepairsperbindingsite. .......................... 18
Table2.1.1.3FreeenergyofanthracyclineantibioticbindingtocalfthymusDNA. ........... 19
Fig. 1.3.1:Structureformulaofcarminicacid. ..................................................................... 20
Fig. 2.2.1Spectrumofelectromagneticradiation:thespectralrangeofopticalspectroscopy
isdepictedinenlargedform. ................................................................................................. 22
Fig.2.2.1.1SchematicrepresentationofaUV/VISabsorptionspectrometer. ..................... 24
Figure2.2.1.2.1Jabłońskitermscheme................................................................................. 25
Fig. 2.2.1.2.2Setupofafluorescencespectrometer.............................................................. 27
Fig2.2.2.1.1Energyschemeoftheelectronicstatesinvolvedinapumpprobe.................. 33
experiment;excitedstatesrelaxationdynamicsdetectedbyabsorptionchangesoftheprobe
............................................................................................................................................... 33
pulseare:1)bleaching;2)excitedstateabsorption;3)stimulatedemission. ....................... 33
Fig. 2.2.2.1.1 Schematic representation of the fs transient absorption spectroscopy
experiment;BS:BeamSplitter;FM:FlipMirror;DS:delaystage;VA:variableattenuator;
P: polarizer; L: lens; WLC plate: rotating fused silica plate for white light continuum
generation;PM:parabolicmirror;SHG:BBOcrystalforsecondharmonicgeneration;BC:
Berek compensator; BD : beam dump; BG: BG38 filter; GG: GG420 filter; ND: neutral
densityfilter. .......................................................................................................................... 36

Fig. 2.2.2.1.4 a) Intensity as a function of time for a Gaussian laser pulse b) Time
dependenceofthefrequencyforapositivenonlinearindexofrefraction,n ....................... 392
Fig. 2.2.2.1.5. Representation of the wlc probe pulse as a composition of the temporally
shifted,differentspectralsubpulseshavingthesamepulsewidthasthepumppulse......... 39
Table 2.2.2.2 Relative quantum efficiencies η (normalized relative to KDP), damageq
thresholdsI ,andcutoffwavelengthsforfluorescenceupconversionwith800nmpumpthr
pulses. .................................................................................................................................... 44
Fig.2.2.2.2.1Femtosecondupconversiontechniqueapparatus;DM:dichroicmirror;HW:
halfwaveplate;CCDvideocameraforthevisualsuperpositionofthebeamsintheBBO
crystal;Mono:monochromator;PM:photomultiplier. ......................................................... 46
Scheme 1:Dissociationreactionofcarminicacid................................................................. 54
Fig. 4.1.1:pHdependenceofthespectralfeaturesofcarminicacidinwatermeasuredby
UV/VISabsorptionspectroscopy. ......................................................................................... 55
Fig.4.1.2:Dualfluorescenceofcarminicacidwithablueemissionpeakat470nm(22700
0 0cm )andanorangeemissionpeakat570nm(15100cm )................................................. 56
0Fig. 4.1.3:Orangefluorescenceofthetautomerat15100cm . ........................................... 57
Fig 4.1.4:Normalizedfluorescencespectraof5JMcarmi nicacidinBPES(dashedline)and
5JMcarminicacidwith5JMDNAinBPES(solidline) ..................................................... 58
Fig. 4.1.5: Absorption ofcarminic acid in BPES; the band structure (thin solidline) was
analyzedbyfittingwithasuperpositionoffourGaussianfunction(dashedline)................ 59
Fig.4.1.6:AbsorptionofcarminicacidinDMSO;thebandstructure(thinsolidline)was
analyzedbyfittingwithasuperpositionoffourGaussianfunction(dashedline)................ 60
Scheme 2:Molecularstructureofthenormalformofcarminicacid(CAH)andthethree
tautomers(CAHT1,CAHT2,CAHT3) .............................................................................. 61
Table4.1.1:Calculatedvaluesofthetotalenergy(E ),thebindingenergy(E ),theabsoluteT B
energy(E ),theenergyoftheS S transition((S S ))andS S transition((S S ))abs 0 m 0 m 0 n 0 m
transitionandoftheoscillatorstrength(f). .......................................................................... 62

Fig. 4.1.7:Thespectraoftheorangefluorescenceof5JMcarminicacidinBPES.Theband
structure of the spectra (thin solid line) was analyzed by fitting with a superposition of
Gaussianfunctions(dashedline)........................................................................................... 64
Fig. 4.1.8: The spectra of the orange fluorescence of 5 JM carminic acid in DMSO; the
bandstructureofthespectra(thinsolidline)wasanalyzedbyfittingwithasuperpositionof
Gaussianfunctions(dashedline)........................................................................................... 65
Fig. 4.1.9: Concentration dependence of bound carminic acid, c , on the DNAB
concentration;theexperimentaldata(dots)wereobtainedfromfluorescencetitrationof6
JM carminic acid with DNA in BPES and were fitted ( solid line) employing the
1 5 0relationshipc =c c /(K +c )withK =5.0×10 (Mnucleotide) . .......................... 67B T⋅ DNA B DNA B
Fig. 4.2.1.1 3Dplotofthetemporalevolutionofthetransientabsorptionspectraobtained
forcarminicacidinBPES. .................................................................................................... 68
Fig. 4.2.1.2 Transientabsorptionspectrum(thinsolidline)recordedatthedelaytimeτ=1
ps; thefit (thick short dashed line) arises from the superposition of8 Gaussian functions
assigned to four different time constants (thick solid, dashed, dashed dotted and dotted
lines). ..................................................................................................................................... 69
Fig. 4.2.2.1Fluorescenceupconversionspectrum(dots)fittedbyasuperposition(thinsolid
line)oftwoGaussians(dashedline)...................................................................................... 70
Fig. 4.2.2.2 3D plot of thetemporal evolution of the fluorescence upconversion spectra
obtainedfor0.6mMcarminicacidinBPES......................................................................... 71
Fig.4.2.2.3: 3D plot of the temporal evolution of the fluorescence upconversion spectra
obtainedfor0.6mMcarminicacidand3.9mMDNAinBPES........................................... 72
Fig. 4.2.2.4: Fluorescence upconversion decays of 0.6 mM carminic acid in BPES (thin
0solidline)detectedat 14910cm inthe parallelpolarization geometry(I(t) )andinthepar
perpendicularpolarizationgeometry(I(t) );bothfitswereobtainedusingtheparameters:perp
a=0.37,b=0.91,ττττ =1.7ps,ττττ =33psandr(0)=0.137. ...................................................... 731 2
Fig.4.2.2.5: Fluorescenceupconversiondecaysof0.6mMcarminicacidinBPES(thin
0solidline)detectedat 16490cm inthe parallelpolarization geometry(I(t) )andinthepar

perpendicularpolarizationgeometry(I(t) );bothfitswereobtainedusingtheparameter:perp
a=0.50,b=0.92,τ =1.5ps,τ =47psandr(0)=0.202. ...................................................... 741 2
Fig. 4.2.2.6: Fluorescence upconversion decays of 0.6 mM carminic acid and 3.9 mM
0DNAinBPES(thinsolidline)detectedat14910cm intheparallelpolarizationgeometry
(I(t) )andintheperpendicularpolarizationgeometry(I(t) );bothfitswereobtainedwithpar perp
theparameter:a=0.50,0.97,τ =1.9ps,τ =48psandr(0)=0.182. .................................... 751 2
Fig. 4.2.2.7: Fluorescence upconversion decays of 0.6 mM carminic acid and 3.9 mM
0DNAinBPES(thinsolidline)detectedat16490cm intheparallelpolarizationgeometry
(I(t) )andintheperpendicularpolarizationgeometry(I(t) );bothfitswereobtainedwithpar perp
theparameter:a=0.50,b=0.97,τ =1.1ps,τ =61psandr(0)=0.243................................ 761 2
Table4.2.2.1: Theamplitudes, aandb,thetimeconstants, ττττ and ττττ ,andtheanisotropy,1 2
r(0),wereobtainedfromthebestfitofthefluorescenceupconversiondecaycurvesthatare
0 1detectedat14910cm and16490cm . ................................................................................ 78











Introduction
1. Introduction

Drugs for treatment of cancer on basis of anthracyclines have been extensively
studiedfordecadesinanefforttooptimizetheirtherapeuticfunction.Thesecompoundsare
believed to develop their cytotoxic effect by penetrating into the tumor cell nucleus and
interactingtherewithDNA[14].TheformationofdrugDNAcomplexesisdeterminedby
the structural features of the anthracyclines composed of a dihydroxyanthraquinone
chromophore with one or two glycosyl side chains. The formation of intercalation
complexeshasbeenobservedtoinhibittheDNAreplicationandtheRNAtranscriptionthat
blocks the gene expression [3]. Irradiation with light enhances the cytotoxicity of
anthracyclines (e.g. daunomycin) by several orders of magnitudes [58]. This photo
activationeffectisunderstoodtooriginatefromanultrafastelectrontransferreactionfroma
GbaseoftheDNAtotheintercalatedchromophore,whichisassociatedwiththeoxidation
of theG base and the reduction of the dihydroxyanthraquinone [7, 8]. This hypothesisis
based on femtosecond spectroscopy studies of daunomycinDNA and adriamycinDNA
complexes, yielding a decrease of the S state lifetime of the drug by three orders of1
magnitudewhenDNAispresent[7].ThelifetimeofthedaunomycinDNAcomplexwith
τ=290fswasascribedtotheoccurrenceofthephotoinducedelectrontransferfromtheG
base to the 1,4dihydroxyanthraquinone chromophore. On the other hand, other
radiationlessdecayprocessessuchasintersystemcrossing,internalconversionandexcited
state intramolecular proton transfer (ESIPT) may also be enhanced by conformational
reorganization that daunomycin experiences in the hydrophobic environment of the DNA
base pair stacking. Despite extensive research activities on the examination of photo
activated anthracyclineDNA complexes [711] to date there exists no unambiguous
evidenceforphotoinducedoxidationoftheDNAandmoreover,theexcitedstaterelaxation
dynamicsaswellasthestructuralmechanismatthemolecularlevelaremorehypothetical
thanreallyunderstood.
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