Mass spectrometric studies of distorted porphyrins : fragmentation pathways and ionization energies

160 Pages·1997·5.9 MB·English

by  Ferguson, James Allen

MASSSPECTROMETRICSTUDIESOFDISTORTEDPORPHYRINS:
FRAGMENTATIONPATHWAYSANDIONIZATIONENERGIES
By
JAMESALLENFERGUSON
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1997
ForSPTHB
ACKNOWLEDGMENTS
Thereareanumberofpeoplewhogavemeassistance,guidance,andsupport
whilecompletingthisdissertation;itismydesiretothankthemall. Thehumancondition
ofbeinglessthanperfectmeansthatIwillinevitablyforgetsomeone. IfIshouldinadver-
tentlyoverlooksomeoneintheseacknowledgments,Ihopetheywillforgivemetheslight
andknowthatIamtrulygrateful,ifforgetful. Thereareseveralindividualswhohave
definitelycontributedmoreassistancethanduty,friendship,orrelationshiprequire. The
wordswithwhichIvoicemythanksbelowcannotadequatelyexpressmyappreciation.
IammostdefinitelyindebtedtoProfessorRichardA.Yostforprovidingthein-
strumentationandtoolswithwhichtoperformthis(largelyunfunded)researchanda
groupinwhichindependenceandexcellentscienceareimportant. Thereare several
"PostYosties"andcurrentYostgroupmemberswhodeservespecialmention: Dr.Jodie
JohnsonforhispatienceinshowingmehowtomaketheTSQ70workwhenIfirststarted
tobeamassspectrometristanddidn'tknowonescanfunctionfromanother,andlaterfor
showingmehowtomaketheblastedthingworkwhenIthoughtIdidknow,butthe
instrumentwasnotbeingcooperative; Dr.UliBernierformanyofthesamereasonsand
forhisperennialoptimismandencouragement; Dr.JohnLaycockfortheinitialworkon
the 5-mononitrooctaethylporphyrinwhich ledto thisdissertationresearch; Mr. Scott
Quarmbyforhishelpwhentheelectronicsoftheinstrumentwereactingup; andthe
wholegroup,pastandpresent,fortheirhelp,encouragement,andfriendshipthroughout
mytimeatUFandforputtingupwithmyidiosyncrasiesandattentiontodetails.
ProfessorJ. MartinE. QuirkeofFloridaInternationalUniversityalsodeserves
bothhighpraiseandmydeepestgratitudeforhishelpandinterestinmywork. Inan
iii
averageweek,Martinhasenoughideasabouthowtousemassspectrometrytoanswer
questionsabouttheporphyrinstokeepanentireresearchgroupbusyforatleastayear.
WhateverquestionsIhadabouttheporphyrins,academicpoliticsandhowtoplaythe
game,pushingelectronsaroundtomakesenseoffragmentations,orlifeingeneral,Mar-
tinwastherewiththeanswer. I am especially appreciative ofhis weekendtripsto
Gainesvilleathisownpersonalexpensetoworklonghourswithmeonthisresearch. His
concernformygrowthasascientistandasapersonhavebeenagreathelpandsupportto
meandIlookforwardtocontinuingourfriendship.
IamthankfulforthehelpandencouragementoffriendsatHolyTrinityEpiscopal
Churchwhichprovidedmeanoutletformymusicaltalentsinthechoir(andpaidmeto
enjoymyselfdoingwhatIwouldhavedoneanyhow). Also,whenthechurchwasrebuilt
afterthefire,forprovidingamarvelousorganonwhichtopracticeand,whenthepres-
sureoftheacademicworldwastoogreat,toventsomeofmyfrustration. Thepeopleof
theChapeloftheIncarnation,especiallyDaveFrancoeurforhiringmeandBonnieRob-
ertsandNancyTiederman forbeing good listeners, alsodeserve mythanks fortheir
encouragementandsupportandforgivingmebothajobasorganistand,sinceAugust,
forprovidingmewithaplacetolive,rent-free. Asimplethankyoucannotexpressthe
thanksIowe.
The encouragement ofmy parents, siblings, grandparents, aunts, uncles, and
cousins(first,second,andhigher)inmypursuitofthisdegree,nomatterhowlongithas
taken,hasbeenuniformlysolid. MomandDadandmygrandparentstaughtmethevalue
ofhardworkandpersistence. MomandDadhavealwaysbeentheretolisten,admonish,
encourage,andsupportmeinmyeducationandcareer. Mysiblingshavealsohelpedme
getthroughthisworkbylisteningandbye-mail. Theloveofmyentirefamilyhasbeen
anever-presentcomfortandsupportnomatterwhat.
Othershavehelpedintoomanywaystomention. Pleaseacceptmyprofound
gratitudeandmythanks.
iv
TABLEOFCONTENTS
ACKNOWLEDGMENTS Hi
ABSTRACT vii
1 INTRODUCTION 1
Porphyrins 1
HistoryofPorphyrinResearch 3
PorphyrinsinAnalyticalChemistry 6
PorphyrinsinInorganic,Organic,andPhysicalChemistry 7
PorphyrinBiochemistry 10
PorphyrinsinGeochemistry 15
PorphyrinsinMedicine 18
PorphyrinsinMaterialsScience 19
DistortedPorphyrins 20
MassSpectrometry 22
TripleQuadrupoleMassSpectrometry 24
PorphyrinMassSpectrometry 29
ElectronIonization 30
ChemicalIonization 33
Thermospray 34
Electrospray 34
IonizationandAppearanceEnergies 35
Definitions 35
MethodsforDeterminingIonizationandAppearanceEnergies 38
OpticalSpectroscopy 38
PhotoelectronSpectroscopy 38
Ion/MoleculeEquilibriumConstantDeterminations 39
Photoelectron-PhotoionCoincidence 40
ElectronIonization 40
ScopeoftheDissertation 42
2 FRAGMENTATIONPATHWAYS 44
Introduction 44
MassSpectrometryofPorphyrins 48
InterpretingFragmentationPathways 49
Experimental 55
SamplesandSamplePreparationforAnalysis 55
MSOperatingConditions 55
v
5
ResultsandDiscussion 57
OEPandOEMNP 57
EI-MS 57
EI-MS/MS 63
HighResolutionEI-MS 73
StructuresandFragmentationPathwaysforSelectedIons 76
OtherNitroOEPs 81
EI-MSSpectra 81
SelectedEI-MS/MSSpectraandFragmentationPathways 85
Surface-InducedThermalDecomposition 94
SuppressionofP-CleavageofMethylGroups 98
Conclusions 98
3 IONIZATIONENERGYDETERMINATIONS 101
Background 101
Thermodynamics 101
IEDeterminationusingEI-MS: ProblemsandSolutions 102
Experimental 109
Samples 109
SamplePreparation Ill
MSOperatingConditions Ill
SampleHandling 115
SingleCompounds 11
PorphyrinMixtures 115
InternalStandardandCompoundExperiments 116
ResultsandDiscussion 116
SingleCompoundResults 117
MixedSampleResults 117
VariableLeakValveResults 119
Conclusions 123
4 CONCLUSIONSANDFUTUREWORK 125
FragmentationPathways 126
Ionization/AppearanceEnergies 127
FutureWork 127
DistortionversusElectronWithdrawingQuestion 128
OtherBiologicallyActiveTetrapyrroles 129
PorphyrinTypeIsomers 130
PorphyrinStructuralIsomers 132
AdditionalExperimentstoImprovetheReliabilityofIEDeterminations 135
REFERENCES 138
BIOGRAPHICALSKETCH 150
vi
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
MASSSPECTROMETRYSTUDIESOFDISTORTEDPORPHYRINS:
FRAGMENTATIONPATHWAYSANDIONIZATIONENERGIES
By
JamesAllenFerguson
May,1997
Chairman: Dr.RichardA.Yost
MajorDepartment: Chemistry
Thisworkdescribesnovelfragmentationpathwaysfortheentiresuiteofmeso-
nitrosubstitutedoctaethylporphyrins(OEPs)asdeterminedbyvariousmassspectrometric
techniques,aswellasdeterminationsofthefirstionizationenergiesofdivalentmetal
complexes ofOEP and the meso-nitrosubstituted OEPs under automated instrument
control.
Porphyrinmacrocycleswhichare stable intheirgroundandionizedstatesare
criticalinbiologicalfunctions. Fragmentationpathwaysresultinginnovelringscissions
ofthemeso-nitroOEPswereelucidated by acombinationofelectron ionizationmass
spectrometry (EI-MS), El-high resolution mass spectrometry (EI-HRMS), collision-
induceddissociation(CID)duringEl-tandemmassspectrometry(EI-MS/MS),andthe
useofisotopicallylabeledcompounds. Newpathwaysandplausibleexplanationsfor
fragmentationspreviouslynotedbutnotexplainedarepresentedandresultsarecompared
withpreviousworkon5-mononitroOEP[Laycocketal,1996]. Scissionsofthemono-,
vii
di-,andtrinitroOEPmacrocyclesresultingineliminationoftwopyrrolicrings,unusualin
EI-MSofporphyrins,wereobserved. Increasednitrosubstitutionwasseentoincrease
thelikelihoodofsurface-induceddissociationofnitrogroupsintheionsourceIncreased
nitrosubstitutionalsoresultedindecreasedP-cleavage,normallyobservedforsubstituted
porphyrins,andincreasedtendencytowardotherfragmentationpathways. Theseobser-
vationsindicateadestabilizationoftheseporphyrinmacrocyclesintheexcited(ionized)
state.
Threetechniquesfordeterminingionizationenergiesusingelectronionizationon
theFinniganMATTSQ70triplequadrupolemassspectrometerarepresentedandevalu-
ated. Theuseofavariableleakvalveforintroductionofastandardofknownionization
energyintotheionsourcesimultaneouslywiththeunknownsampleproducedIEswhich
agreed well with photoelectron spectroscopy results fordivalent metal complexes of
OEP. ThefirstIEsoftheNi(II)meso-nitroOEPswerefoundtocorrelatewiththeroot-
mean-squareout-of-planedistortionsofthemacrocycles,indicatingthatincreaseddistor-
tionoftheporphyrinsmayleadtoincreasedionizationenergy. Thedifficultyinseparat-
ingtheeffectofdistortiononionizationenergiesfromtheeffectoftheelectronwithdraw-
ingcharacterofthesubstituentisalsodiscussed.
Futurework,designedtoanswermanyofthequestionsraisedbyboththefrag-
mentationandionizationenergyresearchpresentedhere,ispresentedintheconcluding
chapter.
viii
CHAPTER
1
INTRODUCTION
Thisdissertationisareportontwoaspectsofmassspectrometricinvestigationsof
distortedporphyrins. Thefirstaspectistheelectronionization(EI)massspectraofthe
weso-nitrosubstitutedoctaethylporphyrins(OEPs)andtheircollision-induceddissociation
(CID)fragmentationpathways. Thesecondisthedeterminationofthefirstionization
energiesofmetallatedOEPsandtheweso-nitrosubstitutedOEPs. Theremainderofthis
chapterisdevotedtotopicswhichwillprovidegeneralbackgroundandpreviouswork
relevanttotheresearchpresentedinthisdissertation. Abriefexplanationofwhatpor-
phyrinsareandhowtheyaresignificantinseveralfieldswillbepresentedfollowedbya
briefintroductiontotriplequadrupoletandemmass spectrometryandthemass spec-
trometryofporphyrins. Thelastsectionofthischapter(ScopeoftheDissertation)will
includeanoverviewofthedissertationandputitintoperspectivewithpreviouswork.
Backgroundinformationspecifictothetopicscoveredinsubsequentchapterswillbe
presentedinthosechapters.
Porphyrins
'Whygrasseisgreene,orwhyourbloodisredaremysterieswhichnonehaue
reach'dvnto.' Thisstatementwasmadein1612bytheAnglicandivineJohnDonnein
his'SecondAnniuersarie',subtitled'OftheProgresoftheSoule' [Donne,1995]. The
questionsDonneraisedhavebeenansweredandtheanswersarechlorophyll(aporphyrin
analog)andhemoglobin,aporphyrin-containingprotein,respectively. However,there
arestillsomanyunansweredquestionsabouttheporphyrinsthatthefieldofporphyrin
1
2
researchhasprovedfruitful a
for many scientists. A
quickcheckofrecentlitera-
ture shows approximately
five thousand publications
everyyearforthelastsev-
eral years involving some
fundamental study of por-
phyrins or some practical
application ofthem. The
wordporphyrincomesfrom
M
theGreekwordforpurple, Figure • tPhoerpIhUinPeA,Cthneusmibmeprlienstgpfoorrpphoyrrpihny,risnhso.wing
7iop(()upa,andstemsfroma
purpledecompositionproductofhemoglobin. Theporphyrinsarehighlyconjugated,
aromatic,cyclictetrapyrroleswithfourone-carbonbridges(themesocarbons)linkingthe
fourpyrrolesubunits. Thesimplestporphyrin,porphine,alongwiththeIUPACnumber-
ingforporphyrins,isillustratedinFigure1-1. Thecarbonsinthepyrrolicringsadjacent
tothenitrogensaredesignatedtobeintheot-position,andtheperipheralcarbonsarein
P-pyrrolicpositions. Thefourpyrrolicsubunitsaresometimesdesignatedbytheindi-
catedletters(A-D). Thehighlevelofconjugationofthesp2carbonsleadstoaplanar
geometryforthemolecule.
Porphyrinsareimportantinmanyfieldsincludinganalytical,inorganic,organic,
and physical chemistry, biochemistry, geochemistry, medicine, and materials science.
Theyarethereactivecentersofseveralbiologicallyimportantmoleculesincludinghe-
moglobin,myoglobin,allofthecytochromes,andthechlorophylls(dihydroporphyrins).
Porphyrinanalogssuchasthecorrinsandchlorinsareimportantinbacterialsystems.