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X-ray absorption spectroscopy as a tool for characterizing sulfur based reactive intermediates Martin-Diaconescu, Vlad 2009

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X-RAY ABSORPTION SPECTROSCOPY AS A TOOL FOR CHARACTERIZINGSULFUR BASED REACTIVE INTERMEDIATESbyVLAD MARTIN-DIACONESCUB.Sc., The University of British Columbia, 2003A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Chemistry)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)August 2009© Viad Martin-Diaconescu, 2009AbstractSulfur K-edge X-ray absorption spectroscopy (XAS) has proven to be a great toolfor the investigation of sulfur oxidation states and sulfur-metal ligand bonding. In thisthesis, XAS has been applied in the detection and characterization of sulfur-basedreactive intermediates and products of photo-reacted sulfur species, with applications inboth bioinorganic and inorganic chemistry.Low molecular weight thiols and their derivatives have important proteinmodulation, signal transduction and antioxidant activities. This includes glutathione(GSH), nitrosoglutathione (GSNO), and lipoic acid (LA), which are involved in complexredox pathways resulting in a variety of intermediates and products that can be difficultto characterize. These compounds have been used as models for thiol nitrosation andoxidation reactions, and their reactivity was probed with sulfur K-edge XAS, which hasbeen developed into a valuable tool for the investigation of sulfur-containing radicalspecies and related non-radical intermediates.XAS was also applied to investigate the reactivity of p-toluene sulfonyl chloride,an initiator in metal catalyzed living radical polymerization, to explore the effect ofhyperconjugation on the reactivity of the S-Cl bond. A series of model compounds ofthe form RSO2G (C = -Cl, -OH, -alkyl) were used to evaluate the effect of aryl versusalkyl R groups on the photo-reactivity and orbital mixing of theS-G bond.IITable of ContentsAbstract iiTable of Contents iiiList of Tables vList of Figures viList of Schemes ixList of Equations xList of Abbreviations xiAcknowledgements xiiChapter 1: X-ray Absorption Spectroscopy (XAS) I1.1 The Synchrotron Light Source (X-ray Source) I1.2 Sulfur K-edge X-ray Absorption Spectroscopy 41.3 Applications of XAS 71.4 Thesis Overview 9Chapter 2: Implications of Thiol Oxidation 102.1 Low Molecular Weight Sulfur Species In Vivo102.2 Mechanism and Control of Thiol Oxidation 132.3 Experimental Rationale 18Chapter 3: Experimental Section 213.1 X-ray Absorption Spectroscopy (XAS) 213.2 Density Functional Calculations 243.3 Nuclear Magnetic Resonance Spectroscopy (NMR) 253.4 Electron Paramagnetic Resonance Spectroscopy (EPR) 253.5 Materials 26Chapter 4: Sulfur K-edge XAS as a Probe of SulfurCentered Radical Intermediates 274.1 Background 274.2 Results and Discussion 284.3 Conclusion 37Chapter 5: Perthiyl Radical and Disulfide Bond Formation inPhoto-irradiated Nitrosoglutathione and Lipoic Acid 385.1 Background 385.2 Results and Discussion 43III5.3 Conclusion.54Chapter 6: Effects of Hyperconjugation on the ElectronicStructure and Photo-reactivity of Organic Sulfonyl Chlorides 556.1 Background 556.2 Results 586.3 Discussion 716.4 Conclusion 74Chapter 7: Concluding Remarks and Outlook 77References 80Appendix I ADF Input Files 88ivList of TablesTable 2.1.1 Biologically important low molecular weight sulfur species 11Table 4.2.1 Principal component analysis of UV irradiated GSH XAS spectra 31Table 4.2.2 TD-DFT calculated sulfur K-edge bound transitions for thiyl andperthiyl radicals 36Table 5.2.1 Half power saturation values for the EPR of LA 45Table 6.2.1 TD-DFT and IiSCF of p-toluene sulfonyl chloride 66Table 6.2.2 TD-DFT and ASCF of methane sulfonyl chloride 66Table 6.2.3 TD-DFT and ASCF of p-toluene sulfonic acid 67Table 6.2.4 TD-DFT and iSCF of methane sulfonate 67Table 6.2.5 TD-DFT and IISCF of ethyl phenyl sulfone 68Table 6.2.6 TD-DFT and IiSCF of methionine sulfone 68VList of FiguresFigure 1.1.1 Schematic diagram of the main componentsfoUnd in a synchrotron 2Figure 1.1.2 Schematic diagram of the main componentsfound in a beamline 4Figure 1.2.1 XAS model spectra of possible S1 transitions 5Figure 1.3.1 XAS fingerprint spectra of varying sulfur oxidation states 7Figure 2.1.1 Thiol oxidation and cell life cycle 10Figure 2.2.1 Thiol oxidation pathways 15Figure 2.2.2 Reduction pathways of oxidized sulfur species 17Figure 3.1.1 XAS experimental setup at SSRL beamline 6-2 23Figure 4.2.1 X-band EPR of UV irradiated glutathione 29Figure 4.2.2 Power saturation study of UV irradiated glutathione 30Figure 4.2.3 Sulfur K-edge XAS of UV irradiated GSH 32Figure 4.2.4 Difference XAS spectra of UV irradiated GSH 33Figure 4.2.5 Time evolution of features at 2468.8eV and 2470.5eV inUV irradiated GSH 34Figure 4.2.6 DFT density contour maps of thiyl and perthiyl radicals 35Figure 4.2.7 Peak fitting of XAS features due to thiyl and perthiyl radicals 37viFigure 5.1.1 GSNO sulfur K-edge XAS spectrum 40Figure 5.1.2 DFT density contour maps ofCH32SNO 40Figure 5.1.3 LA sulfur K-edge XAS spectrum 42Figure 5.2.1 LA sulfur K-edge XAS spectra under photo-irradiation 43Figure 5.2.2 EPR spectra of irradiated LA 45Figure 5.2.3 EPR power saturation spectra of irradiated LA 46Figure 5.2.4 GSNO sulfur K-edge XAS spectra under photo-irradiation 48Figure 5.2.5 Time evolution of features at 2470.2 and 2471.6in irradiated GSNO 49Figure 5.2.6 EPR spectra of irradiated GSNO 51Figure 5.2.7 NMR of irradiated GSNO and GSSG 52Figure 5.2.8 EPR spectra of irradiated GSSG 53Figure 5.2.9 EPR power saturation study of irradiated GSSG 54Figure 6.2.1 Structure of sulfonyl model compounds 59Figure 6.2.2 Geometries of ethyl phenyl sulfone used for TD-DFT 60Figure 6.2.3 XAS and simulated spectra of sulfonyl chlorides 61Figure 6.2.4 XAS and simulated spectra of sulfonates 62viiFigure 6.2.5 XAS and simulated spectra of sulfones 63Figure 6.2.6 Molecular orbitals of benzene 65Figure 6.2.7 Time evolution of the sulfur K-edge features dueto the S-Cl bond with in-situ irradiation 69Figure 6.2.8 XAS with in situ irradiation of sulfonyl chlorides 70Figure 6.3.1 Mixing of ffL and cT into SCl* 72Figure 6.3.2 Effect of turning off4n*/SCla* mixing 73Figure 6.4.1 Fitted XAS of p-toluene sulfonyl chloride 75Figure 7.1.1 XAS and complementary techniques 77VIIIList of SchemesScheme 5.1.1 GSNO photo-reactivity induced reaction pathways 39Scheme 5.1.2 LA photo-reactivity induced products 41Scheme 6.1.1 Metal catalyzed polymerization with p-toluenesulfonyl chloride as an initiator 56Scheme 6.1.2 Sulfonyl chloride photo-cleavage reaction 58ixList of EquationsEquation 1.1.1 Bragg’s law 3Equation 1.2.1 Heisenberg uncertainty principle 6Equation 6.4.1 Percent excited state hyperconjugationin p-toluene sulfonyl chloride 75xList of AbbreviationsA Angstrom°C Degrees CelsiusCysS CysteineCysSSCys CystineDFT Density Functional TheoryDHLA Dihydrolipoic AcidDNA Deoxyribonucleic AcidEPR Electron Paramagnetic Resonance SpectroscopyeV Electron Voltf Oscillator StrengthGPx Glutathione PeroxidaseCR Glutathione ReductaseGrx GlutaredoxinGSH GlutathioneGSNO NitrosoglutathioneGSSG Glutathione Disulfidehr HoursK KelvinLA Lipoic Acidm MultipletMIN MinutesNMR Nuclear Magnetic Resonance SpectroscopyP112 Power of Half Saturation (of EPR signal)Prx PeroxyredoxinRF Radio FrequencyRNR Ribonucleotide ReductaseRS Thiyl RadicalRSNO NitrosothiolRSS Perthiyl RadicalASCF Slater Transition State Self-consistent Field MethodSSRL Stanford Synchrotron Radiation Lightsourcet TripletTD-DFT Time Dependent Density Functional TheoryTrx ThioredoxinTrxR Thioredoxin ReductaseUV-VIS UV-Visible SpectroscopyXANES X-ray Absorption Near Edge StructureXAS X-ray Absorption SpectroscopyZeff Effective Nuclear ChargexiAcknowledgementsFirst and foremost I would like to thank Dr. Pierre Kennepohl, my supervisor andmentor, whose encouragement, patience and support, have not only made this workpossible but have made my time as a graduate student a wonderful experience. Hisdoor has always been open and his help was always gladly given no matter thecircumstance. Secondly, I would like to thank my lab mates, in particular AnushaKarunakaran with whom I have spent countless hours acquiring data, Mario Delgadowith whom I have argued endlessly about everything under the sun and Kendra Gettywho still holds the record for the number of straight hours of beamline data acquisition.Needless to say that I consider the fore mentioned people my friends and they will besorely missed.I would also like to thank Dr. Serena DeBeer George, who has always beenready to help at any time and who was of great help during our data acquisition trips atthe Stanford Synchrotron Radiation Lightsource (SSRL). From experimental setup andrunning the data acquisition, to sample preparation, Dr. S. George was always therewhen needed. On a related note, I would also like to thank Dr. Matthiew Latimer for hishelp with experimental setup for our photo-irradiation studies.Last but not least I would like to thank the UBC Chemistry department supportstaff, in particular Maria Ezhova for her help with NMR data acquisition, Jane Cua forher help with running and maintaining the ADF program, John Ellis for his help withacquisition orders, Ken Love and Razvan Neagu for their help over the years withequipment trouble shooting, and Pat Olsthoorn for his help with sample shipmentstoSSRL.xiiI X-ray Absorption Spectroscopy (XAS)This thesis relies heavily on the application of X-ray absorption spectroscopy(XAS) at the sulfur K-edge to investigate and characterize the bonding, electronicconfiguration, and reactivity of a series of biologically relevant low molecular weightthiols and their derivatives, as well as sulfur species with applications in inorganicpolymer chemistry. Photochemistry was employed to follow the reactivity of thesespecies, and investigate their reactive intermediates and products. XAS proved to be auseful technique in the detection and characterization of the elusive thiyl radical in UVirradiated glutathione. The perthiyl radical intermediate was detected in irradiatedsamples of lipoic acid and glutathione, as well as from irradiated nitrosoglutathionewhere formation of the perthiyl radical was a two step process. To perform theirradiation experiments, new methodologies of in situ photo-irradiation coupled with XASspectra acquisition allowed the photo-reactivity to be observed. Last but not least, therelevance of excited state hyperconjugation was identified and its effect on the reactivityof p-toluene sulfonyl chloride explained as it pertains to the initiation step in the metalcatalyzed polymerization of olefins. In each case, XAS was essential in the detectionand characterization of these products.1.1 THE SYNCHROTRON LIGHT SOURCE (X-RAY SOURCE)X-ray absorption spectroscopy was the primary method used in theseinvestigations. The intensities and the wide energy ranges used for the X-rays in thefollowing experiments require a synchrotron source. A synchrotron light source consistsof a circuit of accelerated electrons that give off electromagnetic radiation in the form ofX-rays. All electrons when accelerated give off energy, and this has many applicationsin everyday life, such as radio signal transmission. In a synchrotron, however, electronsare accelerated to speeds approaching the speed of light (99.9% speed of light). Theserelativistic electrons give off electromagnetic radiation in a parallel path to the directionof propagation of the relativistic electron.A synchrotron light source usually consists of a booster ring where electrons areaccelerated to near the speed of light, from which they are injected into the storage ring(figure 1.1.1). Magnetic fields from “bend magnets” change the direction of theIelectrons in the storage ring allowing them to maintain a closed path. The velocity ofelectrons is maintained by constructively interfering radiofrequency (RF) waves whichtakes place in the cavity. The RF waves are generated by a klystron (RF amplifier)similar to those used in TV broadcasting stations. The RF waves are guided to thecavity by a wave guide and timed to “synchronize” with the electrons entering the cavity.At various positions in the straight sections on the storage ring there are wigglers andundulators. These are a series of magnets with alternating polarities that cause theelectrons to have small deviations in their linear trajectory, in a sense causing them towiggle and undulate. Since the emitted X-rays have no charge they are not affected bythe magnets and their trajectory is linear. X-rays emitted during a “wiggle” have a paththat comes out of the storage ring and it can be delivered to various work stations(hutches) using beamlines. On any one storage ring there can be several wigglers andundulators allowing for multiple work stations or hutches.EMITTED X-RAYSBENDMAGNETELECTRONSWAVE GUIDECAVITY_- KLYSTRON (RF SOURCE)/tSTORAGE RINGWIGGLERFigure 1.1.1 Schematic diagram of the main components found in a synchrotron.2Beamlines screen the broad range of radiation energies (from radio frequenciesto y-rays to X-rays) coming from the wiggler and select those needed for the particularexperiment. They also shape the beam to suit the sample size and help control flux.This is achieved with the help of beamline optics as shown in figure 1.1.2. As the X-raybeam leaves the wiggler it may pass through a series of slits that shape it to have theneeded dimensions. X-rays with the required experimental energies are then selectedusing a double crystal monochromator. X-rays of various energies are selected basedon their wavelengths using Bragg diffraction. Bragg diffraction occurs when theincoming X-rays are scattered by the periodic structure of the crystal lattice according toequation 1.1.1.n2=2dsin6n = harmonic (integer > 1)Equation 1.1.12 = wavelengthd = distance between lattice layers0 = angle of diffractionTherefore the crystal monochromators can be used to modulate the energiesbeing diffracted to the experimental hutch by varying the angle of incidence of the Xrays and by changing the type of crystal used (lattice structure). If only one crystal wasused scanning over a range of X-ray energies would mean moving the sample to matchthe angle of diffraction of the incoming beam. In practice, two crystal monochromatorsare used and moved in concert so that the diffracted beam is parallel to the incidentbeam. It is also evident from Bragg’s equation that a series of harmonics or X-raywavelengths can satisfy the equation for a particular angle of diffraction and crystallattice structure. Therefore beamlines can be equipped with harmonic rejection mirrorswhich can efficiently refract X-rays of a certain energy range, but not those of itsharmonics. These mirrors are coated with elements of higher atomic number becausethe refraction of X-rays of a particular energy is dependent on the material density of thecoating. Mirrors can also be used to focus both the incident X-ray beam and thediffracted beam used for experiments. Once X-rays of the correct energy are isolatedthey are allowed to pass into the hutch. This is where the experimental setup for thevarious experiments is located (see Chapter 3 Methodology).3MONOCH ROMATORI — — — —I IFigure 1.1.2 Schematic diagram of the main components found in a beamline.I .2 SULFUR K-EDGE X-RAY ABSORPTION SPECTROSCOPYX-RAYSTOHUTCHSulfur K-edge (S K-edge) XAS involves detection and characterization of sulfurcore electron excitations, specifically those from the Is core level(Si).Features due tothese excitations, as well as electron ionizations from theSiscore, fall in the 2400-2600eV energy range. The sulfur K-edge spectra is dominated by an ionization edge or“edge jump” due to excitations from the core Is orbital to the continuum (cc—Sis),resulting in ionization (figure 1.2.152.)Features due toSisexcitations into unoccupied(figure 1.2.1 RS.) or partially unoccupied (figure 1.2.1 RS) acceptor orbitals with sulfurcharacter appear as absorption peaks. They fall within what is termed the X-rayAbsorption Near Edge Structure (XANES) region of the spectrum. The intensities ofthese transitions are governed by Fermi’s Golden Rule which states that the intensity ofa transition is proportional to the probability of it occurring1.The probability of atransition occurring is dependent on the coupling between the ground state and the finalstate, therefore transitions resulting from atomic core excitations can only involveexcitations to other orbitals in the same atom. Furthermore, dipole allowed (s4—p; p—d)transitions have a higher probability of occurrence. This means that excitations of Issulfur core electrons into acceptor orbitals with a higherS3pcharacter will be moreSLITSIMIRRORWIGG LERII. — — — — .143pRSIs SCo 3pSCc**3pFigure 1.2.1 XAS model spectra showing the possible transitions and resulting XAS featuresfrom S2 (only ionization is possible), thiolate (ionization and bound transitions to unoccupiedmolecular orbitals is possible) and the thiyl radical (ionization and bound transitions to bothunoccupied and half occupied orbitals possible).FINAL SPECTRUM FOR RS’ENERGYENERGYRS**IsSCa 3pSCa*ENERGYS2-Is5intense than transitions to acceptor orbitals having lower S3 character. In addition, theprobability of a transition is also dependent on the number of ways it can occur. At itssimplest this means that a transition to a half occupied acceptor orbital should be half asintense as a similar transition to a fully unoccupied acceptor orbital. These acceptororbitals are usually the antibonding counterparts of each of the chemical bonds sulfur isinvolved in, within the species being investigated. The intensities of these bound-statetransitions can provide information regarding the amount of sulfur character in aparticular bonding scheme, as well as the electronic configuration.Equation 1.2.12The spectral linewidth of the transitions is dependent on the resolution of themonochromator as well as the core-hole lifetime resulting from the particular transition.The core-hole lifetime is dependent on the energy of the transition according to theHeisenberg uncertainty principle (Equation 1.2.1). Therefore high energy transitionsresult in short core-hole lifetimes and broad features or linewidths, while low energytransitions such as those found at the sulfur K-edge result in long core-hole lifetimesand relatively sharp features. The line shapes for each of the transitions are acombination of a gaussian component as a result of the resolution of themonochromator and a lorentzian component due to the core-hole lifetime and can bedescribed by a pseudo-voigt function which is a linear combination of the twocomponents.Another important feature of XAS transitions is the energy at which they occur.This is dependent on both the energy level of the core electrons, as well as that of theacceptor orbital. The energy level of the core electrons can be a measure of effectivecharge on the sulfur atom, while the energy of the acceptor orbital can give informationabout bond strengths. For example, in the study of perthiyl radicals (RSS) there aretwo transitions from each of the sulfurs to the same SS acceptor orbital. If the twosulfurs were equivalent only one feature should be present because the two transitionswould be overlapping. However, two transitions are visible corresponding to thedifference in effective nuclear charge (Zeff) between the sulfurs which lowers the coreelectron energies in one of the sulfurs versus the other.61.3 APPLICATIONS OF XASR2SO4RSO3Na:III I:1RSONa’:RSH: 1c2: \:2/!\\ !:I I ‘•.., :I \i! \‘ ... -‘r, — — — _. _. —1Ii., ,.../.-.‘ .. -..-...2470 2472 2474 2476 2478 2480 2482 2484 24862488 2490ENERGY (eV)Figure 1.3.1 XAS fmgerprint spectra of sulfur specieswith varying oxidation states.The nature of the transitions observed in sulfurK-edge XAS make this techniquesensitive to the various oxidation states of sulfur(figure 1.3.1). In fact, much of theliterature involving sulfur K-edge XASuse the “fingerprint” method to investigate sulfurspeciation. In this approach, the compositionof the sulfur K-edge spectrum fromaparticular sample is determined by fittingand summing up spectra from similar samplescontaining 100% of a particular sulfuroxidation state2’.This method can be applied toboth non-biological and biological systems.For example, sulfur speciation as it pertainsto the effectiveness of different coaldesulfurization methods was investigatedusing least square fitting of the XANES regionof spectra from various coalsamples that were desulfurized usingeither biological,chemical or caustic leaching methods4.Sulfur K-edge XAS was also usedto assessthe impact of land use by characterizingsulfur oxidation in soil samplesfrom naturalforest, tea plantationsand cultivated fields from various locationsin Ethiopia. Naturalforest were found to have the mostreduced sulfur species, followed by plantationswithcultivated fields being most oxidized5.Most notably, oxidation of reduced sulfurspeciesin the seventeenth-century Swedishwarship, Vasa, was found tobe catalyzed by iron5Na2SO4Cl)zwIzwN-J0zGLUTATHIONE—— METHIONINE SULFOXIDESODIUM METHANE SULFINATE— ---SODIUM METHANE SULFONATESODIUM SULFATE7from corroded iron bolts, leading to the formation of sulfate salts which threaten thepreservation of the ship6.Researchers have also collected the spectra for a variety of biologically relevantsulfur species. The distinct features differentiating between thiol groups and disulfideswere used in a number of studies investigating the thiol redox couple responsible forredox homeostasis, in blood, and separated plasma and erythrocyte samples2’3Extracellular cysteine was found to be mostly in its oxidized cystine form whileintracellular cysteine was found to be more reduced2’.Increased oxidation state ofsulfurs in transthyretin investigated with sulfur K-edge XAS is seen in amyloid fibrils, themain component of amyloid deposits present in such disorders as Alzheimer’s diseaseand Creutzfeldt-Jakob disease7.Features due to S-H bonds (SHa**_Sis),S-C bonds(SC0*€_Si), and S-S bonds (SS*E-_Sls), in samples were identifiedby comparing theXANES features from H2S, H2S and reduced and oxidized cysteine2’8 Investigationofthe dependence of the cysteine spectra on pH showsa dramatic change in the spectralfeatures with deprotonation2’.Another advantage of this technique is its ability to toleratea broad range ofsample types and preparation techniques. For example, the studiesdiscussed thus farhave included solid samples in the formof coal, soil, and wood; liquid samples in theform of blood, plasma and buffered cysteine,and H2S and H2S gas samples. It is notsurprising then, that samples consisting of varioussulfur accumulating bacteria wereprepared and quantitative sulfur speciation studieswere carried out. At least threedifferent forms of sulfur were found in bacterial sulfurglobules with cyclooctasulfur beingprevalent in Beggiatoa alba,polythionates in Acidithiobacillus ferrooxidansand sulfurchains in green and purple sulfurbacteria10.XAS becomes an even more powerfulexperimental technique when coupledwithsimulations of the electron excitationtransitions along with calculationsof the orbitals ofthe donor and acceptor states involvedin the transitions. This was first recognized andexplored in chlorine K-edge XASspectroscopy” and then extendedto sulfur K-edgeXAS where the metal-ligand covalencywas measured in transition metal tetrathiolatecomplexes’2.XAS coupled with densityfunctional calculations (DFT) wereused in thecharacterization of the Cu-S bondin the active sites of blue copperproteins which wasfound to have a high covalency(38% S3 character) and a unique single it-bondbetween the copper and the sulfur13.The methodology was then appliedto the8investigation of oxidation, reduction, and bonding of iron sulfur clusters in ferredoxinsand rubredoxins’3.XAS and DFT were further used to assign the spectral features forS-nitroso proteins where transitions to SC, SNOa* and S-NO acceptor orbitals wereidentified14.The dependence of XAS spectra of disulfides on the dihedral angle wasinvestigated by comparing the experimental and simulated spectra of oxidized lipoicacid to those of less conformationally strained disulfides. The results showed distinctdifferences in both the intensity and peak width of theSS.+-S1 and SC0*+-Sitransitions, emphasizing the importance of accountingfor molecular conformationswhen analyzing spectra using the fingerprint method15.1.4 THESIS OVERVIEWThe above discussion is meantto show the range of applicability of thisexperimental technique. It has been shown thatsulfur K-edge XAS is sensitive to sulfuroxidation regardless of sample preparation.Sulfur speciation and electronic structurecan be gained from the XANES regionof the spectra encompassing bound transitionsto low-lying molecular orbitals withS character. A combination of thefingerprintmethod and density functional calculations canbe used in the assignment of spectralfeatures and characterization of thesulfur bonding manifold. The currentstudyinvestigated in situ photo-reactivityof both biological and non-biologicalsamples. Ineach case DFT calculations were appliedto better assign the spectral features andunderstand the electronic configuration.Additional spectroscopic techniqueswere usedin the characterization of startingmaterials, intermediates and products.Particularlyelectron paramagnetic resonance (EPR)spectroscopy was essentialin the detectionand characterization of radicalintermediates generated duringthe photo-reactivitystudies. The following chapters covera discussion of the experimental setupsusedthroughout the thesis, a briefoverview of the importance of low molecularweight sulfurbased antioxidants in redoxhomeostasis, followed bythe studies involving the mainrepresentatives of these specieschosen for their relevance,reactivity and interestingelectronic structure. Last but notleast, is the investigation of the bondingand electronicconfiguration of p-toluenesulfonyl chloride as it pertainsto its role as an initiator in themetal catalyzed polymerizationof styrene, methacrylates,and acrylates.92 Implications of Thiol OxidationLow molecular weight sulfur species are involved in a variety of processes thatmaintain the oxidative balance in vivo. These compounds (table 2.1.1) are known tohave antioxidant properties which come into play particularly in times of oxidative stress,when increases in oxidizing species disrupt regular cell function. Their interaction withreactive oxygen (ROS) and nitrogen (RNS) species not only have antioxidativeconsequences but are also important in signaling pathways. Low molecular weightthiols and their derivatives could be described both as “middle men” and, due to theirantioxidant functions, as modulators of these signaling pathways. Their antioxidantproperties also make low molecular weight su’fur compounds potential therapeuticagents in various diseases with symptoms of oxidative stress. Of these the mostimportant and abundant is glutathione (GSH), which behaves as a redox buffer withinthe cell16. Outside the cell the most important thiol for maintaining redox homeostasisiscysteine (CysS)16.2.1 LOW MOLECULAR WEIGHT SULFUR SPECIES IN VIVOPROLIFERATION DIFFERENTIATION APOPTOSISINTRACELLULAR (GSH)-2601-230 mV -220/-I 90 mV -1 7OmV[RSH] > LRSSR] [RSH) < [RSSR]EXTRACELLULAR (CysS)<-80 mV -80 mV ‘-80 mVFigure 2.1.1 Intracellular and extracellular disulfide/thiol redox stateat various stages in thelifecycle of the cell as described by the GSSG/2GSH couple(intracellular) and CysSSCysI2CysScouple (extracellular)16’17Thiol oxidation has been linked to severalcellular processes with implicationsranging from cell signaling to progressionof disease. Both the glutathione(GSSG/2GSH) and cysteine (CysSSCys/2CysS)redox states vary over the life cycle ofthe cell (figure 2.1.1). One can see aprogression to a more oxidized state going fromproliferation to apoptosis’6’17The extent of oxidation has been exploredin severalsystems by induction of differentiation in proliferatingcells. Application of thedifferentiating agent sodium butyrateto a HT29 cell line caused a +6OmV shift (from-26OmV in the proliferating cells to -200mVin the differentiating cells)18. Exposure ofslime mold to differentiationstimuli resulted in a decrease inGSH10Table 2.1.1 Biologically important low molecular weight sulfur species.LOW MOLECULAR WEIGHT SULFUR SPECIESCysteine (CysS)0Precursor to GSH and the majorextracellular thiol redox buffer16’19 HS OHMethionine0In addition to being used for cysteinesynthesis, methionine oxidation/reductionH3C OHis involved in metal ion channel gating andNH2neurodegenerative diseases20.Glutathione (GSH)The most abundant low molecular weightthiol, has antioxidant properties,modulates protein function throughS-glutathionylation and maintains redoxhomeostasis2’.Nitrosoglutathione (GSNO)Used in the storage, transport and deliveryof N0 a molecule important in cellsignaling22.Dihydrolipoic acid (DHLA)Lipoic acid (LA) is the oxidized form,has aredox couple of LA/DHLA (-32OmV) andcan directly reduce GSH23. Canchelatemetal ions and prevent lipid peroxidation24.It is an antioxidant in both hydrophilic andlipophilic environments25.11and an increase in the antioxidant enzyme manganese superoxide dismutase(MnSOD), indicating a shift to a more oxidized environment during differentiation26.Variation of thiol oxidation states also occurs within the cell.Compartmentalization allows a more reduced environment within the nucleus where theGSH pool is implicated in protection from oxidative stress of DNA27 and DNA bindingmotifs28,regulation of gene transcription29,DNA synthesis30,and DNA repair31’32 In theendoplasmic reticulum on the other hand, the thiol redox couple favors the moreoxidized state. Here the redox state of the GSSGI2GSH couple is approximately-18OmV33,as opposed to -26OmV in the nucleus16. This facilitates disulfide bondformation and isomerization of incorrectly formed disulfide bonds,a processmodulated in part by protein disulfide isomerase (P01),a thiol disulfideoxidoreductase34’The disulfide/thiol redox state can also be affectedby external factors such asdisease. A prime example is the oxidative pressuresassociated with HIV (Humanlmmunodeficiency Virus). In HIV there isa systemic decrease in reduced GSH339 andGSH synthesis4°along with a decrease in totalcysteine and cystine amounts ([CysS] +2[CysSSCys])38.Chronic increasein thioredoxin (Trx, a family of proteins involved inthe reduction of disulfides) concentrationscan further compromise the innate immuneresponse by inhibition of Iipopolysaccharideinduced chemotaxis, resulting in shorter lifeexpectancy of AIDS victims41.Additionof NAC (N-acetylcysteine) increases survival16and in vitro addition of GSH and NAC inhibits viralreplication42,indicating the potentialfor therapeutic applications of low molecularweight thiols.Last but not least, thiol oxidationcan also affect signaling pathways. Proteintyrosine phosphatase (PTP) hasa catalytically active cysteine residuewhich can beinhibited if oxidized toa sulfenic acid by H2043. Increasedthiol oxidation andGSSGIGSH ratios also seemto stimulate the activity of several kinasessuch as themitogen activated protein kinases(MAPK), JNK (c-Jun N-terminalkinase) and p38,required for tumor necrosis factor (TNF-c)induced apoptosis45.Nuclear transcriptionfactors can also be affectedby GSH oxidation. Forexample, Nil 2 (NE-F2 relatedfactor) which regulates expressionof several genes involvedin antioxidantresponse46’47,is translocated to the nucleus whenits Keap-1 subunit dissociates fromthe main complex due to oxidationand conjugation of its cysteineresidues48.However,within the nucleus, DNA bindingactivity is controlledby Trx49,which maintains Nil 2’s12DNA binding cysteine residue in its reduced form50,highlighting the importance ofcompartmentalization, as discussed earlier.2.2 MECHANISM AND CONTROL OF THIOL OXIDATIONReactive Oxygen (ROS) and Nitrogen (RNS) Species and Thiol OxidationThiol oxidation can be driven by the reactive oxidant pathways of ROS and RNScoupled with disulfide reduction systems such as those of the thioredoxins (figure 2.2.1).Comprehensive reviews on the formation and consequences of reactive oxygen species(ROS) and reactive nitrogen species (RNS) are available5053.These reactive speciescan arise endogenously from cellular processes or be mediated by external factorssuchas chemotherapeutic agents, UV irradiation, and other environmental stimuIi5053.At lowconcentrations, ROS and RNS can be beneficial and manyare involved in signalingpathways50,gene transcription29 and host defenses against infection.Examples areleukocyte release of ROS to attack infecting bacteria, NO’induction of smooth musclerelaxation55’56,and N0 inhibition of platelet aggregation57.Thiolsand thiol redoxpathways play important roles both as antioxidants tomaintain ROS and RNS athomeostatic levels and as intermediary mediatorsin some of the ROS/RNS signalingpathways.In eukaryotes reactive oxygen species arise mainly inmitochondria from aerobicrespiration58’59or enzymatic reactions of NADP(H) oxidasesin leukocytes54.Theproduct of such reactions is thesuperoxide anion (Oj) which is not membranepermeable and thus reacts within whichever cellularcompartment it is created60.Oj isconverted to hydrogen peroxide (H20)by superoxide dismutase (SOD)61. H20 ismembrane permeable and can actas a signaling molecule50’62In the presence ofredox-active metal ions, H20 is rapidly convertedto the hydroxyl radical (HO’) via theFenton reaction6365.HO is highlyreactive and unselective towardsa host of biologicalmolecules. H2O can also oxidize thiols,and in high enough concentrations, it can leadto the generally irreversible sulfur oxidationstates of RSO2H and RS03H66.Three mainenzyme systems are employedin H20 removal: catalases (CAT)67,glutathioneperoxidase (GPx)68 and peroxyredoxins(Prx)69 will degrade H20to yield H20 and 02.The latter two, GPx and Prx involveintermediate oxidation of thiols. GPx reductionofH20 results in GSH dimerizationto GSSG68.Prx is initially oxidizedto sulfenic acid13(RSOH), which in the case of 1-CysS Prx can be reduced by vitamin C70. In 2-CysS Prx(Prx containing two cysteines in its active site) the initial oxidation to RSOH is followedby internal disulfide bond formation which can be later reduced by thioredoxins7’. Prxcan also be further oxidized by H20 to give sulfinic acid (RSO2H), which can bereduced back to the active form by sulfiredoxin7173.N0 production from arginine is facilitated by nitric oxide synthase and similar to02, it is the first product in the RNS chain74.N0 can further react with 02 to give N203,a nitrosylating agent, and with02 to give peroxynitrite (ONOO)52.The reaction of N0with thiols is generally considered too slow to have biological relevance, however N203can nitrosylate a variety of substrates including thiols to give S-nitrosothiols75.0N00contributes to oxidative stress and is scavenged by thiols resultingin disulfide bonds76.Denitrosation is facilitated by dihydrolipoic acid (DHLA) andTrx, which undergooxidation resulting in release of a free thiol and HN077.Similar to the denitrosationmechanism of DHLA and Trx, transnitrosation reactions, followedby S-thiolation anddisulfide bond formation modulate protein activityas is the case of creatine kinase78.Release of N0 can also be achieved by decompositionof two nitrosothiols ornitrosothiol reduction of metals such as copper.Both cases result in formation of adisulfide79’8°Disulfide formation via reduction of metals suchas vanadium, copper and iron isalso possible and reported to proceed through athiyl radical intermediate65’81-83Thiolssuch as glutathione and dihydrolipoic acid can ligatemetal ions and are involved in bothmetal detoxification84 and delivery85. Reducedmetal ions such as arsenic (Ill),chromium (IV) and chromium (V), copper (I),and iron (II) can induce oxidative stress,and their ligation may help in their excretion whichwould prevent them from reacting toinduce oxidative stress63 . Metal reductionby GSH and GSH derivatives is associatedwith lipid peroxidation, which stimulates oxidativestress rather than represses it. Theefficiency of the reduction is modulatedby the ligating abilities of the thiol species andtheir pKa’s87’88 Lipid peroxidation due to Cu2is inhibited by DHLA which is believedtochelate the metal via its vicinal thiols.The stability of the complex however,is pHdependent and the complex becomes destabilizedwith increasing pH24.14RSHOHo=NOSN0ArginineOOXIDASESI— SODH2M1TOCHONDRIA2 2 2Figure 2.2.1 Thioloxidation pathways, invivo.RSH NO0N00RSHRSNOHNOINO15Mechanisms of Disulfide ReductionFrom the discussion of ROS and RNS one can see that thiols tend to becomeoxidized to disulfides when exposed to oxidative pressures. Therefore, a mechanismfor the reduction of disulfides to thiols must exist to rejuvenate the reduced thiol pool.The principal pathways for such processes are the thioredoxin and glutaredoxin enzymesystems (figure 2.2.2).Thioredoxins (Trx) are a family of thiol-disulfide oxidoreductases found withinboth prokaryotes and eukaryotes89.The active form found in the cytosol and nuclei ofcells is thioredoxin-1 (Trx 1)°, while mitochondna has the thioredoxin-2 (Trx 2) isoformwhich contains an N-terminal mitochondrial translocation sequence91.A third form ofthioredoxin is found in spermatozoa, called sperm-specific Trx (Sptrx)92.Allisoformscontain a conserved active site sequence: Cys-Gly-Pro-Cys93.They interactwith avariety of disulfide containing species, most notably ribonucleotide reductase(RNR)involved in DNA repair and synthesis31,transcription factors, PDIin the endoplasmicreticulum, and peroxyredoxins53’.The Trx mechanism of action involves nucleophilic attackof the disulfide speciesto be reduced by the Trx N-terminal cysteine, forminga mixed disulfide intermediate.This in turn is reduced by the C-terminal cysteineof Trx95. The end products are areduced dithiol species and an oxidized Trx nowcontaining a disulfide in its active site95.Trx is ultimately reduced by thioredoxin reductase(TrxR) coupled with oxidation ofNADPH to NADP and formation ofa Sec-SCys bond in TrxR96.TrxR is a homodimerselenocysteine (Sec) enzyme97.TheSec site is located near the C-terminal of theenzyme (Gly-Cys-Sec-Gly-COOH)98.The motifis conserved across species andisoforms of TrxR99. Enzymatic activity ofoxidized TrxR is restored by donation ofelectrons from NADPH via a boundFAD to a thiol/disulfide site (Cys-Val-Asn-Val-GlyCys) present in one subunit of TrxR reducingit to a dithiol. This site is similarinstructure and reactivity to that of glutathionereductase (GR) and is in the proximity ofthe Sec residue on the second subunitof TrxR. This allows the thiolldisulfide site todonate electrons to the C-terminalselenocysteine-cysteine bond(Sec-SCys), reducingit and restoring TrxR reductioncapabilities100’10116RSOHROSRSO2HROSRSO3HRSHRSSR/RSNORNW\RSHROSGRx/TRxNADP—NADPHFigure 2.2.2 Reduction pathways of oxidizedsulfur species, in vivo.17Glutaredoxins (Grx) are another essential family of thiol/disulfideoxidoreductases, which only differ slightly from thioredoxins in their mechanism ofaction. The major isoform, glutaredoxin 1 (Grx I), is found in the cytosol and has anactive site sequence Cys-Pro-Tyr-Cys’°2 Mitochondria have two glutaredoxins,glutaredoxin 2 (Grx 2) with the active site sequence Cys-Ser-Tyr-Cys’°3andglutaredoxin 5 (Grx 5) with only one cysteine in its active site implicated in ironhomeostasis’°4. Grx 1 is associated with dithiolldisulfide exchanges93,celldifferentiation105 and apoptosis’06,dehydroascorbate reduction107,transcription factorregulation108,and as an electron source for ribonucleotide reductase3”‘. Grx can alsocatalyze the formation and reduction of mixed disulfides between proteins and GSH109,which has implications in protein regulation and cellular responses to oxidative stress.Similar to thioredoxins, Grx reduces disulfide bonds by oxidizing its own activesite cysteines to a cystine. However, the Grx active site is reducedby GSH to giveGSSG. First one GSH forms a mixed disulfide with Grx, followed by formation of GSSGwhen a second GSH interacts with the complex”°. The resulting GSSG can bereduced to GSH at the expense of NADPH by glutathionereductase (GR)1’1’3.Similarly, GSSG resulting from the Gpx catalyzedreduction of H20 is reduced to GSHby glutathione reductase. CR is a homodimer’’4with binding sites for NADPH andGSH, which are opposite to each other but onthe same subunit”5.The electrons aretransferred from the NADPH binding siteto the active site of the enzyme via anFADprosthetic group next to the redox active cystineresidues1’3115, 116The active site hasthe Cys-VaI-Asn-Val-Gly-Cys sequence, whichis conserved in humans andyeast, andis similar to lipoamide dehydrogenaseand TrxR”3’117Upon electron donation fromNADPH the active site becomesreduced and catalytically active.2.3 EXPERIMENTAL RATIONALEEvidently, thiol oxidation is central to severalbiological processes, rangingfromredox homeostasis to DNA transcriptionand modulation of enzyme activity.The mostencountered oxidation states are the reduced—SH thiol of cysteines essentialfortranscription factor binding to DNA,and its counterpart the disulfide bondwithimplications in modulation of protein functionas outlined by its effect on translocationoftranscription factors to the nucleus.Enzymatic activity can alsobe affected by thiol18oxidation, as is the case of tyrosine phosphatase, where formation of sulfenic acidinhibits its function. Therefore characterization of the oxidation states of sulfur centersis important in the exploration of enzyme mechanisms as well as metabolic pathways.Unfortunately, there are no conventional methods that can analyze sulfuroxidation. The most common way of detecting thiols is the use of Ellman’s reagent(DTNB, 5,5’-dithiobis-2-nitrobenzoic acid)118. DTNB is a disulfide that exchanges withfree thiols in solution to give a yellow chromophore118 Other chemical derivatizationmethods for detecting thiols involve tagging thiols with maleimides, iodoacetaimides,iodoacetates or thiosulfates, all of which have to be linked either to fluorophores,radionucleotides, affinity labels such as biotin or labels that will change the overallmolecular weight119.This is generally followed by gel electrophoresisand comparisonbetween runs with tagged thiols and runs with untagged thiols to look for band profilechanges. Similarly disulfide bonds are investigated via electrophoresisunder oxidizingand reducing conditions followed by comparison of bandshifts9.Analysis of otheroxidation states is often further complicated bythe short lifetime of the species.Sulfenic acids, are generally not very stable but havebeen detected in vitro by selectivereduction with sodium arsenite or selectively reactingthem with dimedone or NDB-Cl (7-Chloro-4-nitrobenzo-2-oxa-1 ,3-diazole) followedby biotinylatio&’9’120A similar methodinvolves detection of S-nitrosylated thiols by selectivereduction with sodium ascorbatefollowed by biotinylation121.Both methods havethe drawback of requiring denaturingconditions and alkylation of free thiolsprior to treatment with the selectivereducingagents119Therefore, sulfur K-edge XAS is proposedas an alternative and direct methodtolook at thiol speciation, which may proveto be a powerful tool in elucidatingthe catalyticmechanisms of enzyme systems.It has the advantage of being sensitiveto all sulfuroxidations states. Disulfide bondsand free thiols along with methionine, sulfenic,sulfinic and sulfonic acids are readilydetected by XAS3’122Furthermore, spectral fittingof a mixture of thiols with varying oxidationstates allows for quantitative analysisof thesample2’.Most recently XAS was provento be a useful technique for the detection ofS-nitrosylated proteins14.This suggestsXAS could be applied to detectintermediates inthe catalytic cycle of enzymes whichare difficult to see byother means. A primeexample is ribonucleotide reductase(RNR). There are three classesof this enzymeand in each case the reductionof the ribose moiety ofthe nucleotide substrate is19believed to have a thiyl radical intermediate123124Thiyl radicals are notoriously difficultto characterize due to their large spin orbit coupling (=382cm1)125 and broad g11component126, 127Sulfur K-edge XAS, on the other hand, should be very sensitive tothe dipole allowedS3—S1transition from the thiyl radical.XAS can also prove to be a useful source of information on particular bondingmanifolds around the sulfur atom. It was previously proposed based on theoreticalcalculations coupled with X-ray structural data that the sulfonyl moiety (SO2)in RaSO2Rbtype compounds experiences hyperconjugation interactions from the Ra nonbondingorbitals into the S-Rb*antibonding orbital128.A similar effect was also inferred in thecase of aryl sulfonyls, which experience a bathochromic shift in the UV-VIS spectra oftheir benzyl group when the sulfonyl moiety is present129.Because sulfur K-edge XASconsists of transitions to unoccupied or partially unoccupied molecular orbitals it couldbe a direct probe for the detection of hyperconjugative effects which result frommolecular orbital mixing.Thus, this thesis involved development of in situ experimental methods toinvestigate the reactivity, intermediates and products of biologically relevant lowmolecular weight thiols. Furthermore, the electronic configuration around sulfonylgroups was probed to investigate the effects of hyperconjugation on the reactivity ofthese compounds. The findings reported here, and the experimental methods devised,provide steps toward expanding XAS identification of thiol species inmore complexsystems, such as enzyme catalysis and reaction pathways.203 Experimental SectionTo study the reactivity of sulfur model compounds samples were photo-irradiatedwith a Xenon arc lamp and X-ray absorption data was collected. The experimentalsetup for in situ photo-reactivity of sulfur containing model compounds is described inthe subsection below. Time-dependent density functional theory was employed tobetter understand and assign the transitions giving rise to the various features of theXAS spectra. Electron paramagnetic resonance spectroscopy (EPR) was used to helpidentify the sulfur based radical intermediates formed during photolysis while nuclearmagnetic resonance (NMR) was used to characterize the starting materials and finalproducts. The materials used in the following experiments are described in section 3.5.3.1 X-RAY ABSORPTION SPECTROSCOPY (XAS)Experimental SetupThe XAS data was collected at beam line 6-2 of the Stanford SynchrotronRadiation Lightsource (SSRL). The facility is a 3GeV ring with a current of 60-1 OOmA.Beam line 6-2 operates in a high magnetic field mode of 10kG and consists of a 54-polewiggler followed by a Ni coated harmonic rejection mirror, and a fully tuned Si(1 II)double crystal monochromator. The beam-line optics are under vacuum and protectedfrom the pressurized experimental hutch by a127i.tmberyllium window followed by a6.3511mpolypropylene window and a He chamber13.The spot size of the incident beamis controlled by a pair of horizontal and vertical JJ X-ray exit slits followed by a He gasionization chamber, which measures the intensity of the incident beam (lo). The samplechamber is isolated from the ionization chamber by another polypropylene window. Thesample itself is 45° to the incident beam and 45° to a fluorescence ion chamber SternHead-Lytle detector130,which was filled with argon gas and maintained at ambienttemperatures. He flow from a liquid helium cooler provided by Cryo Industries (HFC1645 LHE-Cryocool) maintains the sample below -20°C where it can be photo-reactedwith a Ushio 75W Xenon arc lamp positioned 50cm from the sample. At thesetemperatures the samples did not react in the X-ray beam. The sample chamberencasing consists of a specially adapted transparent glove-bag providing an anaerobicHe atmosphere for data collection (figure 3.1.1). By varying the point to point21increments, number of data points and collection time at each point, the scan rate wasvaried in order to better follow the rate of photo-reactivity131.The spectrometerresolution was -0.5eV’32.Solid samples were mounted as a finely ground powder dusted on the adhesiveside of sulfur-free Kapton tape (polyimide film with silicone adhesive) to minimizefluorescence self absorption effects13.Methyl sulfonyl chloride was mounted as a neatsolution on the sulfur-free Kapton tape and covered with a polypropylene window undera nitrogen atmosphere. Spectra were acquired while the samples were irradiated with a75W Xenon arc lamp at 253K (-20°C) under a helium atmosphere with <1% oxygencontent. Glutathione mounted on the Kapton tape was irradiated with UV light atambient temperature under aerobic conditions. The UV irradiation source was aLonglifeTM Filter 254nm shortwave ultraviolet lamp from Spectroline®.XAS spectra processingEnergies of the Sulfur K-edge XAS spectra were calibrated using hydratedsodium thiosulfate(Na2SO3.5H0)with the first pre-edge feature peak maximum beingcalibrated at 2472.02eV13.The value of 2472.02eV for the first pre-edge feature wasarrived at by repeated experiments on different beamlines and checked against theinflection point of sulfur at 2471.3eV and the X-ray absorption spectra of molybdenumand copper foil132. An alternative value for the first pre-edge feature of sodiumthiosulfate is 2469.2eV determined by calibrating the spectra of sodium thiosulfate tothe first intense feature in the spectra of NiS powder assigned as occurringat2469.8eV’33. Spectra were background subtracted using the method of energysummation of two linearly weighted background terms derived from the backgroundbefore and the background after the edge jump of the XAS spectra. Themagnitudeof the edge jump was normalized to a total intensity of 1. Principal component analysisof transient spectra was carried out using SixPack version0.53135.22—.IThermocoupleCl)SampleIHeFilledGloveBagTablePWPW=polypropylenewindowIonizationChamberLPW——IDetectorSlitsBeWindowXeArcLampF’.) c)3.2 DENSITY FUNCTIONAL CALCULATIONSMolecular orbital calculations to simulate spectrometric parameters and deducemolecular bonding interactions were carried out for the various species investigated.Density functional theory calculations were carried out using the Kohn-Sham Self-consistent field methodology provided with the Amsterdam Density Functional SoftwarePackage2007.01136137Full geometry optimizations were followed by single-point and time-dependentdensity functional theory (TD-DFT) calculations. Calculations were performed using theBP86 functional and a doubly polarized triple-c basis sets (TZ2P) using Slater-typeorbitals (STO) basis functions. TZ2P was necessary to allow for the interactionbetween the sulfur valence (3s, 3p and 3d) orbitals resulting in correct geometries forthe hypervalent sulfur atom. Previous researchers have shown this combination offunctional and basis sets to give the best approximation for calculated spectra andrelative experimental energies and intensities in ligand K-edge XAS1. To calculate thecorresponding sulfur core Is excitations for each of the compounds, theModifyExcitations key was used, along with the no core option for any of the atoms andno symmetry operations were applied. The resulting excitation energies for the sulfurIs electrons were shifted by 76-81eV to account for deviations in the calculated coreexcited-state energies from experiment. These energy shifts fall within range of thosecalculated by other researchers1.In the case of the aryl and alkyl sulfonyl compoundsfragment calculations were necessary to better understand contributions to molecularorbitals from the different “fragments” such as the aryl and sulfonyl moieties. Fragmentcalculations consist of modeling the orbital interactions for each fragment individuallyand then combining them to give the final picture.To assist in the spectroscopic assignment of XAS features and to account forrelaxation effects due to electron excitation,’3814°the transition energies wererecalculated using the Slater transition state self-consistent field method (ASCF) for thefirst 10 transitions of each model compound of interest. XAS spectra were thensimulated using the recalculated energies and the oscillation strengths calculated withTD-DFT. iXSCF was done by having 1.5 electrons in the S1 orbital and one-half of an24electron in the orbital of interest which gives a good estimate for the relaxation energy inmost core ionization/excitation cases’41.The magnitude of the calculated oscillatory strengths were used for the peakareas needed to simulate the features of the various XAS spectra using a pseudo-voigtfunction with 80% lorentziari and 20% gaussian character. The intensities of theoscillatory strength were normalized to fit the normalized experimental spectra. Detailsof further molecular calculations and deviations from the above procedures arementioned in the text wherever applicable. Sample input files are found in Appendix 1.3.3 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR)NMR data was collected on a Bruker AV 300 NMR spectrometer. ‘H NMR datawas collected with a I 5ppm sweep width centered at 6ppm, each spectrum consistingof 128 scans with a time domain of 32K. Samples were dissolved in deuterium oxide(020) solvent with a 99.9% deuterium atom composition, having aproton peakmaximum calibrated at 4.8oppm.3.4 ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY (EPR)X-band EPR data collection at 9.5GHz was carried out using a Bruker Elexsys E500 series continuous wave EPR spectrometer running the Xepr software package.Photo-reactivity studies were carried out using a 75W Xenon arc lamp positioned 50cmfrom the sample. In situ irradiation experiments were carried out while collectingspectra at room temperature or, using a finger dewar filled with liquid nitrogen, attemperatures of 77K. Studies at 195K (-78 °C) were carried out by irradiating thesample while in an acetone-dry ice bath followed by data acquisition at 77K. In situexperiments were not viable in acetone-dry ice bath because of “lossyness” due tomicrowave absorption by acetone. Samples were run either under vacuum or under anargon gas atmosphere. DPPH (g=2.0036) was used as the standard for spectracalibration’42.Power saturation profiles were acquired by varying microwave powersbetween 63mW and 0.002mW. Further details and deviations from the aboveprocedures are mentioned in the text when needed. Simulation of spectra was carriedout using the Bruker WinEPR SimFonia software package.253.5 MATERIALSSulfur-free Kapton tape was purchased from Creative Global Services Inc. andchecked for sulfur contamination (by sulfur K-edge XAS) before use. The irradiationsources consisted of a LonglifeTM Filter 254nm shortwave ultraviolet lamp fromSpectroline® for UV irradiation and a Ushio 75W Xenon arc lamp for full spectrumirradiation. 707-SQ-250 EPR tubes and a large finger dewar flaskl5Oml (Suprasil WG853-B-Q) from Wilmad Labglass were used for EPR experiments. Polycrystallinepowders of reduced L-glutathione, (±)-x-lipoic acid, p-toluene sulfonyl chloride,ptoluene sutfonic acid, phenyl ethyl sulfone, methionine sulfone,and sodium methanesulfonate along with a neat solution of methane sulfonyl chloridewere purchased fromSigma-Aldrich and stored at 4°C until use. S-nitrosoglutathionewas synthesized byreacting glutathione with sodium nitrite under acidic conditionsand characterized usingNMR (chapter 5 figure 5.2.7 p.52) andby its absorption at 545nm using UV-V1S143.Glutathione, (±)-a-Iipoic acid and p-toluene sulfonylchloride were also characterizedusing NMR and no contaminants were found.S-nitroso glutathione (GSNO) synthesisTo a mixture of Sml of deionized water mixed with5m1 IM HCI, 5mmol (1.5g) ofglutathione (GSH) were added and dissolved.The solution was kept in an icebathwrapped in aluminum foil to preventproduct photo-degradation. To thecolorless GSHsolution, 5mmol (O.35g) of sodiumnitrite was added. The reactionimmediately turnedred and was stirred forI hour. Precipitation of the GSNOproduct was initiated withI OmI of -20°C acetone followedby a further 30 minute of stirring.The product was thenfiltered by suction filtration and washedsuccessively with 5 x O.5m1 of ice-cold water,5 xI ml acetone and 6m1 of diethylether added drop-wise. Thepale pink product was driedand stored in the dark at -20°Cuntil use.264 Sulfur K-edge XAS as a Probe of Sulfur-Centered Radical Intermediates4.1 BACKGROUNDSulfur based radical intermediates are involved in various biologicalprocesses1”,most notably the enzymatic reduction of ribose sugars in ribonucleotidereductase (RNR)’45’146Thiyl radicals are postulated to be essential in the catalyticmechanism of all classes of ribonucleotide reductases124 however their detection andcharacterization have been elusive. Paramagnetic species are generally detected usingelectron paramagnetic resonance spectroscopy (EPR), however this is somewhatcomplicated for sulfur because of its relatively large spin orbit coupling (=382cm)125and broad g11 component of thiyl (RS) radicals126’127Recently Lassman and coworkershave generated thiyl radicals using UV irradiation in polycrystalline glasses of bothprotein and cysteine samples. Integrated EPR spectra of UV irradiated bovine serumalbumin (BSA), and the RI subunit of RNR were compared to that of UV irradiated300mM cysteine samples146’147The UV irradiated cysteine samples exhibit a weaksignal at a g, of 2.30 attributed to the thiyl radical. When integrated this gives “a broadascending slope” in the absorption spectrum leading to the main absorption features atlower g values146. Similar “slopes” were also detected in the protein samples andattributed to the thiyl radical even though the g, signal was absent in the1stderivativespectra. Such analysis requires careful background subtraction and the researcherschose the 330mT point in the field domain as the cutoff for the thiyl “absorption feature”so that other features present would not superimpose with the thiyl radical gi signal.Although this approach has proven useful in the detection of free thiyl radicals, propercharacterization of the thiyl radical using this method can be hindered by many factorssuch as incorrect baseline subtraction, imperfections in the EPR tubes, as well as thepresence of other features which may overlap with the absorption signal of theintegrated EPR spectrum of the thiyl radical. Other EPR approaches to the detection ofthiyl radicals involve spin trapping agents. Unfortunately, spin trapping techniquescannot provide information about the g-tensor of the radical, hyperfine couplings, orradical lifetimes147’148Therefore XAS is proposed as a new tool for detecting thiyl radical intermediateswhich has the added advantage of also detecting EPR silent sulfur intermediates such27as sulfenic, sulfinic, and sulfonic acids that generally rely on secondary methods ofdetection such as chemical methods involving thiol derivatization119.These species canoften form as byproducts of the radical generating reactions and their identification andcharacterization is important in the overall reaction mechanism. These studies usedglutathione as a model compound which has a redox couple (2GSH4->GSSG+2e) thatis essential for proper redox balance and homeostasis. It has been shown thatphotochemical one-electron oxidation of GSH forms a multitude of radical productsincluding the short-lived thiyl (GS) and long-lived perthiyl (GSS) radicals, yet the one-electron redox chemistry of GSH and cysteine is still somewhat unclear. Therefore,polycrystalline glutathione is used as a model system for evaluating the use of sulfur K-edge XAS in the study of sulfur-containing radicals.4.2 RESULTS AND DISCUSSIONCharacterization of Irradiated Samples with EPRExperimental conditions were as described in the experimental section.Polycrystalline GSH was irradiated in the UV region (2=254nm) at room temperature(295±5K) giving an EPR spectrum that has been previously investigated and attributedto a complex mixture of radical species (i.e., GS, G, H, and GSS)146.XAS data wascollected for samples irradiated from 2 to 48 hours. EPR of the UV-irradiated samplesremained unchanged even after prolonged exposure to the X-ray beam(>3hours) andwere not affected by the Kapton tape.From the spectra in figure 4.2.1 one can see that the features of the “GSH”spectra and those of “GSH” are comparable. “GSH” was a sample prepared andused for the XAS experiment. It consisted of GSH on Kapton tape irradiated for 48hours and its EPR spectrum was acquired at room temperature. “GSH” is glutathionepolycrystalline powder irradiated for 4 hours and placed in an EPR tube with its EPRspectrum acquired at room temperature. Both samples exhibit features atg values of2.030 and 2.057 that are due to the perthiyl radical, while the feature at 2.009 is in thethiyl radical g± region (g 2.01) as previously reported by Lassmanet a!. and Neese eta!127, 146In addition, the features of the simulated perthiyl EPR spectrum (figure 4.2.1)correspond well to those of irradiated glutathione. Theperthiyl radical EPR spectrumwas simulated with g values at 2.057, 2.03 and 2.07 which match well those derived28from the density functional calculations of the CH3SS model for the perthiyl radical usedto analyze the XAS spectra. The calculated g values for the CH3SS perthiyl radical are2.058, 2.027 and 2.002. The difference in the lower g values at 2.002 and 2.07between the actual and calculated spectra may arise because of the presence ofadditional EPR signals such as those from a possible thiyl radical.I I: : GSHI I III I2.10 2.05 2.oO 1.95Figure 4.2.1: X-band EPR spectra of {JV irradiated glutathione. “GSH” is GSHon Kaptonsulfur free tape irradiated for 48 hours and used for XAS with EPR collectedat roomtemperature (power 2mW, 3scans, modulation amplitude 5G, modulationfrequency 100kHz);and “GSH” is GSH powder irradiated overnight and placed in an EPR tube and runat roomtemperature (power 2mW, 3 scans, modulation amplitude 5G, modulationfrequency 100kHz).The simulated spectrum of perthiyl radical hasg values of 2.062, 2.030 and 2.007._____________09____________________SPECTRUMF PERTHIYL RADICALiGSHg VALUE29The presence of the perthiyl radical was further confirmed with power saturationstudies (figure 4.2.2). The feature at g=2.057 was chosen for these studies because itdoes not overlap with other spectral features. It is found that the signal at 2.057increases linearly with higher microwave powers in the range of 0.002mW to 20mW,which is consistent with the low saturation effects seen in perthiyl radicals146’149The g11of the thiyl radical was not observed in the EPR spectra of either theGSHsor the“GSH” samples but was identified in the XAS spectra.Characterization of Irradiated Samples with )(ASThe XAS data of UV irradiated GSH, shows distinct changes in the GSHspectrum upon irradiation (figure 4.2.3 and 4.2.4). The GSH spectrum is initiallydominated by an intenseCS0—S1at -2473.5eV3.With UV-irradiation, new pre-edgefeatures appear at 2468.8eV and 2470.5eV and the CS—S18feature of GSH broadensand decreases in intensity. The reactivity of GSH can also befollowed by the1.0C’)CD1.0Cl)zLUI—z-JzCDCl)0.5LUN-J0z0.02SQUARE ROOT OF MICROWAVE POWER (mW1)Figure 4.2.2: Power saturation study of the EPR signals from “GSH’” and “GSH” samples.0)ICl)CD>-F—C,)zLUI—z-JzCDCl)LUN-J0z0.50 10.030disappearance of the features at 2476.7eV and 2479.3eV. Second derivative analysisof the spectra shows the presence of an additional feature at 2471.5eV as indicated byan inflection point in this region (figure 4.2.4 B). These pre-edge features coincide withthe appearance of radical species in the EPR spectra and therefore should correspondto transitions to the half-occupied sulfur orbitals of sulfur-centered free radicals. Atlonger irradiation times, a high energy feature at 2482.6eV appears indicative of a highlyoxidized sulfur species. This species has not been characterized, however formation ofstable sulfoxyl radical intermediates along with sulfinic and sulfonic species have beenreported as a result of photo-irradiated sulfur containing amino acids1. Principalcomponent analysis (table 4.2.1) on the transient spectra shows that 3 components areneeded to obtain >0.99 cumulative variance for the low energy pre-edge region of thespectra (2465 - 2475eV) whereas 4 components are needed when the higher energycomponents are included (2465 - 2490eV). This indicates the formation of 3 speciesasa result of irradiation.Table 4.2.1. Principal component analysis of UV irradiated GSH XAS spectra from the variousirradiation time points. Dotted lines show the number ofcomponents needed for>99%cumulative variance over the energy ranges described.COMPONENT ANALYSIS FOR2465eV — 2475eV RANGEComponent Eigenvalue CumulativeVariance1 25.62 0.9072 1.75 0.9693 0.63 0.9914 0.12 0.9955 0.04 0.9976 0.03 0.9987 0.02 0.999COMPONENT ANALYSISFOR2465eV — 2490eV RANGE1 46.42 0.9222 2.24 0.9663 0.89 0.9844 0.33 0.9905 0.25 0.9956 0.14 0.9987 0.05 0.999312.5FIGURE 4.2.3 Sulfur K-edge XAS ofUV irradiated GSH after various time intervals ofirradiation.2.00—-—-—28----2448SchrhrhrhrhrII1’t2475ENERGY (eV)2480 2485320.2Figure 4.2.4 Difference XAS spectra of UVirradiated GSH at various times and GSH control(A) and the second derivative of the difference spectrashowing an inflection point at 2471.3eV(B).2469 2470 2471ENERGY (eV)0.10.024681.00.50.0-0.524722468 2469 2470 2471ENERGY (eV)247233• • • • •3.0Lii —Ui.-a- -.2.0LiiC)1.5 - -.-- (2470.5) GSS• (24688)GS1.0005________________0 •••_.•__0.0.•I • I • I • I0 10 20 30 40 50TIME HOURSFigure 4.2.5 Time evolution of the features 2468.8eV and at 2470.5eV in terms of calculatedpercentage of thiyl and perthiyl radical after various intervals of irradiation.Based on differences in their rates of formation (figure 4.2.5) the two low-energypre-edge features must correspond to different sulfur radical species, most likely GS(thiyl) and GSS (perthiyl), respectively. To better understand the electronic structureand characteristics of these transitions a combination of unrestricted ground state andtime-dependent density functional theory (TD-DFT) calculations were carried out onsimplified models of the expected thiyl (CH3S) and perthiyl (CH3SS) radical species.Electron density maps were generated and the important empty valance orbitals areshown in figure 4.2.6 with relevant transitions listed in table 4.2.2. Spectra at the sulfurK-edge are dominated by transitions with electric dipole allowedS*—S1character;therefore empty orbitals with S3,character are of most importance. DFT results showthat the unpaired electron in GS resides in a singly-occupied S orbital perpendicularto the S-C bond axis127.Two other orbitals, labeled CS. andHCa*, have significant S3pcharacter and account for the two major bound-state transitionsat 2473.7eV and2474.5eV in the Sulfur K-edge spectrum of GS. The HCa* final state obtains much of itssulfur K-edge intensity through intensity borrowing fromthe CS0 state at -1eV higher34energy. The lowest energy transition at 24688eV corresponds to a nearly pure (-92%)S3,,+—S1transition attributed to the singly occupied sulfur 3p orbital.The unpaired electron in GSS is located in a SS orbital (figure 4.2.6). Twotransitions should be observed to this orbital, one from each of the S1 orbitals. TD-DFTcalculations indicate a significant splitting of the two SS transitions of 1.3eV suggestinga more positive effective charge forSA,which lowers its Is core orbital energy. Thea*transitions for GSS include both CS and SSa* contributions that occur at about thesame energy as in the GSH spectrum. It is important to note that the low-energy preedge feature for GSS should occur at higher energy than that of GS. This is becausethe acceptor orbital in the perthiyl species is a higher energy antibonding orbital. Inaddition, a splitting of the pre-edge feature in GSS is expected from the DFT results.cHscssCSc,850*”Figure 4.2.6 DFT calculated electron density contour maps of important valenceorbitals forthiyl and perthiyl radicals (Isovalue = O.075e.A3).CSc,*s3psst35Table 4.2.2. TD-DFT calculated sulfur K-edge pre-edge features of relevant model compounds.The resulting excitation energies for the sulfur is electrons were shifted by +76.5eV in all cases,using the main a’ feature of GSH as a calibration point.Species Assignment Energy (eV)fCS0*+HScr*4_Sis 2472.8 2.Ox1OCH3SH CS,,*—S1 2473.7 i.ixiO3CSc*+HSa*(Sis 2474.4 1.3x103S—S12468.8 3.9x10CH3S HCa4Ss 2473.7 8.1x10CSa*Sjs2474.5 9.3x10SS,_SBi2469.3 2.5x104SS*,_SA12470.6 1.8x1O• SS&±SBs2472.7 3.1x10CH3SS2473.4 5.3x10SS0*4SAi2474.1 2.6x103CS*+SAi2474.8 1.6x103The DFT data is in good agreement with the experimentalsulfur K-edge spectra.The lowest energy pre-edge feature in the UV irradiatedGSH spectra was assigned to aS3,÷-S1transition in GS while the feature at2470.5eV was attributed to the SStransitions of GSS. The GSS pre-edge featurewas fitted using PeakFit v4. 12 (figure4.2.7) and the splitting caused by differences inZeff of the two sulfur atoms, is similar tothat predicted by DFT (—1.1eV vs. 1.3ev).The kinetic behavior of the sulfur K-edgepre-edge features are also consistentwith previously published EPR data, showinginitial formation of the thiyl radical followedby subsequent formation of the longer-livedperthiyl species146.Radical yieldswere estimated using the ratiosof OFT predictedoscillator strengths (t) for pre-edgefeatures of the methyl thiyl (CH3S)and methylperthiyl (CH3SS) radicalswith that of methane thiol (CH3SH).This allowed theextrapolation of the value for theareas of the radical speciescorresponding to a 100%thiyl or perthiyl XAS spectrum. Thecalculated areas correspondingto 100% of aspecies were then comparedto the actual areas achieved at thedifferent irradiationtimes to calculate the percent radicalyields. Maximum yields forthe thiyl and perthiylradical were achieved afterirradiating the sample for 4hours (—0.45%) and 48 hours(—3.1%), respectively (figure4.2.5).36CH3SS(SS1*SAis)Figure 4.2.7 Peak fitting of the difference spectra for the pre-edge region of the GSH sampleirradiated for 48 hours and control (0 hours). The peak ratios for the two perthiyl radical preedge features were kept within 20% of the DFT calculated result. Pseudo-voigt fI.inctions with50% lorentzian and 50% gaussian character were used for each of the fitted peaks.4.3 CONCLUSIONThis study shows the usefulness of XAS as a probe to detect sulfur based radicalintermediates and was the first study to detect and characterize isolated free sulfurradicals using XAS. Within the same model system it is possible to differentiatebetween two sulfur radical intermediates GS and GSS’, which had pre-edge featureswell resolved from each other and the intensea*contributions. An EPR silentbyproduct of the photo-chemical reaction described by the peak at 2482.6eV was alsoidentified. This feature is indicative of a highly oxidized sulfurspecies possibly asulfoxyl radical intermediate or a sulfonic acid. In the followingsections this techniqueis applied to increasingly more complicated systems and reactivity profiles, which provethe usefulness of this technique when used in conjunction with moremain streamspectroscopies such as EPR and NMR.• 48-0 hrSum of fitsThiyl GSPerthiylGSSB----PerthiylGS’SCH3S(S3—S1)/CH3SS(SS÷- SBi)//,2468 2469 2470 2471 2472ENERGY (eV)375 Perthiyl Radical and Disulfide Bond Formation in Photo-irradiatedNitrosoglutathione and Lipoic Acid5.1 BACKGROUNDThe initial study involving glutathione (GSH, chapter 4) showed that XAS can bevery useful in the detection of sulfur based radicals, which have low energy pre-edgefeatures well separated from the typical spectroscopic features of non-radical species.Nitrosoglutathione (GSNO), the S-nitrosylated version of glutathione, and lipoic acid(LA), the oxidized disulfide of dihydrolipoic acid (DHLA), are low molecular weight thiolswhich are also involved in antioxidant defense as previously described. In fact, DHLA isinvolved in the denitrosation of GSNO to yield LA and the free thiol GSH77.Thereactionis believed to proceed via transfer of the N0 moiety to DHLA followedby the formationof an internal disulfide and release of N0. Sulfur K-edge studieswith in situ photoirradiation show formation of new disulfide bondsin GSNO and formation of anadditional pre-edge feature attributed to the perthiyl radical. The transition assignedtothe perthiyl radical is also present in LA. EPR andNMR techniques were applied tobetter understand the reactivity of these species and complementthe XAS experiment.Even though these systems have been previously investigatedthere is still some debateto their mechanism of action. Furthermore, their reactivityseems to be very dependenton the reaction conditions1501.If XAS is to beapplied to more complicated reactionprofiles such as those of enzymes, the reactivityof simpler systems and the conditionsthat govern them must first be investigated.S-nitmso cylutathione (GSNO)The main function of GSNO is as an N0 carrierand delivery system, N0 beingan essential signaling molecule. The mechanismof N0 delivery is still underinvestigation and most recent studies suggestthat at neutral pH N0 is released fromnitrosoglutathione by a one electron reductionmechanism involving a possible GSNO -intermediate155.Still, other research emphasizesthe importance of disulfide bondformation during N0 release asa result of denitrosation reactions, such as thoseinvolving DHLA and thioredoxinto give the corresponding lipoic acid andoxidized38thioredoxin products77.Nitrosothiol (RSNO) decomposition accompanied by release ofN0 and formation of disulfides is also catalyzed by the presence of copper (Cu),application of heat and photo-irradiation156.Based on previous experiments with GSH,GSNO decomposition can be observed with sulfur K-edge XAS and in situ photo-irradiation of the sample. Initial formation of the GS intermediate should be followed bydisulfide bond formation (GSSG) and be proceeded by the disappearance of featuresdue to the GSNO starting material (scheme5jj)152GSNO‘> GS + N0GS + GSNO > GSSG + N02GS >GSSGScheme 5.1.1 GSNO photo-reactivity induced reaction pathways.The sulfur K-edge spectrum of GSNO was previously investigated and assignedby Szilagyi and coworkers; our data are consistent with this previous work (figure5.1.1)14.The dominant features are theSN—S1at 2471.7eV and the SNa*Sitransitions at 2473.4eV due to the S-NO bond and theSC---S1feature at 2474.8eV.This assignment is reinforced further by TD-DFT simulation of the core Sboundtransitions of CH32SNO model system which indicates twoSC*—S1transitionsaccount for the feature at 2472.8eV, while a single transition is observed for theSN0*+_Si feature. TheSNG acceptor orbital for the SN**.Si at 2471.7eV shows asignificant interaction between the p orbitals of allatoms making up the S-N-O bondingmanifold (figure 5.1.2). This is consistent with anS-NO bonding model stabilized byresonance as previously described, with the mostsignificant contribution being due totheSNa resonance structure followed by S-Nt and SNO ion pair157. Therefore, in aseries of 5-nitrosothiols the intensity of the SN**Siand SN0*Si transitions could beused to determine the relative importanceof the S-Na resonance structure, a projectwhich is currently ongoing in the Kennepohl group.392.51.0Figure 5.1.1 GSNO sulfur K-edge XAS spectrum (solid line) withthe main S1 core excitationtransitions assigned’4.The edge jump of the spectra was normalizedto 1. TD-DFT simulatedXAS spectrum of GSNO usingCH32SNOas a model system (dotted line).sca*SN0SNFigure 5.1.2 DFT calculated electrondensity contour maps of important empty valenceorbitalsforCH32SNO (Isovalue = O.05e.A3).2.01.5SNscIy*SN.0.52472ENERGY (eV)40Lipoic acid (LA)Lipoic acid is an important cofactor and antioxidant. The LA/DHLA redox coupleis distinguished from that of other low molecular weight thiols by its ability to function asan antioxidant in both hydrophilic and lipophilic environments and even reduce otherantioxidants such as glutathione23’25In addition, LA can be further oxidized to give adisulfide radical cation1. The photochemical reactivity of LA was previouslyinvestigated. Depending on the conditions the presence of thiyl radicals, perthiylradicals, disulfide radical cations, and triplet states were detected, making LA a versatilesystem to observe by XAS. Photolysis of disulfides in solution results in the formation ofperthiyl radicals149.This reaction is more favourable with increased substitution of theresulting carbon radical which further stabilizes it150’151The presence of oxygen duringphotolysis seems to facilitate thiyl radical formation150.Cage effects also impactphotolysis. The hexacyclic disulfide trans-4,5-d ihyd roxy-1 ,2-dith iacyclohexane whenirradiated (wavelengths between 305-4lOnm) near its absorbance maximum at 280nm(the absorbance spans the 250-325nm range) forms a thiyl radical pair which isproposed to undergo H shifts to form more stable products, including the formationofthe starting material153.Laser flash photolysis of LA at 266nm inaqueous solutionresults in the formation of the disulfide radical cation, while flash photolysis at 355nmyields a triplet state with a lifetime of 75ns154. Therefore,irradiation of LA underanaerobic condition with a Xe arc lamp would be expected to give a combination of theperthiyl radical, thiyl radical or disulfide radical cation intermediates (scheme 5.1.2).RSSRShvRS coupled with H-shifts (giving RSH; R=S; RSSR)‘ RSSR’3RSsRScheme 5.1.2 LA photo-reactivity induced products.The transitions of the sulfur K-edge XAS spectrum(figure 5.1.3) of LA were previouslyinvestigated15.The first and most intense pre-edgefeature corresponds to the41SS—S1at2472.3eV followed by the SC—S1at 2473.9eV. TD-DFT simulated XASspectra for the bound transitions of methyl disulfide (CH3SSCH)is consistent with thisassignment (figure 5.1.3). The feature labeled LA1 at 2475.4eV in figure 5.1.3 isattributed to a collapse in symmetry between the sulfurs of LA due to bonding to aprimary carbon in one case and a secondary carbon in the other. This feature is notpresent in symmetric disulfide systems15. The steric strain in the pentacyclo LAdisulfide is emphasized by a lowering in energy of both the SS0 and SC0* whencompared to cystine where these features appear at -2472.6eV and -2474.2eVrespectively15.This suggests that weaker bonds than generally found in disulfidesresult in LA due to steric strain. The XAS of LA also exhibits broader features whichcould be do to self-absorption effects from non-homogenous grinding of the solidsample158.However, the intensities of the features are consistent from run to run andcomparable to those of other researchers15 indicating that self-absorption should beminimal. Furthermore the features of the spectra are well resolved and self-absorptionshould not pose a significant problem for determining the energy of the varioustransitions of the spectra.11.ss*SC..LA12465 2470ENERGY (eV)Figure 5.1.3 LA sulfur K-edge XAS spectrum (solid line) with the main S1 core excitationtransitions assigned’.The edge jump of the spectra was normalized to 1. TD-DFT simulatedXAS spectrum of LA using CH3SSCH as a model system (dotted line).42Previous assignments of the features from the sulfur K-edge XAS of GSNO andLA give an idea of which features are most important to observe in the irradiationstudies. Disappearance of the SN—S1and SNa*-Sis transitions in GSNO wouldindicate breakage of the S-NO bond, while a decrease in intensity of the SS0*÷—S15 in LAwould suggest bond homolysis. Previously determined reaction pathways helped toexplain the reactivity observed. It was found that although LA and GSNO are “simple”,small sulfur containing compounds their reactivity can be quite complex, and under theexperimental conditions, even related.5.2 RESULTS AND DISCUSSIONPhoto-irradiation of LAc)0.60 2.50.55 __—•—-—-——•-—----.-_______.:::y2.00.40—.-—2470.OV:/I.0 2 4 6 810121416JTIME (MIN) I0 I10<IAl 0Il0.5I . I . I . I .0.02460 2462 2464 2466 2468 2470 2472 2474 2476 2478 2480ENERGY(eV)Figure 5.2.1 LA sulfur K-edge XAS spectrum with in situ photo-irradiation. Dotted arrows aredecreasing features, solid arrows are growing features. Subset: Formation of the feature at2470.0eV attributed to the perthiyl radical.43Irradiation of LA with a Xe arc lamp under anaerobic conditions was followed withsulfur K-edge XAS. In this system the starting compound is a strained cyclic disulfideand as displayed in figure 5.2.1 a feature at 2470.0eV appears after only a few minutesof irradiation. For LA a shift is also seen coupled with a drop in intensity of the featureat 2472.3eV corresponding to the breaking of the cyclic disulfide. However, formation ofa feature at 2472.5eV and an increase in intensity at 2473.9eV with a 1.5eV separationbetween the two is suggestive of new disulfide bonds forming, along with perhaps otherminor products. The higher energies lead to the conclusion that the forming disulfideshave stronger bonds resulting from a less strained conformation. The disappearance ofthe peak at 2475.3eV indicates that if new disulfides are being formed they have sulfurswith similar core excitation energies. Unfortunately, the product from the irradiationforms a white insoluble precipitate which does not lend itself to easy characterization byother spectroscopic techniques such as NMR, and confirmation and characterization ofthe final product formed from the irradiation is still unclear.To confirm the presence of the perthiyl radical, EPR spectra of anaerobicallyirradiated LA were collected and analyzed. The formation of the perthiyl radical wasclearly visible (figure 5.2.2). A rhombic signal was observed with irradiation at 195K and77K with g-values of 2.002, 2.026 and 2.062 matching well the simulated EPR spectrumof the perthiyl radical and the density functional theory calculated values for the CH3SSperthiyl radical (2.058, 2.027 and2.002)146.Table 5.2.1 shows half power saturationvalues (P112) which represent the value at which the signal intensity divided by thesquare root of power drops by 50%. Analysis shows that the features between 2.026and 2.002, as well as those below 2.000, saturate at low powers suggesting they aredue to carbon centered radicals49.Perthiyl radicals however like most sulfur centeredradicals saturate at higher powers and their intensity increases with microwave powerover the ranges investigated, overwhelming the spectra from other species (figure5.2.3)146149•The feature due to the perthiyl radical at g=2.062 is well resolved fromother peaks and has a P112 of 20mW, consistent with previously reported values146.Thefeatures due to the perthiyl at 2.026 and 2.002 overlap features with low saturationpowers (2.026 to 2.002 and below 2.002), which might explain why they have lower P1,2values (6mw vs. 20mW). P112 values of carbon radical species were found to lie in the0.6mW range which is consistent with previously reported values (‘-1 mW)146.LA showsa similar reactivity profile when dissolved in D20 as in the solid state.44Table 5.2.1 Half power saturation values for EPR spectra of solid LA irradiated at 77K (as infigure 5.2.3).g VALUE RANGE P112 mW SATURATION2.062 202.026 62.026—2.002 0.62.002 62.00-1.95 0.612.062 2.026 12.002SIMULATED SPECTRUM OFPERTHIYL RAICALI I II:II IIIIRRADIATED 6OMIN AT 195KI I Ii I I I I I I ISOLID LIPOIC ACID2.10 2.08 2.06 2.04 2.02g VALUE2.00LIPOIC ACID IN D20IRRADIATED 6OMIN AT 77K1.98 1.96 1.94Figure 5.2.2 EPR of irradiated (60mm) LA under different conditions (microwave power0.6mW, modulation frequency 50MHz, modulation amplitude 3.OG, 5 scans) and simulation ofthe spectrum for the perthiyl radical with g values of 2.062, 2.026 and 2.002.452.062 2.026 2.0023 3050 3150 3150 3250 3250 3133 3550 3550 3650 3650 3700• r - -3000 3050 3100 3150 3200 3250 I 3300 I 335J 3450 3450 3500 3550 3600 3650 3700__________________________0.0635mWI I I III I ‘I IITI’’ I I I I I I I I I I I I0 3050 3100 3150 3203 3250I3300I335 3400 3450 3500 3550 3600 3650 37000.201rii/(2 3050 3100 3150 3200 3250 3 335( 3450 3450 3500 3550 3600 3650 37003 30150 3150 3450 3203 3250 3300 335I 3400 3450 8550 3555 3600 3650 3707I I I I I’20C)rT,V’I3 0 3100 3150 3 3200 I 3300 I 3450 3550 3550 3603 3650) 3703—--—-16.33mW30)) 36503100 31I’20.0mW3050 3150) 3150 3450 3450 3501) 35503600 0 3750)1ZZr\..JEJE3000 3050 3100 3150 3200 3250 3300 3350 3400 3450 3500 35503600 3650 3700GaussFigure 5.2.3 EPR power saturation study of irradiated(60 mm) LA solid at 77K. Microwavepowers were increased between 0.002mW and 63.3mW. Perthiyl radicalat g values of 2.062,2.026 and 2.002 is found to increase with increasing microwave powers; (modulationfrequency50MHz, modulation amplitude 3G, lOscans).46Photo-irradiation of GSNOOver the duration of 1 hour, irradiation caused the features in GSNO due to theS-NO bond to disappear as highlighted by the dotted arrows in figure 5.2.4. Ofparticular importance is the peak at 2471.6eV, which is well resolved from otherfeatures and allows one to easily follow the breaking of the S-N bond. Under theconditions of the experiment some minor products indicative of oxidized sulfur speciesform in the 2476.5 — 2477.5eV region where transitions from R2SO and RSOj mayoccur and at 2480.8eV indicative of the formation of RS032The transition at2480.8eV due to RS03 is also present in the initial spectrum, showing that it is formedas a minor product during GSNO synthesis. Appearance of features at 2472.8eV and2474.0eV during irradiation, suggest the formation of disulfide bonds and correspond toSS—S1 andSC*+-S1excitations2’15They are split by 1.2eV and the higherintensity of the first feature over the second further suggests the presence of disulfidebond formation. However, theSS—S1and SC—S1transitions in disulfide bondsare generally separated by 1.5eV. The poor resolution of the separation between theSS—S1and SC—S1transitions indicates the presence of other species whosefeatures are overlapping those of the forming disulfides and could be due to minorproducts. A small shoulder forming at 2470.2eV is consistent with the formation of aperthiyl radical, and forms congruently with S-N bond cleavage reaching a steady statewhen about half the GSNO is reacted (figure 5.2.5). The rate of perthiyl radicalformation and the fact that it reaches a steady state is consistent with previouslyobserved behavior of perthiyl radicals generated in both GSH and LA experiments, It isnot clear from the XAS data by what mechanism the perthiyl radical is formed, but thepresence of features from disulfide bonds suggests a possible secondary process, suchas disulfide bond formation followed by S-C bond homolysis resulting in a perthiylradical. To further investigate this, EPR data was acquired to determine the time ofperthiyl radical generation and NMR data was used to confirm disulfide bond formation.47Cl)zUI1.5UIN-Jln<Li::0zFigure 5.2.4 GSNO sulfur K-edge XAS spectrum with in situ photo-irradiation. Dotted arrowsare decreasing features, solid arrows are growing features.2.5(NLO 0coF—c.JLqCDF(N2.0(N0F(N0.5ENERGY (eV)480.15>-F—CozIJJz.ciwN-J0.05zC’sJ0C”0.001.60z1.2wF—zUwN-J0.8<0zCD0.4Figure 5.2.5 Time evolution of the intensities from sulfurK-edge XAS spectra of the feature at2471.6, indicating disappearance of the S-NO bond, and the featureat 2470.2, indicating theappearance of the perthiyl radical, during in situ photo-irradiationof GSNO.—.— 2470.2---.---2471.6.1 • I • I • I • I • I • I • I • I • I • I • I.-5 05 10 15 2025 30 35 40 45 50 55 60TIME (MIN)49Irradiation of GSNO at 195K or 77K, both as a solid or dissolved in D20, yields asimilar EPR signal, suggesting that S-NO bond photo-cleavage is not sensitive tosample preparation under the conditions of this experiment. The most strikingcharacteristic of the EPR spectra of irradiated GSNO is the absence of features due tothe perthiyl radical such as those that would be expected at a g—2.06 (figure 5.2.6). Theinflection point at 2.012 may suggest the presence of thiyl radicals, but a clearidentification is not possible because of overlapping features with similar powersaturation profiles.However, formation of the GSSG disulfide from photo-irradiated GSNO underanaerobic conditions at 77K and 195K as well as GSNO irradiated at room temperaturewas detected by NMR and is in fact the major product. Low levels of GSSG are presentin our GSNO samples due to sample decomposition as illustrated in figure 5.2.7. TheNMR of GSNO has features at 2.l5ppm (2H, m), 2.47ppm (2H, t) and 3.85ppm (IH, t)due to the glutamyl13,and a protons respectively. This is followed by a singlet at3.98ppm due to the glycyl protons which sits on top of the signals at 4.l3ppm (1 H, m)and 3.99ppm (1 H, m) from the cysteine13protons, followed by the cysteine a proton at4.68ppm (1 H, t)159. GSSG formation can be followed by observing the doublet ofdoublets at 3.Oppm and 3.3ppm due to the cysteine13protons as well as the triplet at2.6ppm due to the glutamyl y protons (dotted square figure5.2.7)160.After irradiation at77K for 1 hour the GSNO sample whose EPR spectra is shown in figure 5.2.6 wasbrought to room temperature and the NMR was acquired. The initial control NMRshows a ratio of 10:3 GSNO to GSSG, derived from the relative peak integrations. Afterirradiation the GSSG features increase in intensity leading to a 2:1 ratio GSNO toGSSG. The sample was further irradiated at room temperature for 5 minutes tofacilitate full conversion of GSNO to the disulfide GSSG. The newly formed GSSG wasthen used to determine if perthiyl radical generation would occur with further irradiationat 77K.50- -- CONTROL— IRRADIATED-VçNITROSO GLUTATHIONE IN D20IRRADIATED 6OMIN AT 77K---SOLID NITROSO GUTATHIONEIRRADIATED 3OMIN AT 195KFigure 5.2.6 EPR of GSNO irradiated under different conditions. Noclear detection of sulfurbased radicals; (microwave power 0.6mW, modulation frequency 50MHz, modulation amplitude3.0G, 5 scans).:2.060 :2.012I I. I: - - - CONTROL:— IRRADIATED—— 4----. -A’I4.V-—.I: :: :--- CONTROL: :— IRRADIATED\_.——I IHRADIATED6OM4AT77K2.10 2.05 2.00 1.95gVALUE51GSSG RADIATEDJIF”-1.,I4.5 .0 I 3.5 3.02.0 1.5I I‘GSNO IRRADIATEDI I300K 5 MIN (GSSG)I II II • I - I II I I4.5 4.0 3.5 3.0L5 2.0 1.50.2 0.2Figure 5.2.7 ComparisonofNMR spectra from samples ofGSNO and GSSG in D20. Dottedrectangle encloses featuresdue to the presence of GSSG./2.01.51.0GSNO IRRADIATED2.077K60M1N0.94.52.00.62.852I I I —I II II II Ia aI II II II Ia II II Ia II Ia •LIPOIC ACID IN D20IRRADIATED 60M(N AT 77K (2mW)2.0622.028GSSG IN D20 IRRADIATED60 MIN AT 77K (20mW)2.10 2.052.00 1.95g VALUEFigure 5.2.8 Comparison of EPR from irradiatedGSSG and LA showing the presence ofperthiyl radicals; (modulation frequency 50MHz,modulation amplitude 3.OG, 5 scans).Irradiation of the GSSG sample in D20 at 77Kgives a rich EPR spectrum (figure5.2.8). When the EPR spectra of irradiated GSSGin D20 is compared with that of LA inD20 the feature attributed to theperthiyl radical at g - 2.062 is present inboth samples.Furthermore, a shoulder at 2.028in the spectrum of irradiated GSSGcorresponds tothe peak maximum of the perthiyl featurein irradiated LA. This is also confirmedbypower saturation studies that showthe feature at 2.062 has a P112 greaterthan 20mWand a linear saturation profile in the 0.002mWto 6mW range (figure 5.2.9). This issimilar to the perthiyl radical behaviorseen in both irradiated GSH and LA.The perthiylradical yield under the conditions investigatedis very low as evidencedby the weakEPR signal. This is mirrored by the XASexperiment, where thepeak at 2470.2eV has avery low intensity. It isimportant to note that after 60 minutesof irradiation the NMR ofthe GSSG sample is still dominatedby the disulfide features(figure 5.2.7).531.8.1.6.1.4.Cl)zLUI1.0.-J0.0-—0.2—• • • • •0.0 0.5 1.0 1.5 2.0 2.5SQUARE ROOT OF MICROWAVE POWER mW112Figure 5.2.9 Power saturation study of irradiatedGSSG feature at g2.O62 attributed to theperthiyl radical; (modulation frequency 50MHz, modulationamplitude 3.0G. 5 scans).5.3 CONCLUSIONXAS spectra of irradiated GSNO and LA underHe atmosphere indicate theformation of the perthiyl radical at —2470.0eV.The presence of the perthiyl radicalwasclearly shown in the EPR spectra of irradiatedLA; however it is proposed that GSNOmust first react to form GSSGbefore being able to account for the perthiylradical signalin its XAS spectrum. Formation ofGSSG from irradiated GSNO is indicatedby the XASspectra as evidenced by the disappearance ofthe features due to the S-NO bond andformation of new peaks at2472.8eV and 2474.0eV. This wasconfirmed with NMRwhere the transition from GSNO toGSSG (figure 5.2.7) occurs with little or nosideproducts. Further irradiation of thesample resulted in formation of a smallamount ofperthiyl radical giving rise toa weak EPR signal. XAS of irradiatedGSNO thereforeshows both the formation of GSSGalong with the weak signaldue to the perthiyl radicalS*+.Si transition....g—2.062 FEATURE OFIRRADIATED GSSG546 Effects of Hyperconjugation on the Electronic Structure and Photo-reactivity ofOrganic Sulfonyl Chlorides6.1 BACKGROUNDThe electronic structure of p-toluene sulfonyl chloride and related organicsulfones has generated some attention because of its role as an initiator in living radicalpolymerization reactions (scheme6.1.1)161.162Living radical polymerization ischaracterized by a faster initiation step than the following propagation reactions and aminimization of termination processes, resulting in a narrower polydispersityindex162’163This is achieved by the presence of actively propagating species due tothe persistent radical effect1. Seen from the perspective of the polymerizationreactions involving sulfonyl chloride, this effect can be explainedas follows. The S-Clbond of the sulfonyl chloride is catalytically broken by CuCI/bpy(bpy = 2,2’-bipyridine) togive the sulfonyl radical and the correspondingCuCI2 organometallic complex. Thesulfonyl radical can further react with the olefinic substrates,which can in turn furtherpolymerize. However, termination events are inhibitedby a rise in the concentration ofthe CuCl2 complex. As mentioned, initiationresults in the formation of the CuCI2complex. These complexes can not react with eachother so they accumulate. If thegrowing chains or initiators react with eachother (“self terminate7disproportionate) theconcentration of the CuCI2 complexes will stillincrease with more initiation events.Further termination steps would involvethe reaction of growing polymers (which arefree radicals) with CuCl2 to givea halogenated alkyl, rather than “self termination”simply due to the increase in the concentrationof the CuCI2 complex. However, thenow dormant alkyl halides canbe reactivated by the CuCI/bpy catalyst.Systemsfollowing the persistent radicaleffect reach a steady state of growingradicalsestablished between the activationand deactivation (propagation and termination)processes. This demandsstoichiometric amounts of catalyst tobe added for properreactivity modulation of “capped”dormant alkyl halide chains163. The S-Clbondcleavage in addition to metalreduction can also be initiatedby thermolysis or photoirradiation162’165-16755Scheme 6.1.1 Metal catalyzed living radical polymerizationwith p-toluene sulfonyl chloride asan initiator.C)C)R+C-)C)+I+CNC)C)IICNC)C)+H2Cz0H2Cc’JC-)C-)•[oRCI-44JRC)z0zz0zwI—w-J(1)w56Both aryl and alkyl sulfonyl chlorides (RSO2CI) have been termed universalinitiators of metal catalyzed living radical polymerization of styrene, methylacrylates andacryIates6”162Regardless of the nature of their R group (alkyl/aryl) or electronwithdrawing or donating effects of substituted aromatic R groups, the polymerizationreactions involving RSO2CI result in faster initiation than propagation and narrowpolydispersity indices161.This has been attributed to several factors such as the fasterformation of sulfonyl radicals vs. carbon centered radicals168,and the low rate ofsulfonyl radical dimerization as compared to carbon radicals161167Most notably theyalso attributed this universality to a lack of effect of the R group on the reactivity of thesulfonyl radical161’169For the aryl compounds this was attributed to poorconjugation between the aromatic ring and the suifonyl moiety. However therate ofoxidation of CuCI2 by aryl sulfonyl chlorides was shownto be impacted by thesubstituent on the aromatic ring: electron withdrawing groupsat the para-substitutedposition enhanced copper oxidation166.Furthermore thereis both computational andexperimental evidence for hyperconjugation in sulfonyl compounds128’129Thecomputational study coupled with X-ray diffractiondata for a series of sulfatemonoesters, sulfamates, and methanesulfonates shows that thesulfur bonding is highlypolarized with the substituents aroundthe sulfur acting both as donor and acceptorsresulting in a sulfonyl bonding manifold composedof polar interactions with reciprocalhyperconjugative bonding128.Secondly,a bathochromic shift in the benzene UV-Visabsorption is observed when a sulfonyl groupis attached to a benzene ring, indicatingconjugative mixing between the orbitalsof the two moieties129.Therefore, the electronic structure of p-toluenesulfonyl chloride (Ia) and relatedsulfonyl species (figure 6.2.1) was explored inorder to better understand their electronicstructure, the importance of hyperconjugation,as well as its impact on sulfonyl radicalgeneration and subsequent radical polymerization.A series of compounds of the formRSO2G were probed using sulfurK-edge XAS spectroscopy to determine the effectonthe SG bond (G = -Cl, -OH, -alkyl)due to the presence of a it system in theR group. Inparticular, aryl (a, R = p-XC6H4-,X= H/CH3)and alkyl (b) R groups were chosentostudy the effect of orbital mixingin the sulfonyl centre. Photo-cleavage(scheme 6.1.2)of the S-Cl bond, via irradiationwith a Xe arc lamp, was investigatedfor the sulfonylchlorides (1ab) and the reactivitywas correlated to the hyperconjugativeeffects57observed. Molecular orbital calculations were carried out to aid in the assignment ofspectral features.RSO2C1hv_> [Rso2cl]*> RSO + C1Scheme 6.1.2. Sulfonyl chloride photo-cleavage reaction (R alkyl, p-tolyl).6.2 RESULTSSuffur K-edge XAS spectroscopyThe sulfur K-edge XAS spectra of 1-3 a,b (figure 6.2.1), are shown along withadetailed analysis of simulated spectra in figures 6.2.3 to6.2.5. The XAS of sodiummethane sulfone was used as the model spectrumfor 2b to facilitate analysis of solidsamples. The spectra of aqueous methanesulfone17°and sodium methane sulfone arecomparable. The pre-edge regions of the modelspectra exhibit clear differences in theirfeatures as a function of the R group andsubstituent G. The aryl compounds (l-3a)show additional features not present in the spectraof their alkyl counterparts (l-3b).Compound Ia has three features, two peaksat 2477.4eV and 2481.2eV and a shoulderat 2479.6eV, which is not seen in lb where onlythe peaks at 2477.6 and 2480.9eV arepresent. The sutfonate 2a hasa main peak at 2481.7eV flanked by two shouldersat2479.9eV and 2483.9eV. In contrast2b has only one shoulder at 2483.1eVin additionto its main peak at 2481.3eV (seefigure 6.2.4). Lastly, 3a exhibitsa peak at 2478.6eVin addition to the main absorptionfeature of the spectrum seen in3b. The mainabsorption feature is also at slightlyhigher energy in 3a (2480.6eV)compared to 3b(2480.1 eV). The XAS spectra showthat the aromatic ring has a significanteffect on theenergy and sulfur 3p characterof the valence orbitals. Thiscould be due to energyredistribution of the existing transitionsin the alkyl compounds, thepresence ofadditional transitions or acombination of the two.58Ethyl Phenyl Sulfonep-Toluene Sulfonyl ChlorideIap-Toluene Sulfonic Acid2a 3a0CI——CH3II0Methane Sulfonyl Chloridelb0IIHO—S—CH30Methane Sulfonate2bH3C’Methionine SulfoneFigure 6.2.1 Structures of la. p-toluene sulfonyl chloride; lb methanesulfonyl chloride;2a p-toluene sulfonic acid; 2b methane sulfonate;3a ethyl phenyl sulfone; 3b methiomnesulfone.3b59DFT calculations and spectroscopic assignmentsSpectral features and electronic transitions were simulated using TD-DFT.Fragment calculations were also carried out on R, SO2 and G for a better description ofthe assigned transitions and to assess the effect of the aryl substituent. The relevanttransitions and their descriptions are listed in Tables 6.2.1 to 6.2.6 for each of thecompounds. The XAS of 3b was simulated using (CH3)2S0 as a model compound. Ithas been previously shown that TD-DFT transitions for (CH3)2Sare in good agreementwith XAS spectra of methionine15.Two conformational models were evaluated for thesimulation of the sulfur K-edge XAS spectra of 3a. The lower energy conformation(linear) is only —O.42kJ/mol lower in energy than the higher energy conformation (bent).This low energy barrier suggests that the molecule is able to interconvert between linearand bent conformations (figure 6.2.2). The XAS spectra were best simulated using thebent conformation and thus this geometry was used in our analysis.LINEARBENTH3C CH2H3CFigure 6.2.2 Geometries of ethyl phenyl sulfone conformations(3a).The TD-DFT simulated spectra (figures 6.2.3- 6.2.5) for the alkyl compounds(1-3b) are in good agreement with experimentaldata. The spectra of the alkylcompounds are dominated by features dueto theoorbitals of the S-C and S=O bondsin the RSO2 molecular fragment. A low energy featurecorresponding to the SCl0*+Sitransition is clearly visible in lb. For the arylcompounds (1-3a) only the calculatedspectrum for 2a is in good agreement with experimentaldata. Spectra from TD-DFT forIa predicts the feature due toSCla+—Sis transition at 2477.4 to have a shoulder, whilethere is no accounting for the actual shoulderat 2479.6eV in the experimental data. For3a, TD-DFT completely fails to predict the featureat 2478.6eV.602472 2476 2480 24842488ENERGY (eV)Figure 6.2.3 Comparisonof simulated spectra using TD-DFTand ASCF, and experimentalspectra for compounds la,b thesulfonyl chlorides.>-H(I)zwIzwN-J0z>-I—Cl)zUiI—z0UiN-J0z2472 2476ENERGY (eV)248861ENERGY (eV)ENERGY (eV)Figure 6.2.4 Comparison of simulatedspectra using TD-DFT and zSCF, andexperimentalspectra for compounds 2a,b the sulfonates.62Cl)zLUIzULUN-J0zCl)zUiI—zLiiN-j0z(I)zLUI—zLUN-J0zCl)zLUIzLUN-J0::0zFigure 6.2.5 Comparison of simulatedspectra using TD-DFT and tSCF,and experimentalspectra for compounds 3a,b thesulfones.632472 2476 2480 24842472 2476 24802484ENERGY (eV) ENERGY (eV)However, TO-OFT generates a spectrum using the ground state calculation as itsonly reference, and thus does not account for possible electronic relaxation in theexcited state. To gauge the importance of this effect, the transition energies wererecalculated using the Slater transition state ASCF approach by populating the acceptororbital with half an electron and removing the same half electron from the S1 coreorbital14”171ASCF can overestimate the relaxation shifts; however, it can be veryeffective in determining which transitions are most susceptible to relaxation effects.Generally, the alkyl sulfonyl compounds were not affected by relaxation effects as muchas the aryl sulfonyls. But even for the alkyl compounds the ASCF spectra showbroadening of the main spectral features and even a splitting into two peaks as is thecase of 3b. For the aryls the relaxation effects seem least important in the case of 2awhere there is not much change in the SCF simulation fromthat of the TD-DFT.Relaxation effects become more pronounced in 3a. Here, IXSCF accounts for the peakat 2478.6eV by shifting the energies for TO-OFT calculated transitions5 and 6 to lowerenergy by -1.1eV. In Ia there is a dramatic splitting betweentransition I andtransitions 2 and 3 (table 6.2.1), pushing the latter transitions to higherenergy. Thisresults in a single peak for the transition at 2477.4eVand suggests that transitions 2and 3 are responsible for the shoulder at 2479.6eV inthe XAS spectra, which TD-DFTcalculations failed to account for. It is apparent that orbitalswith iu antibondingcharacter, especially those of the aryl rings are mostaffected by electronic relaxation.Together, the TD-DFT and tSCF results allowus to assign the features of thesulfur K-edge XAS spectra for each speciesas illustrated in figures 6.2.3 to 6.2.5. Thesulfonyl chlorides exhibit low energy featurescorresponding to the energy of theSCI—S18 transition and higher energy featuresdue to the SO0—S1and SCa*#Sistransitions. The shoulder at 2479.6eV, whichis present only in the aryl (Ia),corresponds to the4*4_Sj transition.cI*andcJare the two lowest energy itorbitals of the aryl ring (figure 6.2.6). i1.has the largest electron density on the carbonbound to the sulfonyl moiety allowing it to mixwith the SCIa* final state. The mixinggives r sulfur p character allowing thistransition to be visible in the sulfur K-edgeXAS spectrum (figure 6.3.1). The XAS spectraof the sulfonic acids (2a,b) do not showa distinct pre-edge feature relating to the SG*,because the SOH0* orbital is higher inenergy than SCla*.and overlaps withthe main feature of 2a,b at 2481.7eV. The lowerenergy shoulder in 2a is attributedto the SOH0*_Si, which is lowered in energy going64from the alkyl to the aryl by mixing with the orbital of the aryl group. Similarly themain features of 3a and 3b correspond to a combination of SO—S1and SC—S1transitions. These transitions are lower in energy than in either the case of the sulfonylchlorides (la,b) or the sulfonic acids (2a,b). Like in the previous examples, thepresence of the aryl group is accompanied by additional features in the spectra. 3a hasa lower energy feature not present in 3b attributed to thecI*final state mixing withSO orbital. This interaction is particularly favorable in 3a because the two energystates are closer in energy than in any of the other aryl compounds.,J2LIFigure 6.2.6 Molecular orbitals of benzene.65Table 6.2.1. Sulfur K-edge DFT calculated transitions for la with major contributors listed first.ASCF calculated energy shifts are referenced to the lowest TD-DFT calculated energy transition.Transition Assignment (Primary + Others)Eneiies(eV)ASCF (eV)f1 SCl0 ,, so2476.99 0.02.3x1032 2477.85 +2.31.1x1043 ,so, sci 2477.94 +3.2 1.3x1034c,S, so 2479.74 +1.83.6x 104so 2480.13 +1.47.9x1046so,, so, ci2480.59 +1.31.Ox 1042480.69 +2.59.6x1048 2480.76+2.9 1.4x1039 SO*5OcJ 2481.14 +2.6 2.4x103Table 6.2.2. Sulfur K-edge DFT calculated transitionsfor lb with major contributors listed first.ASCF calculated energy shifts are referenced to the lowestTD-DFT calculated energy transition.Transition Assignment (Primary, Others)Eneiies(eV)zSCF (eV)f1SC1a* ,2477.50 0.003.8x1W32SCa* , SOa*2479.72 +0.667.2x 1043 SC ,SOa*2480.44 +0.181.3x1034SCcy*2480.72 +1.123.5x1045 SO CHa*2480.92 +0.701.6x1036 CH , SO02481.48 +1.842.4x 1047 CHa* ,SOC.2481.67 +1.687.9x 10466Table 6.2.3. Sulfur K-edge DFT calculated transitions for 2a with major contributors listed first.ASCF calculated energy shifts are referenced to the lowest TD-DFT calculated energy transition.Transition Assignment (Primary, Others)EnV)ASCF (eV)f1ci , SO* , SOH02479.16 0.00 1.3x1032 2479.87 +0.16l.0x1043 , SOHy*,SOa*2479.97 +0.493.6x104 SO,rjo* 2480.93 +1.274.3x1035 so , 2481.71+0.11 3.2x1036 SC* , SO* 2481.92-0.28 8.7x 1047 SCo ,OHa* 2482.25 +1.21 1.2x1038 SC*, SO,, OH0 2482.43 +1.76 3.0x1032482.78 +1.867.4x105Table 6.2.4. Sulfur K-edge DFT calculatedtransitions for 2b with major contributors listed first.zSCF calculated energy shifts are referenced to thelowest TD-DFT calculated energy transition.Transition Assignment (Primary, Others)Energies(eV)zSCF (eV)f1SOHa*, SCy*, SOa* 2479.50 0.002.0xl02SO , SCa* 2480.35 -0.212.7x1033 SCa* ,SOHa* 2480.78 +0.021.6x1W34 SC,SO 2481.18 -0.301.5x1035SO7rJcy*, SO2481.31 +0.371.6x1036 CHa*2481.61 +0.052.3x1037 CHa* ,SO2481.83 +1.047.1x10467Table 6.2.5. Sulfur K-edge DFT calculated transitions for 3a with major contributors listed first.ASCF calculated energy shifts are referenced to the lowest TD-DFT calculated energy transition.Transition Assignment (Primary, Others)Energies(eV)bSCF (eV)f1 2476.95 0.001.8x1042 2477.36 +0.853.6x103 SC,* , 2479.04 -0.864.1x1054 SCa*,CSa* 2479.53 -1.15 1.4x1045 SO, SO , SC, C1$0 2479.81 -1.20 1.0x1036 ,SO0.,SC0., 2480.01 -1.19 6.8x1047 SO* ,SO,. 2480.17 -0.57 l.6x1O8 SO* ,SO* 2480.34 -1.03 6.7x1049 CH0. 2480.42-0.498.8x10Table 6.2.6. Sulfur K-edge DFT calculated transitionsfor 3b with major contributors listed first.tSCF calculated energy shifts are referenced to thelowest TD-DFT calculated energy transition.Transition Assignment (Primary,Others)EnV)tSCF (eV)f1 SC0.2478.53 0.001.9x1042 SC0*2479.24 -0.622.4x1033 CH,. , SO0.2479.59 -0.306.2x1044 SC0*, SO. 2479.69-0.547.Ox5 CH0. , SO0.2479.87 -0.331.Ox6son. , CH0. 2480.31+0.133.4x1037 CH0. ,SOo2480.52 +0.124.7x1048 SO0. ,CH0.2480.75 +0.161.7x10368Photo-cleavage Reactions0zwIzfDwN-J0zTo examine the effect of the aryl ring on the generation of the sulfonyl radical andphoto-cleavage of the S-Cl bond, p-toluene sulfonyl chloride (Ia) and methane sulfonylchloride (1 b) were irradiated with a 75W Xe arc lamp and XAS spectra were collectedas described in the experimental section (figure 6.2.8). Scans were acquiredconsecutively with continuous irradiation and changes in the intensity of the featuresattributed to the S-Cl bond, present at 2477.4eV in Ia and 2477.6eV in Ib, wererecorded. Each scan lasted 5.5 minutes, and over the same time span of irradiation theintensities of both peaks decrease indicating cleavage of the S-Cl bond. The photo-cleavage rate of Ia containing the aryl moiety was much higher (figure 6.2.7) than in thealkyl containing compound lb. This is indicated by the larger decrease inintensity withirradiation attributable to the S-Cl bond feature of Ia.—— —..— —..— —B.— _•. — —..SCIm—S INTENSITIESis—.—la—.— lb2.22.01.81.61.41.21.0I . I I I I — I —-5 0 5 10 15 20 253035 40 45 50 55 6065TIME (MIN)Figure 6.2.7 Time evolution ofthe sulfur K-edge features due to theS-Cl bond in la and lbwith in-situ irradiation.69Figure 6.2.8 XAS spectra within situ irradiation of la (top)and lb (bottom) with a 75W Xearc lamp under anaerobicconditions. Scans were takenevery 5.5min with continuousirradiation.702470 2475 24802485 2490ENERGY (eV)6.3 DISCUSSIONAssigning the sulfur K-edge XAS spectral features of these compounds is thefirst step in determining the effect of their electronic structure on their respectivereactivities. Of particular interest are p-toluene sulfonyl chloride (Ia) and its role as aninitiator in living radical polymerization reactions. The reactivities of the complexesstudied should be affected by electronic coupling such as that due to an aromatic groupbound directly to the sulfonyl moiety. These interactions have been confirmed by thepresence of features in the sulfur K-edge XAS spectra of the aryl compounds which, arenot present in their alkyl counterparts, and are attributable to the mixing ofcJY(aryl1*orbitals) with orbitals containing sulfur 3p character.The XAS data coupled with the molecular orbital calculations give insights intothe nature of bonding in these systems. In the alkyl compounds (I-3b), there is adistinct ordering of the empty valence orbitals with the lowest energy attributed to theSG0followed bySR0,SO, and SO0. The splittings between these states is generallysmall resulting in a single intense broad feature for these species. The exception is themethane sulfonyl chloride (I b) with a very low SG0 feature -3eV below the main peakattributed to theSCl0*÷_Sjtransition. In contrast, the ordering in the aryl compounds isswitched such that the transitions due to SR0*4_Sls are higher in energy than those ofSO—S1,resulting in an energy arrangement resemblingE(SG0*) <E(SO*) < E(SO,*)< E(SRa*). The reordering of SR0 and SO0 final states in the aryl compounds isattributable to the stronger S-C bond due to the sp2 character of the aryl carbons172.This pushes the SC0 orbital to higher energy, which is consistent with the main sulfur K-edge feature for the aryl compounds being —0.4eV higher in energy than that of the alkylspecies.Furthermore, the aryl group has two low-lying ir’ orbitals and that canmix and redistribute intensity in the Sulfur K-edge spectra. Since T* has no electrondensity on the carbon bound to the sulfur, t* is effectively non-bonding with respect tothe sulfonyl moiety (figure 6.3.1). As already mentioned cF’ has good overlap and astrong interaction with the sulfur moiety. This interaction, however, is dependent on thenature of the G group and its bonding interactions. The energy of the SGa*+_Sisincreases from Cl— OH —* CH3,in agreement with expected bond strengths172174.In71the case of Ia, mixing with cI results in a further lowering of the SCIa*. The higherenergy SOHa* orbital in 2a can interact more fully with the aryl group resulting in a highlymixed lowest lying final state in the XAS data. In 3a however, it is the mixing of the arylwith the SO which takes precedence. The absence of a strong SG interaction withcould be attributed to both energetic and overlap considerations. As mentionedearlier, the SO* is closer in energy to the aryl I* and the SO orbitals might morereadily overlap with the aryl t’ than the sp3 hybridized orbitals of the ethyl G group in3a.- SCIa*SCIa*Figure 6.3.1 Mixing of the I*, cJ and SCI0* orbitals resulting in transitions1 and 3 in thesulfur K-edge spectrum of la (top). (Isovalues = 0.060e.A3).To test whether the effect of the aryl group could in fact be aninductive effectrather than a conjugative one, the aryl ring was rotated with respectto the SG bond tosimulate “turning off’ aryl hyperconjugation yet maintaining an inductiveeffect. Theresult of the rotation about theS-C(Sp2)bond on the predicted sulfur K-edge spectrum ofIa as calculated by TD-DFT is shown in figure6.3.2. The starting point for thecalculation has the aryl ring at900to the S-Cl bond and is consistent with the geometryobserved in the crystal structure of Ia where the arylring and the S-Cl bond are almostperpendicular (84.3°). As the aryl ringis rotated from a perpendicular plane to theCI\SCISit4SCI+72SG bond, a situation allowing for maximum conjugation, to a plane parallel to the SGbond, resulting in no conjugation to the SC bond, the splitting between the SCIO*—Sisand cI÷—S1decreases by —1eV. At the same time the intensity of SCl0*÷—Siincreases suggesting increased sulfur p character, while the intensity ofI**—S,decreases indicating a decrease in the sulfur p character. This leads to the conclusionthat as hyperconjugation is “turned off” the mixing betweencJi.and S is also turnedoff. The calculated energy stabilization of the hyperconjugative interaction on SCl* is—0.5eV which is equal to the experimental value for the difference in SCl0*.Sitransition energies going from lb to Ia.Figure 6.3.2 Effect of turning off 3?4ISCl0.mixing on the intensities and energies oftransitions 1 and 3, by rotating the aryl ring..2477 2478 2479 2480 2481 2482ENERGY (eV)73It is important to note that the above mentioned hyperconjugative interactionoccurs between two empty antibonding orbitals; hence it is not a typicalhyperconjugative effect. This excited state hyperconjugation17178 enhances cleavageof the S-Cl bond in Ia over lb in accordance with the postulate that the aryl groupshould have a large effect on the S-Cl bond cleavage. Direct photolysis likely resultsfrom excitation into the SCI0 acceptor orbital with subsequent bond cleavage to formradical products. This study provides a direct assessment of the nature of theSCI0*orbital and the effect of the aryl group on the photolytic process. This is very beneficialfor living radical polymerization since initiation has to be faster than propagation;therefore facile radical generation is key. The aryl group in Ia allows for partialdelocalization of the excited state electron through mixing with theI* orbital. Mixingof the cIi with SCl orbital results in a decrease in the excitation energy as previouslydescribed, but also in a likely increase in the excited state lifetime through chargeseparation. An increased lifetime of theSCI0 excited state allows for a highertransmission coefficient for the overall reaction and a faster rate of photo-cleavage. Itcan also be argued that the rate of photo-cleavage is faster in toluene sulfonyl chloridebecause the aryl group itself can enhance the absorption of photons, however this isunlikely since the source of irradiation passes through a plastic window which shouldremove photons with wavelenghts below --350nm. Because toluene has an absorptionmaximum at —260nm179,no enhancement of photon absorption should occur due to thepresence of the aryl group under the described experimental conditions.6.4 CONCLUSIONIn the work on sulfonyl complexes of the type RSO2G interactions present in thearyl compounds but not seen in their alkyl counterparts were identified. The mixing ofthecI%*aryl orbital into the sulfonyl moiety gives rise to new features in the sulfur K-edge spectra. These features were characterized. Of particular interest is the excitedstate hyperconjugation interaction betweent* and SCl0,resulting ina faster photocleavage rate for the S-Cl bond. Excited state hyperconjugation facilitates photocleavage by lowering the SCI0* energy and allowing delocalization of the excited state,increasing its lifetime and enhancing the photo-cleavage reaction. It is then reasonableto assume that the magnitude of excited state hyperconjugation can be modulated by74% mixing=(i48xlOO%2478 2479ENERGY (eV)Figure 6.4.1 Fitted XAS spectra of p-toluene sulfonyl chloride (la) with intensities calculatedfor the SCl0*Sitransition and the4*+—Sis transition.electron withdrawing or donating groups on the aromatic ring. Future experimentsinclude plans to acquire XAS spectra for a series of para substituted aryl sulfonylchlorides with a variety of electron withdrawing and electron donating substituents.Characterization and quantization of the features arising due to the arylcI* mixing withSCI0* should be a good measure of the total excited state hyperconjugative interactionpresent. A preliminary quantization of this effect in p-toluene sulfonyl chloride Ia wascarried out. The increase in intensity(I_s )of thet4*4_Sl transition coupled withthe decrease in SCl0**—S1 transition intensity(Isci4s)is a direct measure ofcI ESC10 mixing and hence excited state hyperconjugation. Assuming that no othercontributions are present in the features due to theSCl0and torbitals, the percentof excited state hyperconjugation can be calculated by fitting the sulfur K-edge spectra(figure 6.4.1) and applying equation 6.4.1 to the measured intensities. For Ia 10-15%mixing of thecI*into theSCI0*is estimated.Equation 6.4.1248275The question still remains as to why excited state hyperconjugation does notseem to affect the living radical polymerization reactions in which p-toluene sulfonylchloride (Ia) acts as an initiator. By definition the initiation step in such reactions is thefaster than the propagation step, so the formation of the aryl sulfonyl radicalintermediate will not be the major factor to impact reactivity. Furthermore using EPRtechniques, previous research has shown that the sulfur p orbital containing theunpaired electron in the aryl sulfonyl radical is in the plane of the phenyl ring180. Thiswould preclude any interaction of the paramagnetic S3,orbital with the aryl cI4 orbitals.Since it is the half emptyS3porbital which is involved in the polymerization reaction,there would be no major effect on the propagation step due to excited-statehyperconjugation. Also, as the polymer chain grows one would expect the effect of thearyl sulfonyl moiety to diminish. Even if the orientation was optimal for mixing of the S3andi%states in the aryl sulfonyl radicals, preliminary TD-DFT calculation predict thisinteraction to be minimal.767 Concludini Remarks and OutlookIn this thesis, the application of Sulfur K-edge XAS to investigate the photo-reactivity of a series of model sulfur containing compounds was discussed. It wasshown that XAS can be applied to a wide variety of systems with applications both inbiological and inorganic chemistry. XAS proved to be a useful tool in the detection ofthiyl radical intermediates in UV irradiated GSH, which are difficult to characterize usingother spectroscopic techniques such as EPR. Further research investigating thiylradical generation and characterization via XAS may help in the elucidation of themechanism of action of enzyme systems which form thiyl radical intermediates, as isproposed in the case of ribonucleotide reductase123.Additional sulfur based intermediates were also characterized in the form of thestable perthiyl radical, which is a product of photo-irradiation in all biologically relevantlow molecular weight sulfur species investigated, Initially it was somewhat unclear whatthe mechanism of perthiyl radical formation in GSNO is; however, the XAS spectraindicated generation of a disulfide bond upon photo-irradiation. NMR of irradiatedGSNO confirmed disulfide bond formation, and EPR showed that only after disulfidebond generation is the perthiyl radical formed during photo-irradiation.::LLr!i\!/IILIPOIC ACID ‘ NITROSOGLUTATHIONEIvXAS XAS246.0 2482 2484 2464 2448 2470 2472 2474 2476 2478ENERGY (eV)201.51.50z24isb’ 2468 2470 2412Figure 7.1.1 XAS data from irradiated LA and GSNO complementswell the information fromboth NMR and EPR experiments, filling in the gaps when needed.2474 2470 2470 2480 2482 2484ENERGY(eV)77Furthermore it is important to note that in both the case of LA and GSNO, XASdetected the intermediates and products being formed, while detection with EPR andNMR proved difficult (figure 7.1.1). In the case of LA, the intermediate was readilydetected by EPR while the final major product, which is believed to also be a disulfide,could not be characterized by NMR due to its insolubility. For GSNO the story issomewhat the opposite. While the major product was easily characterized by NMR theformation of the perthiyl radical intermediate seen in the XAS spectra required extensiveEPR characterization.The sensitivity of this technique to the bonding configuration is particularlyevidenced in the case of LA, which has features due to SSa*4_Sls and SCa*Sistransitions at lower energy than “linear” disulfides suggesting weaker bonding in thesterically strained pentacyclo-disulfide. GSNO, on the other hand, exhibits sulfurcoreexcitations to the SN* orbital consistent with theoretical calculations, indicatingthepresence of an S-Na resonance form in nitrosylated thiols157.The applicability of XAStocharacterize electronic configurations could be usedto further investigate the bondinginteractions in S-nitrosothiols. Understanding the bonding interactionin thesecompounds could help in the synthesis of S-nitrosothiols inwhich N0 release can bemodulated with the potential to target specific locations withinthe body, and stimulateN0 controlled signaling pathways with beneficial consequences.The power of XAS to elucidate electronic configurationwas also exploited in thecase of p-toluene sulfonyl chloride. Here, it was foundthat p-toluene sulfonyl chloride ismore susceptible to photo-reactivity than its alkylcounterpart methane sulfonyl chloride.This was attributed to an excited hyperconjugationbonding interaction, directlydetectable via XAS, found between the aromatic antibondingorbitals and the SCla*orbital in p-toluene sulfonyl chloride.Excited state hyperconjugation has also beenobserved in a series of ruthenium arene thiolates(Ru(p-cym)(en)S02— Ph) whose roleas therapeutic agents to combat canceris dependent on Ru-S bond dissociation181.Hyperconjugation could play an essential role inthese species and may influencebioactivity of the aryithiolato version ofthese drugs. Modulating this effect could lead tobetter anticancer therapeutic agents. 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SriskandakumarT, Petzold H, Bruijnincx P, etal., JAm Chem Soc, (submitted2009).87CD a x - a ‘1 z 0 .1 ‘1 CDcoA1.1 ADF input file for ground state and time-dependent DFT calculations on CH3SH.UNITIlength Angstromangle DegreeENDATOMS1 C xl yl zi2 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z56 H x6 y6 z6ENDGEOVARxl —0.9323645165yl 0.7538453976zi —0.2554338150x2 —1.358906052y2 1.765267821z2 —0.2847699841x3 —0.1640953568y3 0.7242029073z3 0.5300502049x4 —1.707025428y4 0.1481577134E—0].z4 —0.1134618763E—01x5 —0.7743971035E—0ly5 0.3446657011z5 —1.785980422x6 —1.154233121y6 0.4311598236z6 —2.607633668ENDXCGGA Becke PerdewENDSYMMETRY NOSYM tol=0.001SAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 50ENDSCFiterations 50converge 1.Oe—6 1.Oe—3mixing 0.2ishift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 4 7 789A1FIT 10.0Modi fyExcitat ionsUseOccupiedAlSubEndEndFragmentsH t21.HS t21.SC t21.CEndAl .2 ADF input file for ground state and time-dependent DFT calculations on CH3S’radical.UNITSlength Angstromangle DegreeENDATOMS1 C xl yl zi2 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z5ENDGEOVARxl —0.8955097192yl 0.7461104821zl —0.2655950654x2 —1.354738363y2 1.747210887z2 —0.3146675968x3 —0.1716129346y3 0.7262054524z3 0.5651110213x4 —1.692308581y4 0.1402182236E—01z4 —0.5039963098E—01x5 —0.1075872236y5 0.3483459909z5 —1.799000867ENDXCGGA Becke PerdewENDCHARGE 0 1UNRESTRICTEDSYMMETRY NOSYM tol=0.00l90SAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 50ENDSCFiterations 50converge l.0e-6 l.Oe—3mixing 0.2lshift 0.0dils n=lO ok=0.5 cyc=5 cx=5.0 cxx=i0.0ENDINTEGRATION 4 7 7A1FIT 10.0ModifyExcitationsUseOccupiedAlSubEndEndFragmentsH t21.HS t2l.SC t21.CEndA1.3 ADF input file for ground state and time-dependentDFT calculations on CH3SSradical.UNITSlength Angstromangle DegreeENDATOMS1 C xl yl zi2 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z56 S x6 y6 z6ENDGEOVARxl —0.8166129615yl 0.7670805715zl —0.2042560278E—01x2 —1.256955743y2 1.771247152z2 —0.9940988876E—01x3 —0.7116845060E—01y3 0.7416107139z3 0.7891296540x4 —1.60823205391y4 0.2820444077E—0lz4 0.1690125105x5 O.2966549980E—01y5 0.3608788083z5 —1.548454032x6 —1.314040181y6 0.4088199419z6 —2.953060041ENDXCGGA Becke PerdewENDCHARGE 0.01UNRESTRICTEDSYMMETRY NOSYMtol=0.001SAVE TAPE21TAPE13EXCITATIONDavidsonlowest 40ENDQTENSESRENDSCFiterations 50converge1.Oe—6 1.Oe-3mixing 0.2ishift 0.0diis n=l0ok=0.5 cyc=5cx=5.0 cxx=10.0ENDINTEGRATION4 7 7A1FIT 10.0Modi fyExcitationsUseOccupiedAlSubEndEndFragmentsH t21.HS t21.SC t2l.CEnd92A1.4 ADF input file for ground state and time-dependent DFT calculations ofCH32SNO.* The Molecule“$ADFBIN/adf” <<eorModlfyExcitatlonsUseOccupiedAlSubEndEndUNITSENDlength Angstromangle DegreeATOMS‘C2S3N405C6H7H8H9H10 FlEND0.0932993305561.8018709988902.5646080780903.750776088010—0.541415135994—1.5806598237400.010874680875—0.548506825515—0.4756931540850.1575750732700.376389670055—0.2498391009001.4633532514101.5291497936000.3313664840920. 6880332245360.971075028574—0.689497465788—0.2172967362841.407803333340—0.070212789684—0.035836671374—0.011760165370—0.030834631742—1. 458391795540—1.411767390600—2.158717739280—1.8620158762100.6564354396220.310912848049GUIBONDS1 2 1 1.02 3 2 1.03 4 3 1.04 6 5 1.05 7 5 1.06 8 5 1.07 5 1 1.08 9 1 1.09 10 1 1.0ENDCHARGE 0.0SYMMETRY NOSYM tol=0.001BASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSAVE TAPE21 TAPE1393EXCITATIONDavidsonONLYS INGlowest 10ENDSCFiterations 50converge 1.De—6 1.Oe—3mixing 0.2lshift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 6 6 6A1FIT 10.0eormkdir tapesmy —fTAPE*tapes 2>/dev/null* DENSE’cp tapes/TAPE21 TAPE21“$ADFBIN/densf” <<eorDensity fit transPotential coul transeormy TAPE41 tapes/TAPE41rm -fTAPE*my -ftapes/TAPE*A1.5 ADF input file for ground state and time-dependent DFT calculations ofCH3SSCH.* The Molecule“$ADFBIN/adf” <<eorTITLE Methyl Disulfide SE’ SKUNITSlength Angstromangle DegreeENDATOMS1 C xl yl zi942 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z56 S x6 y6 z67 C x7 y7 z78 H x8 y8 z89 H x9 y9 z910 I-I xlO yb zlOENDGEOVARxl —0.4008420982yl 0.3905396080zi —0.2322155253x2 —0.5126493230E—01y2 0.4627550644z2 —1.268344630x3 —0.4137836966E—01y3 —0.5403383933z3 0.2217839358x4 —1.494155479y4 0.4149537687z4 —0.2059453443x5 0.2480197890y5 1.801338869z5 0.7591664997x6 —1.313268316y6 3.138545139z6 0.7997379556x7 —2.222534719y7 2.734816620z7 2.344050187x8 —2.188392019y8 1.654074759z8 2.524988378x9 —3.264016048y9 3.045193457z9 2.193798612xlO —1.790446556yb 3.276290285zlO 3.194541593ENDBASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSYMMETRY NOSYM tol=0.001SAVE TAE’E21 TAPE13EXCITATIONDavidson95lowest 20ENDSCFiterations 50converge l.Oe—6 l.Oe—3mixing 0.2lshift 0.0diis n=lO ok=0.5 cyc=5 cx=5.0 cxx=l0.0ENDINTEGRATION 4.0 7 7A1FIT 10.0ModifyExcitationsUs eOccupiedAlSubEndEndeormkdir -p tapesmyTAPE*tapesmy logfile tapes* DENSF*my tapes/TAPE21 TAPE21“$ADFBIN/densf” <<eorGrid CoarseDensity fit transPotential coul transUNITSlength Angstromangle DegreeENDeormyTAPE*tapescat logfile >> tapes/logfilerm —f logfileA16 ADF input file for ground state and time-dependentDFT calculations of Ia, ptoluene sulfonyl chloride.“$ADFBIN/adf” <<eorTITLE TosCL single point andskUNITSlength Angstromangle Degree96N<FF)-i-I-N)N)N)HF-00wCO 0)DCO0)010)cO0f-HN)N)N)I—ico.0COCOCOCO.J0‘.0CO0)HN)I—icoO).0I—iN)N)‘.0‘.0‘.0COCOCO—J-JJCOCOCO01Cr101‘U)U)0)N)N)N)F-F-I-LiC)0IN)II0)I00)N)I0IIIIN)ICDN)-00I-0CD0<.F-I-•I—iC)•0.•0•F-0•I—i0•0.•N).•-•CO•01.•CO••—J•N)00)CO0.N)I—ii-—JN)0)‘.001—1HCOCO01N)N)0CO-J0N)I-CO—IN)CO—JCOF-If)—J01COU)COF-CD—JC)CO‘.0-0‘.0J‘.0N)Cr10’N)0’‘.00—3‘0.—3Cr1COCC)-)-COU)COU)I-’01CO010)Cr1CON)N)‘.0F-0)0CON)F-0I—’COU)0)00)COF’I-’JF’00’C)U)CD—)00U)CO‘.0—33U)‘.0‘.0—3I—’CO‘.0—1F’U)F’‘.0—-1—J01—3U)F’CO‘.0I-’010)‘.0F’N)N)0—3CO‘.0—1U)01F’1.0Cr1‘.oN)-.JI-’CO010F’—)—3—1COF’F’0.—31-’1.0F’U)CON)010)N)F’CO1.0COCO010-.JCOCO0.I-C)U)CO01N)U)COCOCOF’U)I-’1.0CO—300)CT)F’F’-1.0U)CX)0.U)N)CO0COI-’1.01.0COCO0F’CDCOF’I-’F’1.0U)COF’—3Cr10U)N)U)1.0F’F’F’U)—ICO0.N)CD—1U)COU)0.CT)LiLiLiIII000 F’F’F’F’I-’F-’F’F’F’1.0CO—3CO010.U)N)F’—3COU’0.U)N)F’0LiLiOC)Qflflflfl000CnLiLiLiLiLi I-’ F’ CO<F’F’I-’F’F’F’F’I—’NNNNNNNNNF’—3CO010.U)N)F’01.0CO—)CO010.0)N)F’CO N F’ COF’ 1.0 N) N) ‘ CO CO CO F’ LiLii—’ ZCO ID,<xxpx>xxx<XXXXXX‘0CO—JCOU’U)N)F’F’F’F’F’I-’F’F’I-’—)COCT)U)N)I-’01<1<1<1<1<1<1<1<1<CDCO—3CO011)SU)N)F’Li ID‘-.3 0 (1)NNNNNNNN F—’F’F’F’F’F’F’I-’—3COU’‘0.0)N)F’0Co -.4x13 1.870581833y13 0.5053014545z13 1.437374330x14 0.2301057166E—01yl4 —1.019022097z14 0.7796858473x15 —2.394325646y15 —0.5434618682z15 —0.7173188957x16 —3.414582372y16 0.2173973014z16 —1.405283391x17 —2.702866410yl7 —1.411087197z17 0.3984018889x18 —1.503222788yl8 —1.793094903z18 —2.168454350ENDBAS IStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSYMMETRY NOSYM tol=0.001SAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 40ENDQTENSESRENDSCFiterations 50converge 1.Oe-6 1.Oe—3mixing 0.2ishift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 4 7 7A1FIT 10.0Modi fyExcitationsUseOccupiedA298SubEndEndA1.7 ADF input file for ground state and time-dependent DFT calculations on 2a,toluene sulfonic acid.TITLE TosOHUNITSENDlength Angstromangle DegreeMcdi fyExcitationsUseOccupiedEndAlSubEndATOMS1 C 0.0612248100002 C 1.0812847200003 C 1.0374797100004 C —0.0464146300005 C —1.0710994200006 C —1.0232435300007 C 2.1909108800008 H —0.0933064600009 H —1.89820617000010 H 1.78856168000011 H 2.72970676000012 H 2.86257574000013 H 1.90320403000014 H 0.10390494000015 S —2.33917488000016 0 —3.59682316000017 0 —2.94534206000018 0 —1.68652257000019 H —2.371345840000END0.0490581300000.9238718700002.2672919900002.7414693000001.8665267300000.5233636000003.2117061400003.7850700400002.2284714000004.1707450500002.7811080800003.3394155600000.557946880000—0. 991976200000—0.6319433400000.172111760000—1.370258650000—1.821075810000—2.4457977500000.4713698700000.8573204200000.472526260000—0.280452020000—0.668321440000—0.2756739500000.873950130000—0.562655630000—1.2647002500001.1659462100001.7070147200000.0409801800001.4546161900000.751537950000—0.736751500000—1.5486047700000.670000590000—1.758960180000—2.010584760000GUIBONDS1 1 2 1.52 2 3 1.53 3 4 1.54 4 5 1.55 5 6 1.56 6 1 1.57 3 7 1.08 4 8 1.09 5 9 1.010 7 10 1.011 7 11 1.012 7 12 1.013 2 13 1.014 1 14 1.015 6 15 1.09916 15 16 1.017 15 17 1.018 15 18 1.019 18 19 1.0ENDCHARGE 0SYMMETRY NOSYM tol=0.001BASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20ENDSCFiterations 50converge 1.Oe—6 1.Oe—3mixing 0.2lshift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=l0.0ENDINTEGRATION 4.0 7 7A1FIT 10.0A1.8 ADF input file for ground state and time-dependentDFT calculations on 3a, ethylphenyl sulfone.TITLE TOSET LTUNITSlength Angstromangle DegreeENDModifyExcitationsUseOccupiedAlSubEndEndATOMS ZMATRIX1 C 0 0 0 0.0000000000000.000000000000 0.0000000000002 S 1 0 0 1.8162788851400.000000000000 0.0000000000001003C4C5C6C7C8C9H10 H11 H12 H13 H14 015 016 H17 H18 C19 H20 H21 HENDGUIBONDS1 3 5 1.52 5 6 1.53 6 7 1.54 7 8 1.55 8 4 1.56 4 3 1.57 9 5 1.08 10 6 1.09 11 7 1.010 12 8 1.011 13 4 1.012 2 3 1.013 14 2 2.014 15 2 2.015 1 18 1.016 1 16 1.017 1 17 1.018 18 19 1.019 18 20 1.020 18 21 1.021 1 2 1.0END106. 899856501000124.006129744000115.271556776000119. 412787694000120.251289779000119.986133910000119.857998085000119.580933997000120.034098705000120.255700276000121.239580671000108.344152765000106.519271414000106.785596764000100.583213043000115.165721573000110. 337319935000109.571867685000112.2726597920000.000000000000z4180.131305356000180.265759188000359. 6766858180000.1036095106580.038623816230179.663108224000180.049979527000180. 0034851660000.304966833300243.861518702000230.512938086000309.937580338000248.295464965000239.72136209300054.089810678900119.408268473000120.160925607000CHARGE 0.0GEOVARz4 270 360ENDBASIStype TZ2Pcore NoneENDXCGGAEND2 1 0 1.8032046346403 2 1 1.3973540885103 2 4 1.4007775678205 3 2 1.3935749091806 5 3 1.3989256760207 6 5 1.3951283650205 3 2 1.0888384822206 5 3 1.0893685478907 6 5 1.0895164676008 7 6 1.0892246601704 3 2 1.0883695316902 1 3 1.4598968035702 1 14 1.4600162975101 2 3 1.0976655879001 2 16 1.0998197091701 2 17 1.52507462733018 1 2 1.09650505037018 1 19 1.09859074787018 1 20 1.097122054340SYMMETRY NOSYM tol=0.001Bec]ce Perdew101GEOMETRYlineartransit 7iterations 30optim All Internalstep rad=0.15 angle=l0.0hessupd BFGSconverge e=1.Oe—3 grad=1.Oe—2 rad=1.Oe—2angle=0.5ENDSAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20ENDSCFiterations 50converge l.Oe—6 l.Oe—3mixing 0.2ishift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0cxx=i0.0ENDINTEGRATION 4 7 7A1FIT 10.0A1.9 ADF input filefor ground state andtime-dependent DFT calculationson Ib,methane sulfonyl chloride.TITLE MESO2CL S—KedgeUNITSlength Angstromangle DegreeENDATOMS1 C xl yl zl2 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z56 0 x6 y6 z67 0 x7 y7 z78 Cl x8 y8 z8ENDGEOVARxl 0.1262384994yl 0.3387232576zi 0.1027941566x2 —0.5022336713E—0ly2 —0.7203779344z2 —0.1223752814x3 1.136879736y3 0.6417103710102z3 —0.1854172113x4 —0.6408014542y4 0.9725787169z4 —0.3512016347x5 —0.1444290617E—O1y5 0.4759333329z5 1.887679646x6 1.092015671y6 —0.2175129557z6 2.505788982x7 —1.384380827y7 0.2318915910z7 2.276125538x8 0.3374298739y8 2.534150464z8 2.119821050ENDBASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSYMMETRY NOSYM tol=0.001SAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20ENDSCFiterations 50converge 1.Oe—6 1.0e-3mixing 0.2lshift 0.0diis n=10 ok=0.5 cyc=5cx=5.0 cxx=10.0ENDINTEGRATION 4 7 7A1FIT 10.0ModifyExcitationstJseOccupiedA2SubEndEnd103A1.1O ADF input file for ground state and time-dependent DFT calculations on 2b,methane sulfonate.TITLE MESO2oh S k-edgeModi fyExcitationsUs eOccupi edAlSubEndEndUNITSENDlength Angstromangle DegreeATOMS1C2H3H4H5S6070809HEND0.003598347641—0.1075230866990.929680712750—0.8715169869850.0846344184041.353420691120—1.1765592108200.1385692813501.0297090481700.257529142080—0.8316622499050.5783735639930.7547469544210.6700274037160.2246275808140.3178259500512.3036189602602.5623850264500.031130043395—0.022514917387—0. 456644480656—0.4000801809441.7703855459702.3166623317802.3760369949201.6901732548302.000187685390GUIBONDS1 1 2 1.02 1 3 1.03 1 4 1.04 1 5 1.05 5 6 2.06 5 7 2.07 5 8 1.08 8 9 1.0ENDCHARGE 0.0SYMMETRY NOSYM tol=0.00lBASIStype TZ2Pcore NoneENDXCGGAENDBecke PerdewSAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20END104SCFiterations 50converge 1.Oe—6 1.Oe-3mixing 0.2ishift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.DENDINTEGRATION 5 7 5A1FIT 10.0Al .11 ADF input file for ground state and time-dependent OFT calculations on modelfor 3b, dimethyl sulfone.TITLE MESO2MEUNITSlength Angstromangle DegreeENDATOMS1 C xl yl zi2 H x2 y2 z23 H x3 y3 z34 H x4 y4 z45 S x5 y5 z56 C x6 y6 z67 H x7 y7 z78 H x8 y8 z89 H x9 y9 z910 0 xlO ylO zlO11 0 xli yli zilENDGEOVARxi 0.2044050125E—01yl —0.9974534674E—01zi 0.1700119418E—01x2 0.9474393788E—01y2 —0.9907425984z2 —0.6171230463x3 0.2855590129y3 —0.3163664456z3 1.058590674x4 —0.9905850021y4 0.3205604226z4 —0.3825563074E—01x5 1.107101128y5 1.186052739z5 —0.6261950291x6 2.747257732y6 0.4456510507z6 —0.5275515740x7 3.428762148y7 1.192506370105z7 —0.9527077002x8 2.999641240yS 0.2546875808z8 0.5220608037x9 2.770874092y9 —0.4718597335z9 —1.126793044xlO 1.067924023yb 2.309656506zlO 0.3023560416xli 0.7877457037yli 1.372914110zil —2.036584888ENDBAS IStype TZ2Pcore NoneENDXCGGA Becke PerdewENDSYMMETRY NOSYM tol=0.001SAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20END5CRiterations 50converge i.Oe—6 i.Oe—3mixing 0.2ishift 0.0diis n=10 ok=D.5 cyc=5cx=5.O cxx=i0.DENDINTEGRATION 4 7 7A1FIT 10.0ModifyExcitationsUseOccupiedAlSubEndEnd106A1.12 Example of ADF input file for ASCF calculations on Ia, p-toluene sulfonylchloride. The same parameters and geometry was used as for the TO-OFT simulatedXAS spectra (A1.6—A1.11).TITLE T0sCL single point and skUNITSENDlength Angstromangle DegreeOccupationsA 2 1.5 94 0.5End000000000ATOMS1 Cl2S3c4C5C6C7C8c9C10 H11 H12 H13 H14 H15 H16 H17 018 0ENDGUIBONDS1 4 5 1.52 5 6 1.53 6 7 1.54 7 8 1.55 8 3 1.56 3 4 1.57 6 9 1.08 7 10 1.09 8 11 1.010 9 12 1.011 9 13 1.012 9 14 1.013 5 15 1.014 4 16 1.015 3 2 1.016 2 17 2.017 2 18 2.018 2 1 1.0END0.0000000000000.000000000000106.496853068000118.557279801000117.874299902000121.352353616000118.587827208000121.377518051000120. 876021882000119.608032709000121.505638841000111.392730897000111.560459348000110.467356737000119.050343813000120.544307863000107.426265138000107.6758095760000.0000000000000.0000000000000.000000000000270.000000000000178.725610588000359.0747618960000.374721592606359.630027239000181.516526858000179.311819726000179. 485630372000216. 941805491000121.386500522000119.548767898000178.717365091000357.343771086000114.941385738000129.994024837000CHARGE 0.0SYMMETRY NOSYM tol=0.001ZMATRIX0 0 0 0.0000000000001 0 0 2.3517421437702 1 0 1.7890428035903 2 1 1.3930410395104 3 2 1.3940847424205 4 3 1.4044687078206 5 4 1.4059264901607 6 5 1.3924766898406 5 4 1.5074095007107 6 5 1.0905771828508 7 6 1.0874680680809 6 5 1.0971142882109 6 12 1.0961173238409 6 13 1.1005109521105 4 3 1.0902852598704 3 2 1.0873823104502 1 3 1.4564614129902 1 17 1.456298745440107BASIStype TZ2Pcore NoneENDxcGGA Becke PerdewENDSAVE TAPE21 TAPE13SCFiterations 50converge 1.Oe—6 1.Oe-3mixing 0.2lshift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 5 7 7A1FIT 10.0A1.13 Example of ADFinput file for fragment analysis calculationson Ia, p-toluenesulfonyl chloride. The samegeometry was used as forthe TD-DFT simulated XASspectra (A1.6 — A1.1 1)# dependency: /home/kennepohl/vlad/fatoscl2b.clfatoscl2b.cl.t21t dependency: /home/kennepohl/vlad/fatoscl2b.so2 fatosci2b. so2 .t21* dependency: /home/kennepohl/vlad/fatoscl2b.tolufatoscl2b.tolu.t2141’! /bin/sh41’ The Molecule“$ADFBIN/adf” <<eorModifyExcitationsUseOccupiedA2SubEndEndTITLE TosCL single pointand skUNITSlength Angstromangle DegreeENDATOMS1 C —0.0005308775740.0515250454580.442496070463 f=tolu2 C 1.0282892512500.9144552749380.810372700763 f=tolu1083C4C5C6C7C8H9H10 H11 H12 H13 H14 H15 S16 017 018 CiEND1.010443793250—0 .074152325904—1. 107046027750—1.0578592897502.138166441250—0.115140542054—1.9373508255001.7634169062502.7199660152502.8272587432501.8586331662500.011061904906—2.435499990920—3.492687950070—2.693599655520—1.4800781225002.2697762523402.7438208833401.8950512243400.5544686775383.1919928673403.7962771053402.2601647848704.1824284033402.7858040263403.3377611483400.529916568838—0.994406982662—0.5708817293010.179634794787—1.481187979180—1.7891082975300.441239067363 f=toiu—0.313417372337 t=tolu—0.708044550237 f=toiu—0.319776804537 f=tolu0.830117228063 f=toiu—0.596035678637 f=toiu—1.281690789420 f=toiu1.117787076060 f=tolu1.665544860060 f=tolu—0.015277594327 f=toiu1.402674269060 f=toiu0.744985786363 f=toiu—0.675445186853 f=so2—1.317476475650 f=so20.418872444354 f=so2—2.181365072020 f=clGUIB0NDS1 1 2 1.52 2 3 1.53 3 4 1.54 4 5 1.55 5 6 1.56 6 1 1.57 3 7 1.08 4 8 1.09 5 9 1.010 7 10 1.011 7 11 1.012 7 12 1.013 2 13 1.014 1 14 1.015 6 15 1.016 15 16 2.017 15 17 2.018 15 18 1.0ENDCHARGE 0.0SYMMETRY NOSYM toi=0.001BASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDFragmentsci fatosci2b. ci. t21so2 fatosci2b. so2 . t21tolu fatoscl2b.toiu.t21endSAVE TAPE21 TAPE13SCF109iterations 50converge l.Oe—6 1.Oe—3mixing 0.2ishift 0.0diis n=10 ok=0.5 cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 4.0 4.0 4.0A1FIT 10.0eorA1.14 ADF input file for linear transitcalculation of Ia, p-toluenesulfonyl chloride.TITLE TosCL single point andskUNITSENDlength Angstromangle DegreeModi fyExcitationsUseOccupi edA2SubEndEndATOMS ZMATRIXid 02S 13C 24C 35C 46C 57C 68C 79C 61011 711H 812H 91311 914 H 9i5H 51611 4170 2180 2END0 0 0.0000000000000 0 2.1122123785101 0 1.7768124176702 1 1.3971240475403 2 1.3922832519104 3 1.4048035633005 4 1.4037711337106 5 1.3939189154705 4 1.5078024007406 5 1.0905123750507 6 1.0877198086006 5 1.0973393761306 12 1.0960928095606 13 1.1003585770804 3 1.0900285232403 2 1.0888563855201 3 1.4467638403401 17 1.4466526823700.0000000000000.000000000000101.791283537000119.314039695000118.703047905000121.275067833000118.481451871000121.271228463000120. 851499036000119. 621769687000121.346627364000111.360452967000111.525362136000110.564242778000119. 197779723000120.359365501000106.376924592000105. 3678527680000.0000000000000.0000000000000.000000000000z4182.0415042140000.642281384535359.979719236000359.183344159000180.770071385000178.992673533000179.882358502000219.215075202000121.260757225000119. 670736484000180.1737930420001.708737756580114.588104452000130.961738470000GUIBONDS1 4 5 1.52 5 6 1.53 6 7 1.54 7 8 1.55 8 3 1.56 3 4 1.57 6 9 1.08 7 10 1.09 8 11 1.010 9 12 1.011 9 13 1.011012 9 14 1.013 5 15 1.014 4 16 1.015 3 2 1.016 2 17 2.017 2 18 2.018 2 1 1.0ENDCHARGE 0.0GEOVARz4 270 360ENDSYMMETRY NOSYM tol=0.001BASIStype TZ2Pcore NoneENDXCGGA Becke PerdewENDGEOMETRYsmooth conservepointslineartransit 7iterations 30optim All Internalstep rad=0.15 angie=10.0hessupd BFGSconverge e=1.0e—3 grad=1.Oe—2rad=1.Oe—2 angle=0.5ENDSAVE TAPE21 TAPE13EXCITATIONDavidsonlowest 20ENDSCFiterations50converge 1.Oe-61.Oe-3mixing 0.2lshift 0.0diis n=10 ok=0.5cyc=5 cx=5.0 cxx=10.0ENDINTEGRATION 3.0 7 7A1FIT 10.0111

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