Chemiluminescences and degradations of b-lactam antibiotics after oxidation by potassium superoxide

228 Pages·1998·7.6 MB·English

by  Sun, Jingshun

THECHEMILUMINESCENCESANDDEGRADATIONSOFp-LACTAM
ANTIBIOTICSAFTEROXIDATIONBYPOTASSIUMSUPEROXIDE
By
JINGSHUNSUN
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1998
...J
Copyright1998
by
JingshunSun
Thisworkisdedicatedtomyparents,andmywife,ShuangYang.
ACKNOWLEDGMENTS
Iwishtooffermysincerethanksandgratitudetothechairmanofmycommittee,
mymentorDr.JohnH.Perrin,whosefriendship,supportandunendingpatiencehas
mademygraduatecareeraveryenjoyableexperience.
IthankallofthegraduatestudentsinMedicinalChemistrydepartmentfortheir
interestinmywork,suggestionsandfriendship.ThanksgotoDr.JamesWinefordner,
Dr.IanTebbett,andDr.RayBergeronfortheirenthusiasmandconsistentsupportofthis
project.SpecialthanksgotoDr.KennethSloanandDr.StephenSchuhnanfortheir
helpfiiladviceandfriendshipoverthelastseveralyears.
Iwouldliketothankmyfamilyfortheirloveandsupportespeciallymyparents;
theydeservemuchofthecreditformakingmethepersonIamtoday.ThankstoJan
KallmanandNancyRosawhohelpedtomakeFloridamyhome.Iwouldalsoliketo
extendmythankstoallfaculty,studentsandstuffatChemistrydepartmentwhose
assistanceisanindispensablepartofthisachievement.Lastly,Iwouldliketooffermy
thankstoShuangYangforherlove,patienceandperseverance.
IV
'
TABLEOFCONTENTS
; page
ACKNOWLEDGMENTS iv
USTOFTABLES : viii
LISTOFFIGURES x
ABSTRACT xvii
CHAPTERS
1 INTRODUCTION 1
ReactiveOxygenSpecies 1
SingletOxygen 1
SuperoxideAnion 4
OxygenToxicityandDiseases 6
RheumatoidArthritis 6
PulmonaryEmphysema 7
Ischemic-ReperfusionTissueInjury 8
Carcinogenesis 8
Aging 9
NeurodegenerativeDisorders 10
Chemotherapy 11
RemovalofFreeRadicalsbySuperoxideDismutaseandVitaminE 11
IncreasingFormationofFreeRadicals 12
ChemiluminescenceandP-LactamAntibiotics 14
p-LactamAntibiotics 14
Chemiluminescence 15
2 OBJECTIVES 32
3 CHEMILUMINESCENTMETHODOLOGYOFSTATICMODE 36
MaterialsandApparatus 36
ExperimentalMethods 37
PreparationsofStockSolutions 38
PreparationofLuminolSolution 38
PreparationofHydrogenPeroxideSolution 39
PreparationofSaturatedPotassiumSuperoxideSolution 39
PreparationofPhosphateBufferSolutions 39
4 THECHEMICALPROPERTIESOFPOTASSIUMSUPROXIDE 47
ComparisonStudies 47
SolventSelectionforPotassiumSuperoxide 48
5 CHEMILUMINESCENCESANDp-LACTAMANTIBIOTICS 54
Introduction 54
ProbeofMaximumEmissionWavelength 55
Discussion 55
6 MECHANISTICSTUDIES 65
TheDeuterationExperiment 65
NMRConsideration 69
NMRSpectraofPenicillinG 70
NMRSpectraofPenicillinV 72
Conclusion 74
TLCAnalysis 74
Introduction 74
Experimental 75
ResultsandDiscussions 76
HPLCDetection 78
Experimental 78
ResultsandDiscussion 80
Conclusion 80
HPLC/ESI-MSandHPLC/APCI-MSAnalysisoftheDegradationof
PenicillinGFollowingtheOxidationbyPotassiumSuperoxide 81
Introduction 81
Experimental 84
ResultsandDiscussion 85
7 SUMMARY 120
GLOSSARY ..:..... :.......:. 123
VI
APPENDICES
A 124
B '. 142
C 173
D ...! 186
E 194
REFERENCE 199
BIOGRAPHICALSKETCH 207
vii
LISTOFTABLES
Table page
Table1-1:Somedeleteriouseffectsofsystemsgeneratingthesuperoxideradical 28
Table1-2:Neurodegenerativedisordersassociatedwithfreeradicals 29
Table1-3:Familyofp-lactamantibiotics 30
Table1-4:Analyticalusefulchemiluminescentemitters 31
Table3-1:Chemicalsandreagentsutilizedinthestaticstudiesofchemiluminescence...41
Table3-2:Chemicalstructuresandsourcesofp-lactamantibioticsexamined 42
Table3-2~continued 43
—
Table3-2 continued 44
Table3-3:Thecompositionofphosphatebuffersolutions 45
Table6-1:Effectsofdeuteriumoxideontheintensitiesofchemiluminescenceof
penicillinG 92
Table6-2:TherespondsofsprayingthethreereagentsonsamplePGKOjandsample
KO2 92
Table6-3:TherunningtimeofTLCdevelopments 92
Table6-4:TheretentiontimesofthereferencecompoundsandsamplePGKO2 93
Table6-5:TheoperatingconditionsofHPLC 94
Table6-6:TheproductsofrecombinationincludingdimerizationafterpenicillinG
reactingwithpotassiumsuperoxide 95
Table6-7:ThemajorproductsofhydrolysisofpenicillinGafterinteractingwith
potassiumsuperoxide 96
viu
Table6-8:ThemajorproductsofoxidationofpenicillinGafterinteractingwith
potassiumsuperoxide 97
Table6-9:Thesixp-lactamantibioticsthatemitnochemiluminescence 98
Table6-10:ThesevenP-lactamantibioticsthatemitchemiluminescences 99
IX
LISTOFFIGURES
Figure page
Figure1-1:Bondinginthediatomicoxygenmolecule 19
Figure1-2:Potentialenergycurvesforthethreelow-lyingelectronicstatesof
moleculeoxygen 20
Figure1-3:Statecorrelationdiagramsforthereactionsofthethreelow-lyingstates
ofmoleculeoxygenwithadienetoproduceendoperoxideintriplet(T)and
singlet(S)states 21
Figure1-4:Possiblemechanismforformationofoxygenfreeradicalduringischemic
reperfusion 22
Figure1-5:Parentpenicillin 23
Figure1-6:Parentcephalosporins 24
Figure1-7:Oralbeta-lactamantibiotics 25
Figure1-8:Nonclassicalp-lactamantibiotics 26
Figure1-9:Reactionschemeofluminolbasedchemiluminescencedetection 27
Figure3-1:Schematicdiagramofsetupforchemiluminescentmeasurementinstatic
mode 46
Figure4-1:Thechemiluminescenceofluminolfollowingtheoxidationby(a)HjOj
and(b)K02 51
Figure4-2:ThechemiluminescenceofluminolreactingwithKOjinCH3OH.(a)at
startingtime,(b)twohourslater,(c)threehourslater 52
Figure4-3:Thecorrelationbetweentimeandthechemiluminescentintensitiesof
lcounmtianionlinafgteor.lreMpeaKtOi2ngitnhteoi2n.j5emctlioonfs1o0f"'5M0^l1uomfin1o:l1eCvHe3ryCN15-CmiHn3uOtHessolution 53
Figure5-1:ThecomparisonofchemiluminescentintensitiesofthirteenP-lactam
antibioticsfollowingtheoxidationbyKOj 58