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

Radu Ciobotea

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 43
• Poids de l'ouvrage: 1 Mo

Extrait

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 carm