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Towards the Synthesis of 1,4-Dibenzyl-1,4,7-Triazacyclononane-7-Monoacetate for a Potential Mimic of Oxalate Degrading E...

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004285/00001

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Title: Towards the Synthesis of 1,4-Dibenzyl-1,4,7-Triazacyclononane-7-Monoacetate for a Potential Mimic of Oxalate Degrading Enzymes
Physical Description: Book
Language: English
Creator: Liang, Alexandria
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2010
Publication Date: 2010

Subjects

Subjects / Keywords: Oxalate
Bioinorganic Chemistry
Ligand Synthesis
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate oxidase and oxalate decarboxylase are enzymes responsible for the degradation of oxalate. These enzymes are present in plants, bacteria and fungi. Consumption of oxalate, by both people and livestock, can result in cardiological and kidney disease. This is because neither contains oxalate degrading enzymes. Small molecule mimics of oxalate oxidase and oxalate decarboxylase have the potential to function as a tool for understanding the activity of the enzymes, testing for oxalate toxicity, probing the relationship between structure and function, and much more. An oxalate oxidase or oxalate decarboxylase mimic requires the synthesis of a ligand for complexation with a manganese ion. Based on previous work in the Sherman lab and with collaborators, the novel ligand 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate is a new target ligand. A variety of routes were attempted to synthesize this ligand. The most successful synthetic route required the attachment of benzyl groups to N-tosyl-1,4,7-triazacyclo onane. However, the deprotection of the 1,4-dibenzyl-7-tosyl-1,4,7- triazacyclononane proved to be very difficult. A ligand nearly resembling the target ligand by 1H and 13CNMR has been synthesized. However, peculiarities in the data suggest that this ligand is likely not 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate. The following work describes the methods used towards the synthesis of the intended novel ligand.
Statement of Responsibility: by Alexandria Liang
Thesis: Thesis (B.A.) -- New College of Florida, 2010
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Sherman, Suzanne

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Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2010 L69
System ID: NCFE004285:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004285/00001

Material Information

Title: Towards the Synthesis of 1,4-Dibenzyl-1,4,7-Triazacyclononane-7-Monoacetate for a Potential Mimic of Oxalate Degrading Enzymes
Physical Description: Book
Language: English
Creator: Liang, Alexandria
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2010
Publication Date: 2010

Subjects

Subjects / Keywords: Oxalate
Bioinorganic Chemistry
Ligand Synthesis
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate oxidase and oxalate decarboxylase are enzymes responsible for the degradation of oxalate. These enzymes are present in plants, bacteria and fungi. Consumption of oxalate, by both people and livestock, can result in cardiological and kidney disease. This is because neither contains oxalate degrading enzymes. Small molecule mimics of oxalate oxidase and oxalate decarboxylase have the potential to function as a tool for understanding the activity of the enzymes, testing for oxalate toxicity, probing the relationship between structure and function, and much more. An oxalate oxidase or oxalate decarboxylase mimic requires the synthesis of a ligand for complexation with a manganese ion. Based on previous work in the Sherman lab and with collaborators, the novel ligand 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate is a new target ligand. A variety of routes were attempted to synthesize this ligand. The most successful synthetic route required the attachment of benzyl groups to N-tosyl-1,4,7-triazacyclo onane. However, the deprotection of the 1,4-dibenzyl-7-tosyl-1,4,7- triazacyclononane proved to be very difficult. A ligand nearly resembling the target ligand by 1H and 13CNMR has been synthesized. However, peculiarities in the data suggest that this ligand is likely not 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate. The following work describes the methods used towards the synthesis of the intended novel ligand.
Statement of Responsibility: by Alexandria Liang
Thesis: Thesis (B.A.) -- New College of Florida, 2010
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Sherman, Suzanne

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2010 L69
System ID: NCFE004285:00001


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TOWARDS THE SYNTHESIS OF 1,4-DIBENZYL1,4,7-TRIAZACYCLONON ANE-7-MONOACETATE FOR A POTENTIAL MIMIC OF OXALATE DEGRADING ENZYMES BY ALEXANDRIA LIANG A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirement s for the degree of Bachelor of Arts Under the sponsorship of Dr. Suzanne Sherman Sarasota, Florida May, 2010

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ii Acknowledgments IwouldliketothanktheNewCollegeFoundation,theAlumniAssociationandtheCouncil ofAcademicAffairsforfunding.Iwouldalsoliketothankafewpeoplewhohavereally shapedmytimehereatNewCollege. Dr.PaulScudderwasmyadvisorduringmyrstyear.ItwashisOrganicChemistry coursethatrstconvincedmetopursuechemistry.SincebeginningresearchatNewCollege,Dr.Scudderhasbeenimmenselyhelpfulwithalloftheorganicsynthesistrialsthat Ihavecomeacross.Hisenthusiasmforteachingisinspirationaltostudents. Imetoneofmycommitteemembers,Dr.StevenShipman,duringmythirdyear. Hewatchedmeounderthroughphysicalchemistryandstillhelpedandsupportedme throughoutmylasttwoyearsatNewCollege.Additionally,heguidedmethroughmy graduateschoolapplicationsforwhichIwillalwaysbegrateful. Asachemistrystudent,youlearnthatsometimesthehardestthingtodoiscomeinto lab.IwouldliketothankKaitlinLoveringandErinnBrighamforbeingamazinglabmates, fordecoratingthelabandforbeingtherewhenthingsgotrough.Iwouldalsoliketothank DanielKaplan,whotookmeunderhiswingduringmysecondyear,showedmehowto useablastshield,forewarnedmeaboutthepitfallsofsynthesisandtaughtmethatlabis whatyoumakeofit. NextIwouldliketothankZekeBrustkernforsacricingmanyweekendsandlatenights tostayinthelabwithme.Itcanbehardtounderstandandsympathizewiththeupsand downsofchemistryresearchifyouaren'tinthelab,butZekehasbeentherethrougheach synthesis,goodandbad.Hisunderstandingandcompanionshipwerevitaltomyprogress inthelabandmymaturityasapersonduringthelasttwoyears. Finally,IwouldliketothankDr.SuzanneSherman,mythesisadvisor.Dr.Sherman hasbeenincrediblysupportivethroughoutmytimehere.Shehasbeenthepositivevoicein thebackofmyheadurgingmetopushthroughdifcultiesinthelab.EvenasIstruggled throughmythirdyear,shemadeanenormousefforttokeepmeengagedintheresearch.It wasthroughherguidancethatIfoundmypassionforsynthesisandlabwork.

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iii Contents ListofFiguresv ListofTablesviii 1Introduction1 1.1 GeneralInformation .............................1 1.2 OxalateOxidaseoxox ...........................2 1.3 Oxalatedecarboxylaseoxdc ........................13 1.4 ModelChemistry ..............................20 1.4.1 ManganeseChemistry .......................22 1.5 PreviouslyStudiedComplexes .......................23 1.5.1BerreauLabResearch........................23 1.5.2PecoraroandShermanLabResearch................25 1.6 ProposedComplex ..............................32 2Experimental37 2.1 RouteOne ..................................38 2.1.1 SynthesisofTosylbis[2-tosyloxyethyl]amine[ DEAts 3 ] .....38 2.1.2 Synthesisof1-Tosyl-1,4,7-triazacyclononane[ TACNts ] .....39 2.2 RouteTwo ..................................41 2.2.1 SynthesisofDiethylenetriamine-N,N',Ntristosylate[ DETAts 3 ] 42 2.2.2 Synthesisof1,2-Bistosyloxyethane[ EGOts 2 ] ..........43 2.2.3 Synthesisof1,4,7-tritosyl-1,4,7-triazacyclononane[ TACNts 3 ] ..43 2.2.4 RouteTwoA ............................45 2.2.5 RouteTwoB ............................47 3ResultsandDiscussion54 3.1 RouteOne ..................................58 3.2 RouteTwo ..................................60 3.2.1RouteTwoA.............................62 3.2.2 RouteTwoB ............................66

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iv 4Conclusion75 AAppendix:SpectraandPredictedSpectra77 Bibliography87

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v ListofFigures 1.1OxalicAcidProtonationStates........................1 1.2OxoxReaction................................2 1.3Oxox:HexamerStructure...........................3 1.4Oxox:JellyrollStructure...........................4 1.5Oxox:ManganeseFirstCoordinationSphere................6 1.6Oxox:SecondaryCoordinationSphere....................7 1.7Glycolate...................................11 1.8OxdcReaction................................13 1.9Oxdc:Structure................................14 1.10OxDC:ManganeseFirstCoordinationSphere................15 1.11Oxdc:SecondaryCoordinationSphere....................16 1.12X-rayCrystalStructure:OxdcwithOxalate.................20 1.13Bpppa.....................................24 1.14X-rayCrystalStructure:[bpppaMnCH 3 CNH 2 O]ClO 4 2 ........24 1.15X-rayCrystalStructure:[ f bpppaMn g 2 m )]TJ/F17 11.9552 Tf 9.289 0 Td [(C 2 O 4 ]ClO 4 2 ........25 1.16ShermanandPecoraro:PreviousLigands..................26 1.17X-rayCrystalStructure:[MnTCMAH 2 O]CF 3 SO 3 ............27 1.18X-rayCrystalStructure:[Mn i Pr 2 TCMAH 2 O 2 ]NO 3 ...........27 1.19X-raycrystalstructure:[MnBPZGH 2 O]NO 3 ...............28 1.20EPRSpectroscopy:Scarpellinietal.2008..................30 1.21CyclicVoltammetry:Scarpellinietal.2008.................31 1.22RejectedLigands...............................33 1.23[Bn 2 TCMA ].................................34 1.24[Ph 2 TCMA ].................................35 1.25ProposedLigandFamily...........................36 2.1QuickFilterunderNitrogen..........................46 2.2HBrWaterTrap................................48 3.1 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:HuangmethodcrudeTACNts.............59 3.2 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:TACNts 3 ........................61

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vi 3.3 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:TACN 3HCl.....................65 3.4 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:TACNalternateworkup................66 3.5 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:TACNtsproductfromHBr/AcOHandPhenoldetosylationofTACNts 3 ..............................69 3.6 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:Bn 2 TACNH......................70 3.7PredictedNMRSpectrum:Bn 2 TACNH...................70 3.8 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:Bn 2 TACNH......................71 3.9 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:CrudeFinalLigand..................73 3.10PredictedNMRSpectrum:Bn 2 TACNH...................73 3.11 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:CrudeFinalLigand..................74 A.1 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:DEAts 3 ........................78 A.2 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:HuangmethodpolymerTACNts...........79 A.3 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:DETAts 3 ........................80 A.4 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:DETAts 3 .......................81 A.5 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:EGOts 2 ........................82 A.6 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:EGOts 2 ........................83 A.7 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:StatisticalbenzylationofTACNts...........84 A.8 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:DibenzylationofTACNts...............85 A.9 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRSpectrum:DibenzylationofTACNts...............86

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vii ListofSchemes 1.1ProposedOxoxMechanism:Whittakeretal.2002..............8 1.2ProposedOxoxMechanism:Opaleyeetal.2005..............10 1.3ProposedOxoxMechanism:Whittakeretal.2007..............12 1.4ProposedOxdcMechanism:Richardsgroup2003..............17 1.5ProposedMechanism:OxoxandOxdc....................19 1.6[Ph 2 TCMA ]LigandSynthesis.......................35 2.1LigandSynthesis:RouteOne.........................38 2.2ProposedLigandSynthesis:RouteTwo...................41 3.1TCMASyntheticRoute:PreviousStudents.................55 3.2TCMPSyntheticRoute:EllenWolfgang...................56 3.3UnexploredSyntheticRoute1........................57 3.4UnexploredSyntheticRoute2........................57 3.5Huang:ProposedMechanism.........................58 3.6UnexploredSyntheticRoute3........................58 3.7LigandSynthesis:RouteOne.........................59 3.8ProposedLigandSynthesis:RouteTwo...................60 3.9RouteTwoA.................................62 3.10RouteTwoB.................................67

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viii ListofTables 3.1DetosylationMethods.............................63 3.2BenzylationMethods.............................68

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Towards the Synthesis of 1,4-Dibenzyl-1,4,7-Triazacyclononane-7monoacetate for a Potential Mimic of Oxalate Degrading Enzymes Alexandria Liang New College of Florida, 2010 Abstract Oxalate oxidase and oxalate decarboxylase are enzymes responsible for the degradation of oxala te. These enzymes are present in plants, bacteria and fungi. Consumption of oxalate, by both people and livestock, can result in cardiological and kidney disease. Th is is because neither contains oxalate degrading enzymes. Small molecule mi mics of oxalate oxidase and oxalate decarboxylase have the potential to function as a tool for understanding the activity of the enzymes, testing for oxalate toxicity, probing the relationship between structure and f unction, and much more. An oxalate oxidase or oxalate decarboxylase mimic requires the synthesis of a ligand for complexati on with a manganese ion. Based on previous work in the Sherman lab a nd with collaborators, the novel ligand 1,4-dibenzyl-1,4,7-triazacyclononane-7-m onoacetate is a new target ligand. A variety of routes were attempted to synthesize this ligand. The most successful synthetic route required the attachment of benzyl groups to Ntosyl-1,4,7-triazacyclononane. Howeve r, the deprotection of the 1,4-

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dibenzyl-7-tosyl-1,4,7-triazacyclononane proved to be very difficult. A ligand nearly resembling the target ligand by 1H and 13C-NMR has been synthesized. However, peculiarities in the data suggest that this ligand is likely not 1,4-dibenzyl-1,4,7-tri azacyclononane-7-monoacetate. The following work describes the methods used towards the synthesis of the intended novel ligand. Dr. Suzanne E. Sherman Thesis Sponsor Natural Sciences Academic Division

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1 Chapter1 Introduction 1.1GeneralInformation Oxalateisasmallmoleculeproducedinmanyorganisms,suchasplants,fungiandbacteria.Thisimportantdicarboxylicacid,featuredingure1.1,isinvolvedinthesignaling anddefensepathwaysofplants[1,2].Whileplantsarecapableofdegradingthisubiquitous Figure1.1. Oxalatehasthreepossibleprotonationstates.Fromlefttoright:oxalicacid pKa=1.27,singlydeprotonatedoxalicacidpka=4.28anddoublydeprotonatedoxalate. Thetermoxalatespecicallyreferstothedoublydeprotonatedformontheright,butis oftenusedtodescribeanyofthethreecompounds. compound,peopleandlivestockareincapableofefcientlymetabolizingoxalate.Thusthe consumptionofplantsrichinoxalatecanresultinkidneyandcardiologicaldiseases[3]. Unfortunately,suchplantsincludemanythatarecommonlyconsumed,suchasrhubarb, buckwheatandspinach[3].Theamountofoxalateinsomeplantsishighenoughtonoticewheneating.Anexampleofthisisthechalkyprecipitatethatformsinthemouthif onedrinksmilkaftereatingfoodrichinoxalatesuchasrhubarbpie.Thisprecipitateis

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2 calciumoxalate,theprimaryconstituentinkidneystones[4].Duetothetoxicityofthis smallmolecule,theenzymesthatdegradeoxalateareofchemical,biological,industrial andmedicinalinterest. Threeenzymesareresponsibleforthedegradationofoxalateinplants:oxalateoxidase, oxalatedecarboxylaseandoxalyl-CoAdecarboxylase.Whilebothoxalateoxidaseandoxalatedecarboxylasecontainmetalcofactors,oxalyl-CoAdecarboxylasedoesnot.Basedon theirsimilarity,thefocusofthisresearchisonoxalateoxidaseandoxalatedecarboxylase. 1.2OxalateOxidaseoxox Oxalateoxidaseoxoxismostcommoninhigherplants,suchasbarley,beets,riceand oats[2].Thisplantsubstituentisanoxalatedegradingenzymethatconsumesdioxygen duringcatalysis[5].Intheoverallreaction,oxoxconvertsoxalatetocarbondioxideand hydrogenperoxidethrougharedoxreaction,showningure1.2. Figure1.2. Theoverallreactioncatalyzedbyoxoxinvolvessinglyprotonatedoxalate, dioxygenandaproton.Oxalateisdegradedtoformhydrogenperoxideandtwocarbon dioxidemolecules. NativeoxoxshowsoptimalactivityatpH4.0[6].BasedonthepK a ,singlyprotonated oxalateistheactivesubstrateratherthanthefullyprotonatedorthedoublydeprotonated formsofoxalate.Whiletheoverallreactionconditionsareknown,theexactmechanismof oxoxhasnotbeenelucidated.Thisispartiallyduetotheinitialdifcultiesproducingthe recombinantenzyme.Recombinationofenzymesin Escherichiacoli isthemostcommon methodfortheexpressionofactiveenzymeinlaboratories.Enzymeobtainedthroughrecombinationcanoftenhaveloweractivitythannativeenzymes.However,thesetechniques allowbiochemiststomutateaminoacidresidues,overexpressproteinsandremovecofactorsorotherbiologicalmatterthatcanpurifywiththeenzyme.Unfortunately,oxoxisnot

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3 activewhenobtainedthrough E.coli .Itwasnotuntilnewermethodswereexploredthat recombinantoxoxwassuitableforenzymaticstudiesandsitedirectedmutagenesis[7]. Duetothisdifculty,progresstowardsmechanisticelucidationhasonlyrecentlybegun. However,basedonstudiesofwildtypeoxox,muchisnowknownaboutthestructureof theenzyme. Whilethenerarchitectureofoxoxvariesfromeachorganism,theoverallstructureis consistentthroughouttheorganismsstudiedthusfar[2].Oxalateoxidasebelongstothesuperfamilycupins.Thoughthissuperfamilyisdistinctiveinprimarystructure,theenzymes belongingtothisgrouparefunctionallydiverse.Thesuperfamilyincludesbothenzymatic andnon-enzymaticproteins.Moreover,thefunctionsofcupinsrangefromisomerases enzymesthatcatalyzeisomerizationtonon-enzymaticstorageproteinsinplants[8]. ThroughX-raycrystallography,ithasbeenestablishedthatoxoxformshexamers[9]. Thedimerswithinthesehexamersbindtightly.Thustheoverallstructureisreferredto Figure1.3. Theoverallarchitectureofoxoxisahexamercomposedofthreetightlybound dimers.Eachofthemonomers,distinguishedbyseparatecolors,containsthejellyrolldomainandamanganeseionshownasagreysphere.ThisgurewastakenfromSvedruzic etal.[2]. asatrimerofdimers.Thisarchitectureisshowningure1.3[2].Earlyenzymestudies revealedthatoxoxisextremelyheatstableandresistanttoproteasedegradation.Bothof thesefeaturesareoftenattributedtothistightlyboundquaternarystructure.

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4 Withineachmonomer,thereisaninterestingbetabarrelfoldthatdenesthesuperfamilycupins:thejellyrolldomain.Ajellyrollisanarchitecturalfeatureinwhichabetabarrel foldstocreateacavityinthecenterofaprotein,similartoajellyrollpastry.Withineachof thesepastrymotifs,thereisamanganeseioncoordinatedtoconservedaminoacidresidues. Themanganeseionsareboundtightlytotheproteinanddonotexchangeinsolution.The jellyrollmotifandmanganeseionareshowningure1.4. Figure1.4. Thejellyrolldomain,inred,ofbarleyoxoxcontainsamanganeseion,shown ingrey,buriedinthecenter.Thisdomainisacommonmotifinthesuperfamilycupins. ThisgurewastakenfromSvedruzicetal.[2]. EPRspectroscopyofrecombinantoxoxconrmsthatmanganeseismostlydivalent intherestingstate.However,roughly5%oftheenzymecontainsMnIII.Thissmall amountofMnIIIisoftendisregardedinmechanisticandstructuralstudies[6,7].When oxalateisaddedtooxoxatpH4.0,theEPRspectrumchangesdramatically.Thehyperne couplingofthemanganeseionincreases[7].Hypernecouplingoccursintwosituations: ithecouplingofthemagneticmomentoftheparamagneticelectrontoitsownnucleus oriithecouplingoftheunpairedelectrontothenucleusofadifferentatom.Becausethe hypernecouplingincreasesupontheadditionofoxalateandthemanganeseionsdonot dissociate[6],itislikelythatoxalateisbindingtotheMnII.Thusthemanganeseionsare theproposedsiteofcatalysis. Thisassertionisfurthersupportedbyearlierstudies.Requenaetal.demonstratedthat

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5 oxoxactivityiscorrelatedwithincreasingmanganesecontent[6].Additionally,asolution ofpermanganatewithasmallamountofMnIIwasfoundtoproducehydrogenperoxide andcarbondioxidewhenitreactswithoxalate.Asshownbelow,theactiveoxidationstate inthisreactionisMnIII[10]. MnC 2 O + 4 )497()496(! Mn 2 + + CO 2 + CO )]TJ/F79 8.9664 Tf 6.966 0 Td [( 2 CO )]TJ/F79 8.9664 Tf 6.967 0 Td [( 2 + Cl 2 )497()496(! CO 2 + Cl + Cl )]TJ/F17 11.9552 Tf -131.904 -27.716 Td [(Cl + Mn 2 + )497()496(! Cl )]TJ/F57 11.9552 Tf 9.125 -4.937 Td [(+ Mn 3 + Theredoxreactionobservedwithamanganesesalt,oxalateandchlorineshowsthatin thepresenceofMnIII,oxalatecanbedegradedtoaformylradicalanionshowninthe rststep.Subsequently,thisanionisoxidizedtocarbondioxidebychlorine.Theatomic chlorineoxidizestheMnIIiongeneratedintherststeptoformMnIII.Thereaction shownaboveisofconsiderableinterestbasedonthemechanismsofoxoxandoxdc.This schemewastakenfromBasoloandPearson[11].Althoughthisislessdirectevidence fortheenzymecatalysis,itclearlydemonstratesthatamanganeseionhasthepotentialto catalyzetheoxidativedegradationofoxalate. Duetothecatalyticroleofthemanganeseioninoxox,thegeometryandcomposition aroundthemetalareacriticalaspectofoxoxfunction.Themononuclearmanganeseion adoptsadistortedoctahedralcoordinationsphere,showningure1.5.Intherestingstate, thecoordinationspherecontainsthreehistidineresidues,oneglutamateresidueandtwo watermolecules.Theaminoacidsofthecoordinationspherebindthemanganeseion verytightlytotheenzyme.Eventhroughdialysis,itisoftenverydifculttoremovethe manganeseions[6,13].However,thewatermoleculesboundtothemetalarelabile.During catalysis,atleastoneofthetwowatermoleculesarepredictedtobedisplacedbyoxalate andpossiblydioxygen. Thecompositionoftherstcoordinationsphereiscriticaltoenzymecatalysis.The coordinatedaminoacidsareresponsibleforstabilizingtheoxidationstateandthechange inoxidationstateinthecatalyticmechanism.Oftentimes,thecoordinationenvironment changestheredoxpotentialoftheboundmetalbydonatingorwithdrawingelectrondensityfromthemetal.Thisisthecaseforoxox.ThepotentialfortheMnIII/MnIIreductionintheenzymeiscalculatedtobebetween+0.4and+1.0Vvs.thenormalhydrogen

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6 Figure1.5. Inoxox,themanganesecoordinationsphereisinadistortedoctahedralgeometry.Thesixcoordinationsitesarelledbythreehistidineresidues,onemonodentate glutamateresidueandtwowatermolecules.ThisgurewastakenfromScarpelliniet al.[12]. electrode[14].Fortheaquatedmetalionthispotentialisdramaticallyhigher,+1.5Vvs. thenormalhydrogenelectrode.Thecoordinationenvironmentaroundthemetalisableto lowerthepotentialinordertoaidincatalysis.Thenitrogenatomsofthethreehistidine residuesandthenegativelychargedoxygenatomofthecarboxylatedonateelectrondensity tothemanganeseion.Thisenvironmentisresponsibleformuchoftheredoxactivityand catalyticturnoverintheenzyme. Althoughitisfurtherremovedfromtheactivesite,thesecondcoordinationsphereis alsoimportanttoboththestructureandfunctionoftheenzyme.Thecationicandanionicresiduesofthesecondarycoordinationspherefrequentlystabilizetheintermediates formedduringcatalysis.Mutationsinsecondcoordinationsphereresiduescanoftenalter orcompletelyinhibittheactivityofanenzyme.Ingure1.6,theresiduesofthesecond coordinationsphereareincludedwiththeresiduesboundtothemanganeseion.Ithas beenfoundthatmutationsineitheroftheasparagineresiduesofthesecondarycoordinationspherearedetrimentaltotheenzymeactivity.WheneitherAsn 75 orAsn 85 were mutatedtoalanine,theactivitywasreducedto2.4and0.5%fromthenativeenzyme,respectively[15].Thustheseasparagineresidues,whichcontainprotonscapableofforming

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7 Figure1.6. Thesecondarycoordinationsphereofoxoxcontainsseveralresidues:one phenylalanine,oneglutamine,andtwoasparagineresidues.Thesecondasparagineresidue isnotshowninthisgureitwouldbelocatedabovetheGln-139residueinpositionto stabilizeoneendofaboundoxalatemolecule.Boththeglutamineandtheasparagine residuesarehydrogenbonddonors.Thusthenegativelychargedintermediatescanbestabilizedbythesurroundingenvironment.Atomsarecoloredasfollows:darkgrey,carbon; red,oxygen;blue,nitrogen;lightgrey,MnIIorhydrogens.Thisgurewastakenfrom Svedruzicetal.[2]. hydrogenbonds,mustbeinvolvedinthecatalyticmechanism.Basedonthisevidence, manycatalyticmechanismsproposedforoxoxshowtheasparagineresiduesstabilizingthe negativelychargedintermediates. Thoughseveralmechanismshavebeenproposed,nonehasbeenagreedupon.Most havebeenproposedbythegroupofJamesW.Whittaker.Tworepresentativemechanisms, showninschemes1.1and1.2,depicttheuncertaintyinthebindingmodesandtimingof attack. Themechanisminscheme1.1wasproposedbytheWhittakergroupin2002[7].The mostdistinctivefeaturesofthismechanismaretheinteractionbetweenmanganese,oxalate andoxygen,theredoxcycleandtheformylradicalanionproduced.Intherstframeof thisproposedmechanism,monodentateoxalateisshownboundtoMnII.Theoxalatehas

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8 Scheme1.1. AboveisthemechanismproposedbyWhittakeretal.in2002.ThemechanismbeginswiththeoxalateboundtoMnII.Thedioxygenmoleculeisnotbound.When MnIIisoxidizedtoMnIII,theunboundoxygenisreducedtoasuperoxideradical.With thelossofcarbondioxide,MnIIIisreducedtoMnII.Thenewformylradicalanion isquenchedbythehydroperoxyradicaltoformpercarbonicacid.Thepercarbonicacid iscleavedtoreleasecarbondioxideandhydrogenperoxide.Thisgurewastakenfrom Svedruzicetal.[2]. displacedoneofthewatermolecules.However,thesecondwatermoleculeisnotdisplaced bytheincomingdioxygen.Insteadthedioxygenmovestowardstheactivesiteobtaining aprotonandanelectronbyoxidizingMnIItoMnIII.Thereductionofdioxygenis coupledwithaprotontransferfromtheacidicwatertothenewlyreduceddioxygen,which formstheunboundhydroperoxyradical.Inthefollowingstep,MnIIIisreducedtoMnII. Thisreductionisaccomplishedbythecleavageofoxalatetoreleasecarbondioxide.The remainingformylradicalanioncombineswiththehydroperoxyradicaltoformpercarbonic acidboundtoMnII,showninthebottomrightcorner.Thepercarbonicacidisthen brokenintohydrogenperoxideandcarbondioxideuponreleasefromtheMnIIion.The nalresultistheregenerationofaboundhydroxylgroupinsteadofasecondboundwater molecule.Thisspeciescanbeprotonatedtocompletethecycle. Evidencefortheoxidationstatewasestablishedthroughopticalabsorptionspectroscopy. MnIIIenzymecontentwasalsofoundtobepresentat5%abundance.TheMnIIIwas

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9 proposedtoreactwithoxalateinanon-catalyticreaction.Theformationoftheformyl radicalanionwasproposedbasedonspintrappingexperimentswhichemployedEPRas themethodofradicaldetection.Nootherradicalswerefoundwhichindicatesthateither thehydroperoxyradicalpredictedmustbequenchedextremelyrapidlyorisboundtightly totheenzyme.Basedonthisevidence,itseemsoddthattheproposaldoesnotsuggestthat dioxygenisbounddirectlytothemetalduringcatalysis.Moreover,theassertionthatoxalateismonodentatehasnoevidentiarysupportinthepaper.Itismorefeasibletoenvision oxalateasabidentateligandratherthanamonodentateligand.Someofthesefeaturesare addressedinthesubsequentmechanismsproposedbytheWhittakergroupvideinfra. Whenassessingmechanismsininorganicchemistry,itisimportanttonotethatligands areoftendepictedwithoutachargethatonewouldexpect.Asageneralguide,negatively chargedligandsareoftenshownasneutralwhenboundtoametalcenter.Inthemechanism showninscheme1.1,alldeprotonatedoxygenatomscoordinatedtoametalarenegatively charged.However,thisnegativechargeisnotshownbyconventionininorganicchemistry. Subsequentmechanisticschemesalsofollowthisinorganicconvention. Scheme1.2showsaverydifferentmechanismproposedbytheWhittakergroupin 2005[15].Unlikethepreviousmechanism,thereisnounboundhydroperoxyradicalformation.Thelackofthisfreeradicalseemstobeadramaticimprovementoverthe2002 mechanism.However,therearesomecleardrawbackstothisproposal.Intherststep, monodentateoxalatebindstoMnIIdisplacingonewatermolecule.Thisisfollowedby thedirectadditionofdioxygenandtheoxidationofMnIItoMnIII.Thesuperoxide radicalisnowboundtoMnIII.Thissuperoxideradicalattacksthenearestsp 2 carbonto formavememberedchelateringwithanattachedoxygenradical.Stepveshowsthe reductionofMnIIItoMnIIviathecleavageofcarbondioxidefromthesubstratering. Thefollowingstepisunclear,butproduceshydrogenperoxideandcarbondioxide. InresearchfromtheWhittakerlabin2005,evidenceformonodentatecoordination ofoxalateisbasedonX-raycrystallographyoftheenzymewithglycolateboundtothe manganeseoftheactivesite.Glycolateisshowningure1.7.Althoughglycolateissimilar tooxalate,theoxalateshouldbeabetterchelatingagentthanglycolatebasedontheacidity ofthehydrogenatoms.Becauseofthis,theconclusionthatoxalateismonodentateseems suspect.Themostimportantoutcomeofthisresearchwasthediscoveredsignicanceofthe

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10 Scheme1.2. ThemechanismproposedbyOpaleyeetal.in2005.Itisstarklydifferent fromthepreviousmechanismbecausenounboundradicalisformedhere.Thismechanism alsobeginswithMnII.However,afteroxalatebindstotheMnIIion,dioxygenbinds aswell,oxidizingthemetaltoMnIII.Thesuperoxideradicalformedattacksthebound oxalatetoformachelatering.Adisproportionationreactionoccursinstepvetoresultin carbondioxidereleaseandreductionofMnIIItoMnII.Percarbonicacidremainsbound toMnII.Stepsixisunclear,butresultsinthereleaseofhydrogenperoxideandcarbon dioxide.Thelledincirclesrepresentthewatermoleculespresentintherestingstate.This gurewastakenfromOpaleyeetal.[15]. Asn 75 andAsn 85 residues.Mutationsintheseresiduessignicantlydecreasetheactivity oftheenzymevidesupra.Duetothisnding,itisproposedthatthereisnoformylradical anionintermediate.Itisunclearhowthisnewevidenceexcludestheintermediate,butthis isonlythebeginningofthecontradictionsnotaddressedbetweendifferingmechanisms. However,ifCO 2 andaprotonwerelostbeforestepthreeinthe2005mechanism,aformyl radicalanionwouldhaveformed.Thusitispossiblethatsomefusionofthisproposaland thepreviousproposalisclosetotheactualmechanism. Fromthesetwoproposedmechanisms,itisevidentthatmuchoftheactualenzyme mechanismisunclear.Moreover,muchoftheinformationseemstostandonlessthan solidevidenceexample:bidentatevs.monodentateoxalatecoordination.However,itis certainthatthereactionisdependentondioxygenbasedontheworkofDr.Shimazono

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11 Figure1.7. Glycolate,shownabove,isoftenusedtomimicoxalate.However,theproton onthealcoholgroupofglycolateisnotasacidicastheprotonofmonoprotonatedoxalate. Duetothesedifferences,theuseofglycolateasananalogofoxalatemaynotbeideal. inthemidtolate50s[5].Themanganeseionisalsoestablishedastheactivesitebased ontheEPRspectroscopymentionedabove.Additionally,itismostcommonlyagreed uponthatthemanganeseionundergoesaredoxreaction,changingfromMnIItoMnIII duringthecatalysis.ThisconclusionwasbasedonEPRspectroscopyofbothnative[6] andrecombinant[7]enzymewhenincubatedwithoxalate. Thatbeingsaid,in2007,theWhittakergrouppublisheddatasuggestingthattheresting stateofthemanganeseionwasMnIIIinsteadofthemorewidelybelievedMnII.The mechanisticproposalthatresultedfromthisresearchisshowninscheme1.3.Thesteps throughoutthemechanismaresimilar,buttheoxidationstatecyclesfromtherestingstate MnIIId 4 ,highspin,JahnTellerdistortiontothereducedMnIId 5 ,highspin,roughly octahedral.Thisproposalseemsintriguingbasedontheknownreactionofpotassium permanganatewithoxalate,inwhichMnIIIisthecatalyticallyactivespecies[10]. Intheresearchthatproducedthisproposal,Whittakeretal.studiedthreesetsofrecombinantoxoxeachcontainingonlyoneofthefollowingoxidationstates:MnIV,MnIII andMnII.Fromburstkineticsexperiments,opticalabsorptionspectroscopyandEPR spectroscopy,itwasdeterminedthatoxoxcontainingeitherMnIVorMnIIIareboth activespeciesfordioxygendependentdegradationofoxalate.However,enzymeswith onlyMnIIwerenotabletocatalyzethisreaction.Duetoenzymestabilityandprevious researchofmanganesecontainingenzymes,MnIIIwasdeemedthebiologicallyrelevant species[14].Thisresearchseemsthoroughandconvincing.Itwasdenitivelyestablished thatassayscontainedenzymeswithuniformmanganeseoxidationstatesratherthanthe 95%MnIIand5%MnIIImixtureseeninotherexperiments.Additionally,thelackof catalyticactivityinassayswithonlyMnIIcontainingenzymesuggeststhatitwouldbe impossibleforthereactiontocyclefromMnIItoMnIII.

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12 Scheme1.3. Thismechanismisthemostuniquepublishedintheliterature.Whatismost notablydifferentfromothermechanisticproposalsistheoxidationstatesofthemanganese ion.TheactiveoxidationstateisproposedtobeMnIII.Instepone,oxalatebindsto MnIIIinamonodentateorbidentateshowninparenthesescoordination.Beforeoxygen enterstheactivesite,oxalate reduces MnIIItoMnII.Thisresultsinthereleaseofcarbon dioxideandtheformationofaboundformylradicalanion.Thendioxygenquenchesthe radicalaniontoformasecondcarbondioxidemoleculeandaprotonated,hydroperoxy radicalshownunbound.TheMnIIisoxidizedtoMnIIIbytheradical,toformthe initialcomplexandhydrogenperoxide.Theopencirclesrepresentwatermolecules;the lledcirclesrepresentwatermoleculesthathavecoordinatedinthemostrecentstep.This gurewastakenfromWhittakeretal.[14]. DespitetheconvincingevidencepresentedbyWhittakeretal.intheresearchfrom 2007,allotherresearchisbasedonMnIIactivityinoxox.Moreover,muchofthisresearchwasconductedbythesameresearchgroup.Frustratingly,theWhittakergroupdid notaddressthediscrepanciesbetweenthismechanism,the2005mechanism,andthe2002 mechanism,allofwhichoriginatedfromtheWhittakerlabwithinaveyearspan.Despite that,itisclearfromthisnewresearchthattherestingstateofmanganeseionsinoxox ismuchlesscertainthanpreviouslythought.Duetothe5%MnIIIcompositioninpreviousrecombinantoxoxstudies,itisfeasiblethatthisimportantmechanisticfeaturehad beenpreviouslyoverlooked.Onlyonesubsequentpaperhasaddressedtheissue:laterin

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13 2007,theBornemannlabmaintainedthattheMnIIIoxidationstateisnotalikelystartingpointforthereactionbasedonthemechanismshowninscheme1.5videinfra[13]. NopaperfromtheWhittakergroupnoranyotherresearchgrouphassupportedtheir2007 mechanisticproposalasofspring2010. Basedonthelackofagreementbetweenthesemechanisms,itisevidentthatthereare manyaspectsofoxoxstructureandactivitythatarenotclearlyunderstood:thecoordination ofoxalatemonovs.bidentate,roleofdioxygencoordinationtomanganeseoradjacency tometalandtheoxidationstateofthemetalareamongthemainaspectsstillinquestion. Muchworkislefttodoinordertoexplorethedetailsofenzymecatalysis. 1.3Oxalatedecarboxylaseoxdc Oxalatedecarboxylateoxdccatalyzesthedecompositionofoxalateintoformateand carbondioxide,showningure1.8.Whileoxoxandoxdcbothdegradeoxalate,thedistinctioninproductssuggeststhattheenzymessupportdifferentfunctionsinorganisms. Likeoxox,oxdcisdioxygendependent[16].However,inoxdc,thedioxygenisonlya Figure1.8. Theoverallreactioncatalyzedbyoxdcinvolvessinglyprotonatedoxalateand catalyticdioxygen.Oxalateiscleavedtoformformateandcarbondioxide. cofactor.Thusthecarbonatomsofoxalatedonotundergoanetchangeinoxidationstate toformthenalproduct.Additionally,thecatalyticroleofoxygenmeansthatoxygenis notconsumedbythereaction. Unlikeoxox,oxdcismostcommoninfungi.Earlysequenceanalysissuggestedthat oxdccontainedthemanganesebindingsequenceofoxoxintwodifferentlocations.This curioussimilaritybetweenthetwoenzymesleadtoearlyspeculationthatthetwowere evolutionarilyrelated.Eventually,crystallographicstructuresconrmedthatoneoxdc monomercontainstwooxoxjellyrolldomains.Thenatureoftheirsimilarityinbothstruc-

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14 tureandfunctionindicatesthatoxoxandoxdcareprobablyevolutionarilyrelatedthrough genedeletionorduplication[2]. Thequaternarystructureofoxdcisalsosimilartothatofoxox.Theoverallgeometry Figure1.9. Oxdcisverysimilarinstructuretooxox,exceptthateachmonomerofoxox appearstwiceinthequaternarystructure.A:Thegureonlyshowsoneofthetwohomotrimersthatcompriseoxdc.Thesecondhomotrimerwouldstackontopoftherst, sothatthetwofacesintheplaneofthepagealignedwithoneanother.B:Thereare twojellyrolldomainsineachmonomer.Thustherearetwomanganeseionspermonomer. Whetherornoteachsiteiscatalyticallyactiveremainsacurrenttopicinresearch.This gurewastakenfromSvedruzicetal.[2]. ofoxdcisasetoftwohomotrimers,packedtogethertoformahexamer.Becauseeach monomerofoxdcresemblesadimerofoxox,theresultofthisstructureiswhatlookslike twohexamersofoxox.Similarlytooxox,thejellyrolldomainsofoxdcarecapableof bindingmanganeseions.Thejellyrolldomainsandoncefaceoftheoxdchexamerare showningure1.9. Becauseoxdchastwojellyrolldomainsineachmonomer,therearetwopotentialbindingsitesformanganesewithineachofthemonomers.ThesearereferredtoastheCterminalbindingsiteandtheN-terminalbindingsite.Bothsiteshavethesamerstcoordinationsphere,showningure1.10.Theaminoacidresiduescoordinatedtothemanganese ioninoxdcarethesametypeasthosecoordinatedtothemanganeseioninoxox:threehistidineresidues,oneglutamateresidueandtwowatermolecules.Additionally,thehistidine residuescoordinatefacially,andthetwowatermoleculesoccupy cis positions.Fromthis

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15 Figure1.10. TheN-terminalcoordinationsphereofoxdc,shownabove,isverysimilarto thecoordinationsphereforoxox.Eachmanganeseionisboundtothreehistidineresidues andoneglutamateresidue.Inthegureshownhere,thetworemainingsitesintheoctahedralcoordinationarelledbyawatermoleculeandamoleculeofformate.Theformateion wasarticiallyaddedforX-raycrystallography;inthebiologicalenzyme,awatermolecule wouldoccupyeachofthesolventsites.ThisgurewastakenfromScarpellinietal.[12]. smallpicture,itisdifculttoseewhyoxoxandoxdccatalyzedifferentreactions.Thuswe mustmoveoutwardsandexaminethesecondcoordinationsphere. Thesecondcoordinationspheresofthemanganeseionsinoxdchavesomesignicant differencesfromthatofoxox.Moreover,therearesomedifferencesbetweenthetwomanganesebindingsitespresentineachoxdcmonomer.Figure1.11showstheN-terminal leftandC-terminalrightmanganesebindingsites.Glutamateresidues162and333are orienteddifferentlytowardsthelabilewatermolecules.Additionally,twophenylalanine residuesoftheN-terminalsitearereplacedbyatyrosineresidueandavalineresiduein theC-terminalsite.Althoughthesechangesaresmall,itispossiblethattheyresultinvery differentchemicalactivities. Whencomparingthesesecondcoordinationsphereswiththecoordinationsphereof oxox,themostnotabledistinctionisthepresenceofthearginineresiduesand270 andtheglutamateresiduesand333inoxdc.Figure1.6showsthatinsteadofthese residues,oxoxcontainsanasparagineresidueandaglutamineresidue.Anarginineresidue wouldbeexpectedtobeabetterhydrogenbonddonorthananasparagineresidue.But

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16 Figure1.11. Thesecondarycoordinationsphereofoxdcissimilartothatofoxox.It containsonephenylalanineresidueandonearginineresidue.TheN-terminalmanganese bindingsiteontheleftisoftenproposedastheprimarycatalyticcite.TheC-terminal siteontherightmaintainsacontroversialroleincatalysisandisoftenreportedtobe purelystructural.Oxdccontainsconservedglutamate-162andglutamate-333residues.It isproposedthattheseglutamateresiduesareresponsibleforthedifferenceincatalytic activityofoxoxandoxdc.ThegurewastakenfromMoomawetal.[17]. moreimportantly,aglutamateresidueiscapableofacid/basecatalysis.Glutaminedoes nothavethisability.Itisreasonabletospeculatethatthedifferencesinthesecondary coordinationspherescontributetothedifferencesincatalyticactivity. Althoughbothofthemanganesebindingsitesofoxdcarechemicallysimilar,thereis muchdebateastowhetherbothactivesitesarelledandfunctionalincatalysis.Because ofthesimilarity,spectroscopicanalysisisdifcult.However,sincethedevelopmentof recombinantoxdc,mutagenesisstudieshaveshedlightonthetopic. IthasbeenfairlywellestablishedthattheN-terminalmanganeseionisresponsible forcatalysis[13,18].However,questionsastotheroleoftheC-terminalmanganeseion withrespecttobothstructureandfunctionarestillhighlydebated.Themostpopular conclusionisthattheC-terminalresidueplaysastructuralroleanddoesnotparticipate incatalyticturnover.However,in2009,NigelRichards'groupestablishedthatthereisa linearrelationshipbetweenmanganeseincorporationandoxdcactivityfromlessthan0.1

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17 to1.9Mn/monomer[17].Thiswouldonlybeexpectedifbothmanganesebindingsites werecatalyticallyviable. Moreover,thereisnoagreementastotheoxidationstateofthecoordinatedmanganese.SpeculationhassuggestedthattheenzymecyclesbetweeneitherMnII/MnIII orMnIII/MnIV[2].However,todate,noconclusiveevidencehasbeenestablishedfor eitherofthesehypotheses.Becausethisisacriticalfeatureofcatalysis,itisimportantthat moreresearchexploretheredoxcycleofmanganeseinoxdc. Althoughtherearefarfewermechanismsproposedforoxdcthanoxox,therearesome. In2003,theRichardsgroupproposedamechanismforoxdcbasedondatacomparingthe rateofreactionofoxalatewithanaturalabundanceofheavyisotopes 13 Cand 18 O.The Scheme1.4. ProposedbyReinhardtetal.in2003,thisreactiondepictstheactivityofoxdc. InA,MnIIIisshownboundtooxalateandsuperoxide.Areversibleprotonabstraction occursthroughtheconservedGlu-333residue.Atthesametime,anelectronistransferred fromoxalatetoMnIII.Thenegativelychargedoxalatethenlosescarbondioxidetoform aformylradicalanionthisissimilartotheintermediateformedinWhittaker's2007mechanismforoxox.ThiscarbenedeprotonatestheGlu-333residueandremovesoneelectron fromMnIItocreateformateboundtoMnIII.Theformateanionislosttoregeneratethe catalyst.ThisgurewastakenfromReinhardtetal.[19]. reactiondeterminedtheamountofheavyisotopesinoxalatebeforeincubationwith

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18 oxdc,CO 2 producedduringincubationofoxalatewithoxdc,formateproducedduringincubationandunreactedoxalatefromincubation.Fromthedata,itwasdetermined thatanon-isotopesensitive,reversiblestepoccursbeforeCO 2 release.Additionally,the CO 2 releasedisspecicallyfromtheprotonatedendoftheboundoxalatemolecule.The mechanismshowninscheme1.4wasproposedbasedonthecollecteddata. Theconservedarginineandglutamateresiduesinthesecondcoordinationsphereare shownascriticalfeaturesofcatalysis.InA,oxalateisshownboundtoMnIIIwithmonodentatecoordination,andsuperoxideisboundtotheopen cis siteonthemanganeseion. Thearginineresidueisstabilizingtheelectrondenseoxygenofthecoordinatedcarboxylate.Theglutamateresidueabstractstheprotonfromthemonoprotonatedoxalate,forming doublydeprotonatedoxalate.Concurrently,oxalatetransfersanelectrontoMnIIItoform MnII.Fromthisintermediate,carbondioxideiscleavedtoformaboundformylradical anion.Thedecarboxylationwasdeterminedtobetheratelimitingstep.Aftertheratedeterminingstep,theadjacentglutamateresidueisdeprotonatedbytheradicalanion.This resultsintheformationofformateboundtothemanganeseion.Theformateanionisthen releasedtoregeneratethecatalyst.Theproposalstatesthattheoxidationstatesgivenare hypotheticalandremaintobedetermined[19].Otherthantheproposedoxidationstates, themechanismseemsextremelyclearandwellsupportedbypreviousevidenceregarding thenecessityoftheGlu-333residue[17]. Basedonthestructuralsimilarityofoxoxandoxdc,itispossiblethatseveralmechanisticaspectsaresharedinthetworeactions.In2007,theBornemanngroupproposed amechanismforbothoxoxandoxdcbasedonasingledivergencepoint,seescheme1.5. Thismechanismlinksthekeystructuraldifferencesofoxoxandoxdctothefunctional differencesineachenzyme. Thetoptraceshowstheconservedmechanismuntilthebottomrightcorner.Herethe functionsofoxoxandoxdcdivergefromoneanother.Oxox,whichdoesnothaveapossible acidcatalystneartheactivesite,allowsthesuperoxideandtheformylradicalaniontoform achelatingring.Oxdc,whichcontainsanacidcatalyst,deprotonatestheacidichydrogen oftheadjacentglutamateresiduetocreateformate. Interestingly,themechanismwasproposedbasedonresearchshowingthatmutations inkeyresidues,includingtheglutamateresidueofthesecondcoordinationsphere,can

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19 Scheme1.5. ThismechanismproposedbyBurrelletal.isinterestingbecauseitcombines theinformationfromoxoxandoxdctocreatetwosimilarmechanismswithonedivergence point.Themiddlepathdescribesoxoxandthebottompathdescribesoxdc.Theoxoxpath ismostidenticaltotheOpaleyemechanism.TheoxdcpathissimilartotheReinhardt mechanism.ThisgurewastakenfromBurrelletal.[13]. convertoxalatedecarboxylasetoanoxidaseenzyme.Thesekeyresidueswerereferredto asthelidofoxdc.Theaminoacidsofthelidareneartheactivesite,buttheglutamate residueistheclosesttotheactivesite. Themostcompellingfeatureofthismechanismistheclearlinkbetweenstructureand functionofeachenzyme.Theoxidationstatesareassumed,butnotaddressedinthis study.Theoxdctraceofthismechanismisverysimilartothemechanismproposedby theRichardsgroupin2003.Additionally,theoxoxtraceissimilartoacombinationof theWhittaker2002and2005mechanisms.However,ifMnIIIisthecatalyticspeciesas indicatedbytheproposalfromtheWhittakergroupin2007,itisdifculttoenvisionthis mechanismundergoingtheMnIIItoMnIIredoxcycle. Althoughmanyoftheproposedmechanismsshowoxalateasamonodentatesubstrate, thereismuchdiscrepancyaboutwhetheroxalateismonodentateorbidentate.Acrystal structureofaputativeoxdcenzymefrom Thermotogamaritima ,suggeststhatoxalateis abidentatesubstrateoftheenzyme.Figure1.12showstheX-raycrystalstructureofthe

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20 enzymecoordinationsite.Inthegure,oxalateisclearlyboundinabidentatemodeto Figure1.12. Thecrystalstructureofaputativeoxdcenzymein Thermotogamaritima .The resolutionofthecrystalstructureis1.95 A .Theoxalateisshownasabidentatechelator. ThisgurewastakenfromSchwarzenbacheretal.[20]. themanganeseion.Interestingly,inthisresearch,oxalatewasnotaddedtocrystallization buffersorintentionallyisolatedwithenzyme[20].Thusitislikelyaremnantofpurication fromthebacteria. Despitetheconsiderableresearchintooxdcstructure,functionandmechanism,there ismuchlefttobedetermined.Theoxidationstatehasnotbeenestablished,norhasthe mechanismofthisfascinatingcarbon-carbonbondcleavage.Mostnotably,therelationship betweenthestructuralandfunctionalcharacteristicsofoxoxandoxdcispartofalarger, morefundamentalresearchtopicinbioinorganicchemistry. Becausethereissomuchnotyetknownaboutbothoxoxandoxdc,theseenzymesare ofcriticalinteresttothebioinorganiccommunity.Moreresearchisnecessaryinorderto elucidatetheintricaciesoftheseenzymes. 1.4ModelChemistry Studyingthechemistryofenzymescanbetrickyduetothenatureofsuchchemically complexstructures.Asnotedabove,eventheoxidationstateofthecatalyticmanganese

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21 ioninbothoxoxandoxdcisnotcompletelyclear.Inordertocircumventthisdifculty, smallersyntheticsimulationsofenzymesdevelopedasawaytostudythenerdetailsof enzymecatalysisinamorecontrolledmanner.Whilemethodsforthestudyofenzymesare continuallyadvancingcuttingdownonthetimeanddifcultyofenzymeinvestigation, theuseofsmallmoleculemimicsremainsavaluableadditiontotheunderstandingoflocal enzymestructureandfunction.Functionalenzymemimicscanalsohaveusefulchemical, industrialandmedicinalapplications.Afunctionalmimicofoxoxoroxdccouldpotentiallybeusedforthedetectionandpossiblyeliminationofoxalateinpeopleandlivestock. Whileoxalatedegradingenzymescanbeusedfordetection,itisatahighcost.Small moleculemimicsareoftensignicantlymorestable,lessexpensivetoisolate,morereadily distributableandeasiertocharacterize.Additionally,enzymesarenotusuallyequipped tosurviveingestionorinjection.Acarefullybuiltmimiccouldpotentiallybeusedas medicine.Morebroadly,smallmoleculemimicsallowfortheexplorationoffundamental chemistry...withtheimmediategoalofestablishingpossibilitiesandmethodologiesthat ultimatelywillleadtocorroborativesyntheticanaloguesandachemicalunderstandingof theinspirationalsystem[21].Undoubtedly,smallmoleculemimicsstillhaveanimportant roleinbioinorganicchemistry. Developingastructuralandfunctionalsynzymecanbedifcult.Ifcrystallographicdata arenotavailable,thistaskisevenmoredemanding.However,thestructuresofbothoxox andoxdcaremoderatelywellunderstood.Severalfeaturesofanenzymeareimportantto considerwhenconceivingamimic:theactivesiteoftheenzyme,thetypeofmetal,the oxidationstateofthemetalthroughoutcatalysis,theatomscoordinatedtothemetal,the coordinationnumber,andtheorientation/geometryoftheligandsaroundthemetal. Basedonthestructuresofoxoxandoxdc,aligandshouldbeselectedthatincorporates asmanyofthefeaturesoftheactivesitesaspossible.Thefollowingfeaturesmimicthe mostbasicaspectsofoxoxandoxdc,and,therefore,areanecessaryaspectofanysmall moleculemimicdiscussedinthecurrentresearch: mononuclearMnIIorMnIIIion octahedralordistortedoctahedralgeometryaroundMn

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22 foursitesoccupiedbyanN 3 Ocoordinatingligand faciallycoordinatednitrogendonorsN 3 theremainingtwositesmustbe cis forsolventandsubstratecoordination Thoughthereare,ofcourse,otherfeaturesthatareimportant,forexampleanabilityof manganesetochangeoxidationstates,manyoftheseaspectswillbecomemoreevident throughexaminingpreviousworkintheeld. Inordertodevelopasmallmoleculemimicthathasamononuclearmanganeseion,the coordinationchemistryofsmallmoleculesmustalsobeaddressed.Therstconsideration ispreventingthepolymerizationofthecompounds.Polymerizationoccurswhenthecoordinatingligandsformbridgesbetweenthemetalions.Chelateringswithsevenmembersor moreareoftenpronetobridging.Bothveandsixmemberedringsareconsideredthemost stableandtheleastlikelytoformbridgesbetweencomplexes.Additionally,stericeffects areoftenacontributingfactorinpolymerization.Smallligandsystemsaremoreproneto polymerization.Larger,bulkierligandspreventbridgingthroughsterichindrance.Finally, thekineticstabilityoftheligand-metalsystemisimportantforsmallcatalyticmolecules.If theliganddissociatesfrequently,thenumberofeffectivecollisionswiththesubstratewill decreasedramatically.Allofthesefeaturesareoftendifculttondinoneligandsystem. Trialanderrorisanimportantaspectofbiomimetics. 1.4.1ManganeseChemistry Becausebothoxoxandoxdceachdependonmanganese,thechemistryofthemetal isimportanttothedevelopmentofasmallmoleculemimic.Theelectronconguration ofmanganeseis [ Ar ] 4 s 2 3 d 5 .Typically,thecoordinationnumbervariesbetweenfourand eight.Oxidationstatesupto+7areuncommon,butachievable.However,loweroxidation statesaremorecommoninbiologicalenzymes.Ingeneral,divalentmanganeseisthe moststableoxidationstate,thoughappropriatecoordinationenvironmentscanstabilize otheroxidationstates[22].Additionally,manganesecanbeeitherhighspinorlowspin dependingontheligandeldandtheoxidationnumber.OxoxbindshighspinMnIIand

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23 MnIII[7,14].ThereisnoligandeldstabilizationenergyforhighspinMnIIcomplexes, becausethereareveunpairedelectrons[22]. Ofthetransitionmetals,manganeseisoneofthemostubiquitousinnature[22].Biologically,manganeseispresentinmanyenzymes:superoxidedismutase,oxalateoxidase andoxalatedecarboxylasetonameafew.Oxoxandoxdcspecicallybindmanganese. Themetalbindingsiteinoxoxhasshownsomeabilitytobindzinc,butithasalowafnity forzincandisnotcatalyticallycompetent[6].Zincissimilarinnaturetomanganese,but itwouldnotbeabletoundergoaredoxreaction.Thustheredoxactivityofmanganese inoxoxandoxdcislikelyacriticalaspectofenzymefunction.Basedonthisknowledge, buildingacomplexthatcanundergoredoxchangesatthemetalcenterisacriticalaspect ofbuildingafunctionalmimic.Frominformationregardingbothliganddesignandmanganesechemistry,rationaldesignofsmallmoleculemimicscanbemorereadilyunderstood andachieved.Areviewofpreviouslycharacterizedcomplexesisalsopertinentbecauseinformationfromthesestudiescanfurtherclarifythedemandsregardingliganddesignand manganesecoordination. 1.5PreviouslyStudiedComplexes Threeresearchgroupshavepreviouslystudiedmodelcomplexesofoxoxandoxdc:the Berreaulab,thePecorarolabandtheShermanlab.Thelattertworesearchgroupswork incollaboration.Complexesfromthetwodistinctlinesofresearchareimportanttothe understandingofoxoxandoxdcmodels. 1.5.1BerreauLabResearch In2005,Fulleretal.studiedcomplexesofN-benzyl-N--pivaloylamido-2-pyridylmethyl-N--pyridylmethylamine[bpppa]withMnII.Bpppa,showningure1.13,is aN 3 O-ligatingcompound.Twoofthethreenitrogendonorsarearomatic.Thisnitrogendonorsetissimilar,butnotidentical,tothenitrogenatomsintheimidazoleringsof thethreehistidineresiduesofoxoxandoxdc.Additionally,theoxygendonorispartof anamide,whichisdifferentthanthecarboxylicaciddonorofthecoordinatedglutamate

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24 Figure1.13. BpppaisaligandusedbytheBerreaulabforoxoxandoxdcmodelcomplexes.Theligandcontainsfournitrogenatoms.Threeoftheseareexpectedtocoordinate tothemanganeseion.Theamidenitrogenisnotexpectedtocoordinatebecausethefour memberedchelateringthatwouldariseisstericallyandenergeticallyunfavorable.The oxygenoftheamideisexpectedtocoordinatetothemetal. residueinthenativeenzymes.However,theoxygenisagooddonorduetotheresonance fromthenitrogenoftheamide.Therefore,thedonatingpowerofthecoordinatedligands shouldbesimilartothatseeninnativeoxoxandoxdc.CoordinationofbpppatoMnII Figure1.14. TheX-raycrystalstructureof[bpppaMnCH 3 CNH 2 O]ClO 4 2 shows thatthecomplexisamonomer.Thecoordinationsphereissimilartothebiologicalsystem becausethethreenitrogenatomscoordinatefaciallyandthetwosolventsitesare cis toone another.ThisgurewastakenfromFulleretal.[23]. resultedinamononuclearmanganesecomplexshowningure1.14[23].TheX-raycrystalstructureof[bpppaMnCH 3 CNH 2 O]ClO 4 2 showsthatthecomplexisamonomer. Eachchelatingringhaseitherveorsixmembers.Theoverallgeometryisdistortedoctahedral.Thustheligandmetalcomplexsatisesalloftheprojectedrequirementsforagood

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25 oxoxoroxdcmimic.Oxalatewasaddedtothecomplexforcrystallizationtoexaminethe binding.Thisisshowningure1.15[23].Unfortunately,theX-raycrystalstructureofthe [ f bpppaMn g 2 m )]TJ/F17 11.9552 Tf 9.289 0 Td [(C 2 O 4 ]ClO 4 2 complexshowsthatthecomplexis2:1ligand-metal tooxalate.Itispossiblethatinsolutiontheoxalateisonlyboundtoonecomplex.This Figure1.15. OxalatecrystallizedwiththeMnBpppacomplextoform [ f bpppaMn g 2 m )]TJ/F17 11.9552 Tf 9.289 0 Td [(C 2 O 4 ]ClO 4 2 .Unfortunately,theoxalateactedasabridging ligandtoformadinuclearcomplex.However,thisdoesdemonstrateanexampleofoxalate bindingtoanenzymemodelinabidentatemode.ThisgurewastakenfromFulleret al.[23]. researchisanotherdemonstrationofoxalatebindinginabidentatemodetothemanganese ion. 1.5.2PecoraroandShermanLabResearch In2008,Scarpellinietal.publishedtheirstudiesoftwotriazacyclononanederivatives triazacyclononanemonoacetate:[HTCMA];andpotassium1,4-diisopropyl-1,4,7-triazacyclononane-N-acetate:[K i Pr 2 TCMA]andatripodalligandpotassiumN,N-bis,5-dimethylpyrazolylmethylglycinate:[KBPZG][12,24].Allthreeoftheseligandsareshown ingure1.16.EachligandwascoordinatedtoMnIIandtheresultingcomplexesstudied byX-raycrystallography.BothHTCMAand[K i Pr 2 TCMA]containasmallheterocyclic macrocyclecalledtriazacyclononane[TACN].Thethirdligandstructurewaschosenbased onitsgreatersimilaritytothebiologicalsystem.

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26 Figure1.16. ThesethreeligandswerecomplexedwithMnIItoexaminethestructuralandfunctionalcharacteristics.TriazacyclononanemonoacetateHTCMAandpotassium1,4-diisopropyl-1,4,7-triazacyclononane-N-acetateK i Pr 2 TCMAarebothderivates ofacommonlystudiedligand,triazacyclononaneTACN.PotassiumN,N-bis,5dimethylpyrazolylmethylglycinateisstructurallydifferentfromtheothers,butcontains thesameN 3 Oligationschemeastheothertwoligandsshown.Thisgurewastakenfrom Scarpellinietal.[12]. TheX-raycrystalstructureof[MnTCMAH 2 O]CF 3 SO 3 isshowningure1.17. WhencoordinatedwithMnII,triazacyclononanemonoacetateTCMAorHTCMAforms apolymericchainofsevencoordinateligand-metalcomplexeswithadistortedpentagonalbipyramidalgeometry.Thesevencoordinationsitesarelledwiththethreenitrogen donors,onewatermolecule,andthebidentate,bridgingcarboxylatependantarm.Allthree nitrogendonorsarecoordinatedfacially.ByEPRandmassspectroscopy,ithasbeendeterminedthatthecomplexisbothmonomericanddimericinsolution[12].Thischange wouldlikelyresultinthemonomerchangingtoadistortedoctahedralgeometry.Because K i Pr 2 TCMAissimilarinmanyways,itisimportanttodirectlycomparethegeometriesof thetwocomplexes. Interestingly,[Mn i Pr 2 TCMAH 2 O 2 ]NO 3 isnotmultimeric,itisamonomer.Moreover,ithastheidealdistortedoctahedralgeometry.Ingure1.18,theX-raycrystalstructureofthemonomerisshown.Thethreenitrogenatomscoordinatefaciallytothemanganeseion,andthecarboxylatependantarmisonlymonocoordinate.Thetwounligated cis positionsarelledwithwatermolecules.Thusthiscomplexsatisesthebasicgeometric constraintsforasmallmoleculemimic.Whatmakes[MnTCMAH 2 O]CF 3 SO 3 different from[Mn i Pr 2 TCMAH 2 O 2 ]NO 3 ?TheadditionalbulkonK i Pr 2 TCMAprovidessteric hindrancetowardstheformationofmultimericcomplexeswithMnII.Therefore,steric

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27 Figure1.17. TheX-raycrystalstructureof[MnTCMAH 2 O]CF 3 SO 3 revealsthatthe complexformspolymerslinkedbythebidentatecarboxylatependantarm.Threesubunits ofthecontinuouspolymerareshowntodepicttheoverallgeometry.Thethreenitrogen atomsoftheTACNringcoordinatefacially.Whilethisisadvantageous,thesevencoordinateMnIIisundesirable.Purple-ManganeseII;Red-Oxygen;Blue-Nitrogen;GreyCarbon.ThisgurewastakenfromScarpellinietal.[12]. Figure1.18. X-raycrystallographicdatashowthatthe[Mn i Pr 2 TCMAH 2 O 2 ]NO 3 complexformsmonomericstructures.Thegeometryisdistortedoctahedralwiththenitrogen atomscoordinatingfacially.Unlikethe[MnTCMAH 2 O]CF 3 SO 3 complex,thecarboxylatependantarmisonlymonodentateanddoesnotbridgetwomanganeseions.Thisgure wastakenfromScarpellinietal.[12].

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28 bulkisanimportantfeatureinMnIIcoordination. Finally,thetripodalligandKBPZGisthemostdistinct.Theoverallstructureofthe complexwithMnII,[MnBPZGH 2 O]NO 3 ,ispolymeric.Figure1.19showsthreeunits ofthepolymer.Againthecarboxylatependantarmisbridging.However,thegeometry aroundMnIIissixcoordinatewithN 3 O 3 donorgroups.Oneofthecoordinatedoxygenatomsisfromawatermolecule.Theothertwoareoxygenatomsfromtwodifferent carboxylategroups. Figure1.19. TheX-raycrystallographicdatafor[MnBPZGH 2 O]NO 3 showthatthe complexformsacontinuouspolymer.Aswiththe[MnTCMAH 2 O]CF 3 SO 3 complex, thecarboxylatependantarmformsabridgebetweentwoMnIIions.However,each manganeseisonlysixcoordinatewiththenitrogenatomsoccupyingmeridionalsites.This gurewastakenfromScarpellinietal.[12]. AcomparisonofthethreeX-raycrystalstructuresshowsthattheKBPZGcoordinates toMnIIsuchthatthenitrogendonatinggroupsaremeridional.Therefore,thisstructureis theleastrepresentativeofoxoxoroxdc,whichbothcontainthreehistidinedonorscoordinatedtomanganeseinafacialconformation.Additionally,[Mn i Pr 2 TCMAH 2 O 2 ]NO 3 formstheonlyoctahedralmonomericcomplexinthesolidstate.Thus,theK i Pr 2 TCMA ligandproducesthebeststructuralmimicofthesethreecandidates. InadditiontoX-raycrystallography,EPRspectroscopywasalsoexploredforeach complex.Figure1.20showstheEPRsignalsdetectedforeachofthecomplexesat77K,

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29 andforoxoxandoxdc.Theg-factoriscalculatedusingthefollowingequation h n = g m e B Where n isthefrequency; m e istheBohrmagneton;and B isthemagneticeld. Inthebottommostgure,trace1istheMnTCMAcomplex;trace2istheMn i Pr 2 TCMA complex;andtrace3istheMnBPZGcomplex.AlthoughthespectraforboththeMnBPZG andtheMn i Pr 2 TCMAcomplexeshavetheexpectedsixlinepatternfor 55 MnI=5/2,the spectrumfortheMnTCMAcomplexhasanadditionalbroadsignaloverlayingtheexpectedsharpsixpeaks.Thisbroadfeatureisattributedtothepresenceofabinuclear manganesecenter.ThetopleftgureistheEPRsignalofwildtypeoxoxfromWhittaker etal.2002[7].Thesixlinepatternisalsopresentatg 2.0.Finally,thetoprightgure isofoxdc.Thisgureexhibitsthesamelinepatternshownforoxox.Thisisexpected sincetherstcoordinationsphereisstructurallysimilar.TheseEPRspectraindicatethat manganeseionsinboththeMn i Pr 2 TCMAandtheMnBPZGcomplexaresimilarinnature tothemanganeseioninoxox.Additionally,theyprovideevidencethattheMnBPZGcomplexismonomericinsolution,buttheMnTCMAcomplexexistsasbothadinuclearanda mononuclearcompoundinsolution. Becauseoxoxandoxdcundergoachangeinoxidationstateduringcatalysis,theredox potentialsofeachofthemanganesecomplexesareanimportantaspectoffunctionality. Cyclicvoltammetrystudieswereconductedoneachcomplex.Figure1.21showsthedata acquiredusingferrocene/ferrociniumstandard. TheMnIII/MnIIreductionpotentialfortheioncoordinatedtotheenzymeiscalculatedtobebetween0and+0.6Vvs.theferrocene/ferrociniumelectrode+0.4and+1.0 Vvs.thenormalhydrogenelectrode[14].TheMnTCMAcomplexcontainstwoquasireversibleredoxprocessesatE 1/2 +333mVand0mVvs.theferrocene/ferrociniumelectrode.TheMn i Pr 2 TCMAcomplexcontainsone'poorlyreversible'processat+330mV vs.theferrocene/ferrociniumelectrode.TheMnBPZGcomplexcontainsonereduction peakandtwooxidationpeaksat+727mVand+1070mVvs.theferrocene/ferrocinium electrode.ThismeansthatboththeMnTCMAandtheMn i Pr 2 TCMAcomplexeslowerthe reductionpotentialofthemanganeseiontoadegreesimilartotheenzymeactivesite. ThecyclicvoltammogramsshowthattheredoxcycleofMnTCMAismorereversible thantheredoxcycleofMn i Pr 2 TCMA.Preliminaryfunctionalstudieswereperformed onMn i Pr 2 TCMA.Althoughthecomplexappearstobecapableofbindingoxalatefrom

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30 Figure1.20. AcomparisonoftheEPRspectraofthethreemanganesecomplexesbottom, oxoxtopleftandoxdctopright.Boththeoxoxandoxdcspectraexhibitthesixline patternindicativeofthepresenceof 55 MnI=5/2.TheEPRspectrumfromoxoxcontains aninsetwhichexpandsthepeaksatg 2.Inthebottomgure,theEPRspectraofthe threemanganesecomplexesareshown.Trace1istheMnTCMAcomplex;trace2isthe Mn i Pr 2 TCMAcomplex;andtrace3istheMnBPZGcomplex.Trace2and3correspond welltothesixlinepatternatg 2.0for 55 MnI=5/2.However,trace1hasabroader featureoverlayingthesamepattern.ThisisproposedtobethebinuclearmanganesestructurepresentintheMnTCMAcomplexinsolution.Theunitconversionis0.001KG=1 G=0.1mT.AllofthesespectraweretakenwithanX-bandspectrometeratthefollowing frequencies:oxoxat9.16GHz;oxdcat9.67GHz;synthesizedcomplexesat9.24GHz. ThebottommostgurewastakenfromScarpellinietal.[12].Thetopleftgurewastaken fromWhittakeretal.[7].ThetoprightgurewastakenfromTanneretal[25].

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31 Figure1.21. Thecyclicvoltammogramsforeachcomplexareshownabove.Spectra werecollectedinmethanolwithferroceneasanexternalstandard.Thedatacollectedused thefollowingconditions:Ag/AgClreferenceelectrode,platinumwireauxiliaryelectrode, glassycarbonworkingelectrodeandascanrateof75mVs 1 .Trace1isMnTCMA; trace2isMn i Pr 2 TCMA;andtrace3isMnBPZG.MnTCMAhastheprocessthatis closesttoreversiblequasi-reversible.Thelargeirreversiblepeakathigherpotentialis attributedtoligandredoxchemistry.Thisgurewastakenfromthesupplementarymaterial ofScarpellinietal.[12].

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32 cyclicvoltammetryexperiments,nocatalyticturnoverwasdetected[26]. Whilethecomplexsatisesthegeometricconstraints,thecomplexappearstobeunable tocatalyzethedegradationofoxalate.Thiscouldbeduetoanyofseveralfactors: sterichindrancetosubstrates incorrectsubstratebindingmodebidentatevs.monodentate substratebindingafnity redoxpotentialofmanganese incorrectoxidationstateformanganese Themodeofbindinginthenativeenzymesmaybeeitherbidentateormonodentatebased ontheliterature[7,20].Itispossiblethatthebindingmodeoftheoxalatewiththecomplex isnotthesameasthebindingmodeintheenzyme.Twoscenariosexplainhowanincorrect bindingmodewouldleadtolackofreactivity.Inscenarioone,oxalateismonodentate inthenaturalenzyme,anddioxygencouldbindtothemanganeseionintheenzymatic reaction.Ifthecomplexfavorsabidentatemode,insteadofthescenarioonemonodentate mode,dioxygenmaynotbeabletoaccessthemanganeseion.Inscenariotwo,oxalateis bidentateinthenativeenzyme.Itispossiblethatanaminoacidresidueintheenzyme facilitatesthereactionwithdioxygen.Thelackoftheseaminoacidresiduesinthemimic mayinhibitreactionturnoverinthemimic. Mostimportantly,ifMnIIisnottheactiveoxidationstate,thelackofcatalysiscould beremediedbycoordinationwithMnIII.However,itisoftentimesdifcultforaligand tostabilizemultipleoxidationstates. 1.6ProposedComplex Movingtowardsafunctionalsyntheticmodel,anewcomplexthatmaintainstheimportantaspectsofoxoxandoxdcmustbeexplored.Additionally,acomplexthatbindsMnIII wouldbeuniqueandquiteinteresting.

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33 Animportantconsiderationofmodelchemistryissimilaritytothebiologicalsystem. However,itisalsoimportantforthemodeltobechemicallystableandsuitableforcatalysis.Itiscriticalfortheligandtobekineticallyinert,butalsoforthesolventtobekinetically labileforcatalyticreactivity. Therstligandpossibilityexploredwasatripodalligandwiththreeimidazolerings joinedbyanalphacarbonofacarboxylgroup.Thisstructureisshownontheleftin gure1.22.Thecoordinationofeachoftheseligandswithmanganesewasexaminedusing Figure1.22. Theaboveligandswereconsideredformodelsofoxoxandoxdc.Each allowsforaN 3 Oligationpattern.Theseligandswereconsideredbecauseofthesimilarity tothebiologicalcoordinationenvironmentofoxoxandoxdc.Thethreeimidazolerings representthethreehistidineresidues,andthecarboxylatearmrepresentstheglutamate residue.However,thethreeligandsdonothavetheappropriategeometryforoptimal coordination,asseenonSpartan'04[27]andinspace-llingmodels. space-llingmodelsandthecomputerprogramSpartan'04[27]. TheseN 3 Odonorligandswouldbeverysimilartothebiologicalactivesitemorethan anyoftheligandsdescribedinthepreviouswork.However,theligandonthefarleft isnotgeometricallysuitedtotthemanganeseion.Thecarboxylategroupistooshort tocoordinatetothemetal.Additionally,addingacarbontothecarboxylatependant,as showninthesecondstructureofgure1.22,wouldresultinalongchainthatwouldmore likelyformabridgebetweentwometalionsduetotheunfavorableformationofaseven memberedchelatering.Thiswouldleadtoextensivepolymerization.Moreover,ifsp 3 carbonsareplacedbetweenthecentralcarbonandtheimidazoleringsasshowninthe thirdstructureofgure1.22,thecarboxylatehasmoreroomtocoordinate.However,the additionofthesecarbonsforcesthethreenitrogenatomstoadoptameridionalcoordination

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34 modeonthemetal.Thiswouldleadtotoogreatadiscrepancybetweentheenzymeandthe complex,andthusitwouldnegatethepointofusingsuchaligand.Alloftheseobservations weredeterminedthroughSpartan'04[27]andspacellingmodels.Forthesereasons,the ligandsshowningure1.22werenotsyntheticallyexplored. TACNisacommonlyusedligandsynthesizedintheearly1970s[28].Throughoutthe decade,anabundanceofresearchwasconducted.QuicklythenewmacrocycleTACNbecameimmenselypopularincoordinationchemistry.Thiswasduetotheincrediblechemicalstabilityrevealedinearlyresearch.Thecavitysizeoftheligandisidealforcoordination withmetals.Additionally,thedonatingabilityofthethreenitrogenatomsandthechelate effectconferkineticandthermodynamicstabilitytotheligandmetalcomplex[29,30]. EarlystudiesonTACNcomplexeswithnickelII,copperIIandzincIIrevealedthatthe complexesaredurableinbothacidicandbasicconditions[31,32].Moreover,ligandmetal complexeswithTACNareoftenhighlyheatstable.Forcoordinationpurposes,TACNis alsoveryversatilebecauseitisabletostabilizehighandlowspinmetalswithdifferent oxidationstates[29]. Finally,becausetheligandiskineticallyinert,thecomplexesformedareoftenutilized inbiomimeticchemistrytoformactivecatalysts[29].Thethreecoordinatingnitrogen atomsareoftenusedtosimulatehistidineresidues.Becausetherearethreecoordinating histidineresiduesinoxoxandoxdc,TACNcomplexesareanexcellentchoiceforcomplexationwithmetals.BasedonthestructuralsuccessofK i Pr 2 TCMA,aligandcontaining Figure1.23. 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate[Bn 2 TCMA ]isthe newtargetligand.Itwaschosenforitsabilitytoaltertheredoxchemistryofacoordinated manganeseionandforthestericbulkthatthebenzylgroupsoffer. TACNwaschosenforfurtherstudies[12].Figure1.23showsthenewligandtarget:1,4dibenzyl-1,4,7-triazacyclononane-7-monoacetate[Bn 2 TCMA ].Thebulkybenzylgroups

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35 willallowforbothsterichindranceandatweakingoftheredoxchemistryinthecoordinatedmanganeseion.PhenylgroupswerealsoconsideredfortheN,Npositions.This complexisshowningure1.24.Whilephenylgroupswouldcauseagreaterchangein redoxchemistry,phenylringsareplanaranddonotprovideasmuchstericbulkinthree dimensions.Thisisbecausethephenylgroupsareplanar,buttheCH 2 groupofthebenzyl Figure1.24. The1,4-diphenyl-1,4,7-triazacyclononane-7-monoacetate[Ph 2 TCMA ]ligandisalsoanotherpossibilityformodelcomplexstudies.Amongotherreasons,itwasnot usedbecausethesynthesisrequiresanexpensivecatalystseescheme1.6. hasbulkinmorethanthetwodimensionsoftheplane.Additionally,phenylgroupswould likelydecreasetheabilityofthenitrogenatomstodonatetoacoordinatedmanganeseion. ItisunlikelythatphenylgroupscouldstabilizeMnIII.Alastconcernwasthatthesynthesisof[Ph 2 TCMA ]requiresapalladiumcatalyst,whichisexpensive[33].Thesynthetic schemeforPh 2 TCMAisshowninscheme1.6.Duetothesefactors,benzylgroupswere Scheme1.6. Theproposedsyntheticroutetowards[Ph 2 TCMA ].TsCl/Ether,H 2 O; TsCl/THF,H 2 O;60%NaHinoil,DMF;HBr30%inAcOH,dryphenol; PhBr,PdOAC 2 ,rac-BINAP,t-BuONa,toluene;Unknown;chloroaceticacid, lithiumhydroxidemonohydrate,methanolandwater.

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36 consideredmorechemicallyinterestingthanthephenylgroups,andlessexpensivetodevelop.However,comparisonofthe[Ph 2 TCMA ]ligandwiththe[Bn 2 TCMA ]ligand wouldprovidemoreinformationaboutthekindofstericbulkrequired. Smallchemicalalterationscanbemadeinboth[Bn 2 TCMA ]and[Ph 2 TCMA ]to modulatethechemistryoftheligandmetalcomplex,asshowningure1.25.Thesealterationsincludebutarenotlimitedtoituningthemetal'sredoxpotentialbyaddingelectron donatinggroupsorelectronwithdrawinggroupstotheortho,metaand/orparapositionsof thearomaticrings,iiaddinghydrogenbonddonorstosimulatetheasparagineresiduesof Figure1.25. Fromtheinitialligands,afamilyofligandsthatalterstheredoxchemistryandstericeffectswouldbeinterestingtoexplore.Inboththe[Bn 2 TCMA ]and the[Ph 2 TCMA ]ligands,electronwithdrawinggroupsandelectrondonatinggroupsrepresentedabovebyXcanbeaddedtotheortho,metaandparashownabovepositionsto altertheredoxchemistryofthecoordinatedmanganeseion. theactivesiteandiiiaddinglargeresiduestoexaminewhenoxalatebindsinabidentate ormonodentateconformation. Thistypeofstudywouldbeveryrigorousandprovideawealthofinformationabout fundamentalchemistryoftheligandsandtheresultingcomplexes.Aprojectofthismagnitudeistoogreatforoneundergraduatethesis.Thusthisworkwillfocusonprogress towardsthesynthesisof[Bn 2 TCMA ]forthecoordinationofthisligandtoMnIIand/or MnIII.

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37 Chapter2 Experimental Allofthefollowingreactionsusedchemicalspurchasedcommerciallyfromeither Sigma-AldrichorAcros.Thechemicalsusedwerenotfurtherpuriedunlessotherwise indicated.ABrukerAC250MHzspectrometerwasusedforallNMRstudies.ChemDraw 8.0wasusedtogeneratethepredictedspectra[34].Meltingpointswereobtainedona MeltempII.Allreactionswererunundernitrogenunlessnotedotherwise.Forheatedreactions,aPEGoilbathisrecommendedforheatingbecauseitevenlydispersesheatandmaintainsasteadytemperature.Solventsweredriedinthefollowingmanner:dichloromethanedistilledoverCaH 2 ;acetonitrile-distilledoverCaH 2 ;tetrahydrofuran-distilledoversodium andbenzophenone.Allextractionswithchloroformandbasicwaterweredonequicklyto avoidcarbeneformation. Commonabbreviationsused:diethylenetriamine[DETA];p-toluenesulfonylchloride [TsCl];tosylgroup[ts];N,N',N-tritosyldiethylenetriamine[DETAts 3 ];ethyleneglycolbistosylate[EGOts 2 ];1,4,7-tosyl-1,4,7-triazacyclononane[TACNts 3 ];1,4,7-triazacyclononane[TACN];monotosyl-triazacyclononane[TACNts];1,4-dibenzyl-7-tosyl-1,4,7triazacyclononane[Bn 2 TACNts];1,4-dibenzyl-1,4,7-triazacyclononane[Bn 2 TACN];1,4dibenzyl-1,4,7-triazacyclononane-N-acetate[Bn 2 TCMA];diethanolamine[DEA];tritosyl diethanolamine[DEAts 3 ].*IndicatesanimpurityintheNMRspectrum.Integrationof peaksfromimpuritiesin 1 H-NMRspectraweredeterminedbycomparisontoareference peakinthespectrumbelongingtothedesiredproduct. Tworoutestotheproposedligandwereexploredseeschemes2.1and2.2.Addition-

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38 ally,thesecondroutesubdividesintotworoutes. 2.1RouteOne RouteoneiscomposedofatwostepprocesstoTACNtsshowningure2.1. Scheme2.1. TsCl,triethylamine,methylenechloride;ethylenediamine,K 2 CO 3 acetonitrile. 2.1.1SynthesisofTosylbis[2-tosyloxyethyl]amine[ DEAts 3 ] ThispreparationwastakenfromHuangetal.2009[35]. Athree-neckroundbottomaskwasequippedwithanitrogeninlet,asolidadditionfunnelandathermometerandadaptor.Underanitrogenatmosphere,diethanolamine.474 g;328mmol;105.14g/molwasdissolvedinfreshlydistilleddichloromethaneml. Afterthesolutionwasplacedonice,triethylamineml;1.10molwasaddedtothe stirredsolution.Oncethesolutionwaswellmixed,p-toluenesulfonylchloride.352g; 1.030molwasaddedtothestirredsolutionviaanadditionfunneloveraperiodofthree hours.Thetemperaturedidnotrisehigherthan20 Cduringtheaddition.Aftertheadditionwascomplete,thesolutionwasremovedfromiceandthereactionwasstirredatroom temperaturefor20hours.Inthemorning,thereactioncontainedalightyellowliquidwith alargeamountofwhite,uffyprecipitate.Themixturewasplacedoniceinordertocompletelyprecipitatethetriethylaminehydrochloride.Oncethesolutionhadreached5 C,the whitesolidtriethylaminehydrochloridewaslteredfromthesolution.Theyellowltrate waswashedinaseparatoryfunnelwith1Mhydrochloricacidmlx8toremovethe triethylaminehydrochloride.TheorganiclayerwasthenwashedwithDIwatermlx 6,followedbyasolutionofsaturatedaqueoussodiumcarbonatemlx6toremove thefreetriethylamine.Theresultingorganiclayerwasdriedoveranhydrousmagnesium

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39 sulfate.Afterroughlythirtyminutes,thewhitesolidwaslteredoff.Theltratewasrotaryevaporatedtoyellow-pinkoilwhichwasallowedtositintherefrigeratorovernight hours.Uponrefrigeration,theoilbecameawhitesolid,whichwasrecrystallizedfrom boilingethanol.Theyieldforthisrepresentativereactionwas77%.985g.Thereaction wascompletedasecondtimescalemultipliedby2.5.Theyieldwas77%.388gfor thesecondreaction. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.85d,0.1H,TsCl,7.75d,3.5H,OTs-Ar, 7.60d,2.5H,NTs-Ar,7.40d,4H,OTs-Ar,7.30d,2H,NTs-Ar,4.15t,4H,CH 2 3.40t,4H,CH 2 ,2.45s,9.5H,CH 3 Ar,1.65s,0.5H,H 2 Oppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 : 145.433,132.606,130.240,128.179,127.464,68.496,48.667,21.887,21.763ppm.See gureA.1. Experimentalnotes: Ifthereactionisnotcooledinanicebathbeforeltering,thewasheswillnotbe enoughtoremovethetriethylaminehydrochloride. Duringthewashes,awhitishphasesometimesformedbetweentheaqueousphase andtheorganicphase.Thiswhitephasewasincludedintheorganicphasewithno problems. Onalargescaletherecrystallizationwasneeded,howeveronsmallerscalesitwas notnecessary. Inthesecondreactiontherewasaspillofroughly10-20%ofthesolution. 2.1.2Synthesisof1-Tosyl-1,4,7-triazacyclononane[ TACNts ] ThisprocedurewasheavilymodiedfromHuangetal.2009[35].Thereactionwas completedthreetimeswithmodicationsindicatedintheexperimentalnotesandonce usingtheoriginalprocedure. Thefollowingapparatuswassetup:asingle-neckroundbottomaskwithaClaisen adaptor.TotheClaisen,awatercondenserwithanitrogeninletandamechanicalstirrer wereattached.Themechanicalstirrerusedaglassrodand24/40adaptorwhichwassealed withasmallamountofmineraloil.Thiswastime-consuminganddifculttoassemble.

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40 Underanitrogenatmosphere,DEAts 3 .000g;53mmol;567.6986g/molwasdissolvedin150mLoffreshlydistilled,dryacetonitrile.Anhydrouspotassiumcarbonate .137g;304.88molwasaddedtothestirredsolution.Thereactionmixturewasreuxedwithveryvigorousstirring.Asolutionofethylenediamine.52ml,52.6mmolin 50mLofdryacetonitrilewaspouredintoanadditionfunnel.Theadditionfunnelwasthen attachedbetweentheClasienadaptorandthecondenser.Overtwohours,theethylenediaminesolutionwasaddedtothestirredreactionmixture.Aftertheadditionwascomplete, thesolutionwasallowedtoreuxfor18hours.Inthemorning,thereactionmixturewas lteredwhilsthottoremoveawhitesolid.Theltratewasreducedtoayellowoilthat wascharacterizedtobethecorrectproductwithmultipleimpurities.Theyieldofthisrepresentativereactionwas46%.806gofthisveryimpurematerial.Withthemodied procedure,theaverageyieldwas41.8%;thescaleofthesereactionsvariedfrom32mmol to53mmol.However,whentheexactliteratureprocedurewasfollowed[35],theyield was5%. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.75*m,0.5H,7.70*s,0.5H,7.60d,2H,Ar,7.30d, 2H,Ar,7.15*d,0.25H,4.10*t,0.5H,3.75*m,0.5H,3.40*m,0.25H,3.25-3.10* m,0.5H,3.10-2.90*m,4H,2.80-2.60*t,1.75H,2.50*t,4H,2.45*s,3Hs,2.30* s,broad,3H,2.05s,3H,CH 3 CN,1.25*s,0.25H,1.15*t,0.25Hppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMR CDCl 3 :143.947,132.484,129.876,128.084,55.577,52.789,52.485,46.297,46.144, 21.734ppm.Seegure3.1. Thelteredsolidwasaddedtoroughly1LofDIwaterinanErlenmeyerask.The mixturewasallowedtositfor12hours.Inthemorning,smallamountsofthesolution wereheatedtoreuxandltered.Thisresultedinawhiteprecipitate.Theprecipitatewas leftinvacuoovernighttodry.Theresultwascopiousamountsofcleanwhitepowder.The meltingpointwasdeterminedtobe261-270 C.Thisindicatesthattheproductisapolymer. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.70d,2H,Ts,7.30d,2H,Ts,3.20m,4H,CH 2 ,3.10m,4H, CH 2 ,2.90s,5H,CH 2 ?,2.42s,3H,CH 3 Ts,1.80s,3H,H 2 Oppm.SeegureA.2. Experimentalnotes: Modicationsfromliterature:Eachofthesemodiedreactionswererunattwice thedilutionreportedintheliterature.Additionally,veryslowadditionofethylenediaminewasutilizedforthemodiedprocedure.

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41 Thesemodicationswereexpectedtodecreasethepossibilityofpolymerization,but theydidnotseemtohelpagreatdeal. Dr.PaulScuddersuggestedusingthedualslowadditiontechnique.ErinnBrigham hadgreatersuccesswiththismethod,buttheimpuritylevelwasstillveryhigh. 2.2RouteTwo Scheme2.2. ProposedLigandSynthesisRouteTwo:Thechemicalsusedforeachstep areasfollows:TsCl/Ether,H 2 O;TsCl/THF,H 2 O;60%NaHinoil,DMF; H 2 SO 4 ;HBr30%inAcOH,dryphenol;benzylchloride,K 2 CO 3 ,acetonitrile; benzylchloride,sodiumcarbonate,acetonitrile;400WHglamp,MeOH/H 2 O,NaBH 4 p )]TJ/F17 11.9552 Tf 9.289 0 Td [(methoxybenzene;chloroaceticacid,lithiumhydroxidemonohydrate,methanoland water. Routetwo,showninscheme2.2,isextensiveandsubdividesintotworoutes.

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42 2.2.1SynthesisofDiethylenetriamine-N,N',Ntristosylate[ DETAts 3 ] ThisreactionwasadaptedfrompreviousstudentsintheShermanlab[36].The reactionwasnotconductedunderanitrogenatmospherebasedoninformationfrompreviousstudents.Thereactionwasruntwiceonalargescale.Olddiethylenetriaminewas adequate.Amechanicalstirrerisidealonlargescales. A2-neckroundbottomwasttedwithamechanicalstirrerandapressureequalizing additionfunnel.Intheroundbottom,sodiumhydroxide.100g;728mmolwasdissolvedin188mlofdeionizedwater.Oncedissolved,diethylenetriamineml,223mmol; 103.17g/mol,0.96g/mlwasaddedtotheroundbottomslowly.Heatwasevolvedfromthe solution.InanErlenmeyerask,p-toluenesulfonylchloride.578g,701mmol;190.64 g/molwasdissolvedin625mlofdiethylether.Thetosylchloridesolutionwasaddedto thediethylenetriaminesolutionthroughtheadditionfunnelover1.5hoursatroomtemperature.Somesolidprecipitatedinthefunnel.Thiswaswashedwithdiethyletherandadded tothereaction.Thereactionwasstirredvigorouslyfor24hours.Thenextmorning,the mixturewaslteredtoproduceawhitecottage-cheese-likesubstanceandaclearyellow liquidthatseparatedintotwophases.Thelteredsolidwasaddedto1.5litersofdeionized waterandstirredfor24hours.Thenextdaythewhitesolidwaslteredfromtheclear lightyellowliquid.Afterwashingtheltercakewithcoldethanolanddiethylether,the wetsolidwasdriedinvacuotoyieldawhitetocreamcoloredsolid.Ofthetworeactions, onlyonemostlydryyieldwastaken:80%.763g.Notethatthiswascontaminated withTsCl,whichwasincludedinthereportedyield. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.75d,4H, Ar,7.62d,2H,Ar,7.30d,integrationcomplicatedbyCHCl 3 ,Ar,5.30s,broad,1H, NH,3.10-3.00m,8H,NCH 2 CH 2 N,2.45s,9H,CH 3 Ar,1.80*s,broad,4H,H 2 O, 1.30*m,2H,unknownppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :144.430,136.851,130.237,130.056, 127.542,127.388,50.785,42.864,21.769ppm.SeeguresA.3andA.4. Experimentalnotes: Diethyletherevaporatedfromtheadditionfunnelleavingsolidp-toluenesulfonyl chlorideinandaroundthetipofthefunnel.Paralmaroundthejunctionhadlittleeffect.

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43 Theproductwasalmostalwayswet,despitepumpinginvacuoformultipledays. 2.2.2Synthesisof1,2-Bistosyloxyethane[ EGOts 2 ] ThisreactionwasadaptedfrompreviousstudentsintheShermanlab[36].Thereactionwasnotconductedunderanitrogenatmospherebasedoninformationfromprevious students.Thereactionwasruntwiceonalargescale.Eachrunrequiredamechanical stirrer.Oldethyleneglycolprovedtobeadequate. InalargeErlenmeyerask,sodiumhydroxide3.800g;845mmolwasdissolved in167mlofdeionizedwater.Oncethesodiumhydroxidewasdissolved,ethyleneglycol .5ml,296mmol;62.07g/mol,1.113g/mlwasaddedtothesolution.TheErlenmeyer askwasplacedinanicebath,andtheliquidwasstirredwithamechanicalstirrer.In aseparateErlenmeyerask,p-toluenesulfonylchloride.254g,552mmol;190.64 g/molwasdissolvedin200mloftetrahydrofuran.Thetosylchloridesolutionwasadded tothechilledethyleneglycolsolutiondropwiseover2hours.Afterprecipitatebeganto form,theicebathwasregenerated,andthesolutionwasstirredfor24hours.Thenextday, themixturecontainedyellowliquidwithwhiteprecipitate.Deionizedwatermlwas addedtotheask,andthemixturewasstirredonicefor30minutes.Thewhitesolidwas thenlteredfromthelightyellowliquid.Theltercakewaswashedwithice-coldsolvents: 60mlofethanol,60mlof0.1Msulfuricacid,60mlofdeionizedwaterandthen60mlof ethanol.Thesolidwasthenplacedinvacuotodry.Theproductwasawhitetooff-white solid.Ofthetworeactionsonlyonedryyieldwastaken:70%.907g.Theproduct containedasmallamountofether,butwasotherwiseveryclean. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.69 d,4H,Ar,7.30d,4H,Ar,4.15s,4H,OCH 2 CH 2 O,3.70*q,Hs,ether,2.49s,6H, CH 3 Ar,1.60*s,0.5H,H 2 O,1.25*t,0.5H,etherppm. 13 CNMRCDCl 3 :145.492, 132.545,130.176,128.177,66.886,21.886ppm.SeeguresA.5andA.6. 2.2.3Synthesisof1,4,7-tritosyl-1,4,7-triazacyclononane[ TACNts 3 ] ThisreactionwasadaptedfrompreviousstudentsintheShermanlab[36].Veryold NaHwasusedforthisreactioneachtime.Theyieldwassufcient,buthigheryieldswould

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44 likelyresultifnewNaHwereused.Alarge,football-shapedstirbarwasoftensufcient evenonlargerscales. A3-neckroundbottomaskwasequippedwithacondenser,nitrogeninlet,thermometerwithadaptorandanadditionfunnel.Totheroundbottom,6.288gsodiumhydridein 60%oilmmolNaHwasdissolvedin450mlofN,N-dimethylformamide.Asthe mixturewasstirredwithastirbarunderanitrogenatmosphere,hydrogengaswasevolved. Oncethehydrogenevolutionwascomplete,DETAts 3 .676g,75mmol;565.73g/mol wasaddedtotheroundbottom.Whenheatedto100 C,theDETAts 3 solutionbecamea brightyellowcolor.Foraminimumof15minutes,thereactionwasstirredat100 C.In anErlenmeyerask,0.281gofsodiumhydridein60%oilmmolwasdissolvedin50 mlofN,N-dimethylformamide.Oncethegasevolutionwascomplete,EGOts 2 .800 g,75mmol;370.44g/molwasaddedtotheErlenmeyer.TheEGOts 2 solutionturned lightpeach.Usinganadditionfunnel,thislightpeachsolutionwasaddeddropwiseover twohourstotheDETAts 3 solution.Oncetheadditionwascomplete,theresultinggolden yellowsolutionwasstirredfor18hoursat100-115 C.Thenextmorning,thereactionwas addedslowlyto1literof vigorously stirreddeionizedwater.Thisaqueoussolutionwas subsequentlystirredfor24hoursandplacedonicethenextmorning.Afterroughly30 minutesonice,themixturewasltered.Thelighttansolidwaswashedwithice-coldportionsofwater,ethanolandether.Theresultingsolidwasdriedinvacuo.Theyieldfor thisrepresentativereactionwas75%yieldofalighttansolid.136g.Thereactionwas completedfourtimesintotalwithscalesfrom31mmolto75mmol.Theaverageyield was70%witharangefrom56%to77%.Ineachreaction,thetansolidcontainedlarge amountsofDETAts 3 inadditiontothedesiredproduct.Itwasnotfurtherpuriedbefore useinallofthedetosylationmethodsforbothroutestwoAandB. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 : 7.80*d,1.6H,DETAts 3 ,7.70d,8.0H,TACNts 3 ,7.60*d,1.6H,DETAts 3 ,5.1s,1H, 3.45s,12H,CH 2 ofTACNts 3 ,3.20*m,6H,CH 2 ofDETAts 3 ,2.45s,18H,ArCH 3 of DETAts 3 andTACNts 3 ,1.80*s,4H,H 2 Oppm.Seegure3.2. Experimentalnotes: TheDMFwasnotdriedbeforeuseandtheNaHwasold.Thiscouldbewhythereis somuchDETAts 3 leftunreacted.

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45 Severalprocedurescallforrotaryevaporatingthereactionmixturebeforeaddingto thedeionizedwater.Thiswouldbeverytimeconsumingandunpleasantduetothe highboilingpointofDMFdegC.InsteadofremovingtheDMF,theamount ofdeionizedwaterusedtoprecipitatetheproductwasincreased. Theethanolwashiswhiteandcloudy.Inafewreactionsthiswashwascollectedand ltered.However,thesolidismostlyunreactedDETAts 3 2.2.4RouteTwoA TACNSynthesis:SulfuricAcid ThispreparationwastakenfromSearleandGeue[39].Thoughthisreactionwasnot conductedundernitrogenatmosphere,itshouldbeinfuturereactions. Athree-neckroundbottomwasequippedwithawatercondenser,athermometerwith adaptorandaseptum.ImpureTACNts 3 ,seeabove,.99g;47mmol;591.77g/molwas dissolvedin76mloffresh98%sulfuricacid.Thetan/orangesolutionwasheatedto100 C andstirredforthreedays.Afterthistime,thesolutionwasbroughtto0 Cinanicebath. Veryslowly,40mlof100%ethanolwasadded.Themixtureturnedamuddybrown color,afterwhich100mloffreshanhydrousdiethyletherwasslowlyadded.Themixture waslteredundernitrogenusingapowderfunnelattachedtothenitrogenline,asshown ingure2.1.Thegreyhygroscopiccrystalsweredissolvedin30mlofdeionizedwater. Thesolutionwasboiledwithactivatedcharcoalfor5minutes.Thehotmixturewascooled inawaterbath.Oncetheliquidreachedroomtemperature,thecharcoalwaslteredfrom theliquid.ThesolutionwasroughlypH2,thusnodilutionwasnecessary[39].TheblackgreyliquidwasabsorbedontoacolumnofDowex50W-X2,H + form,cationexchange resin-400mesh.Thecolumnratiowas2cmindiameterand10cminheight.After thegrey-blackliquidwasabsorbedontothecolumn,thecolumnwaswashedwithwater, thenwith1MHCl,2MHCl,3MHCland6MHCl.Eachfractionwasrotaryevaporated. The2Mand3Mfractionswerebothdarkeryellowbeforeevaporation,andeachyielded ayellowsolid.Theyieldwas47%pureTACN 3HCl. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRD 2 OwithTSP:3.40s,CH 2 ppm.Seegure3.3.

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46 Figure2.1. Abuchnerfunnelwithlterpaperisplacedoveralteraskwiththevacuum on.Apowderfunnelisattachedtotheschlenklinewithnitrogenowingoutofit.The liquidisquicklypouredintothebuchnerfunnelandthepowderfunnelisplacedover to'seal'thereactioninanitrogenow.Thelteredsolidisthenquicklyremovedand dissolvedinwater. Experimentalnotes: Thelowyieldislikelybecausethereactionwasnotundernitrogen. Ethanolshouldbeaddedslowly;theadditionisveryexothermic. Thequickltermethodrunsahigherriskofwaterexposurethanaschlenklinefrit, butwasmuchmoreconvenient. Afterlteringawaythecharcoal,theprocedurefromSearleandGeuecallsforthe solutiontobedilutedtopH2.Thiswasnotnecessaryforthereactioncompleted,but thepHshouldbecheckedtoverifythatitisroughly2. Iffreshdowexisused,itshouldbewashedwithdeionizedwaterinafrittedfunnel overvacuum.Thisisduetoanorange,water-solubleimpuritythatispresentin newDowex.Whenmakingthecolumn,acolumnwithafritwasusedtoallowfor recyclingofthedowex.Oddly,thedowexpackedmoretightlyovertime,soallowing ittositbeforeuseisimportant.

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47 Synthesisof1,4-Dibenzyl-1,4,7-triazacyclononane[ Bn 2 TACN ]fromTACN 3HCl TheTACN 3HClsaltwasbasiedtogreaterthanpH13withaqueoussodiumhydroxide.Thegoldensolutionwasextractedwithchloroformx8mL.Theextractions weredriedoveranhydrousmagnesiumsulfatefor30minutes.Afterlteringthemixture, theltratewasrotaryevaporatedtoanoil.Someoftheoilwaskeptaside.Inatwo-neck roundbottomask,therestoftheoil.450g,3.5mmolwasdissolvedin15mLoffreshly distilledacetonitrile.Theaskwasequippedwithareuxcondenserandaseptum.Thesolutionwasheatedtoreuxundernitrogenandstirredvigorouslywithastirbar.Anhydrous potassiumcarbonate.962g,7.0mmolwasaddedtotheroundbottom.Thetemperature was75 C.Benzylbromide.82mL,1.179g,6.9mmolwasdissolvedin11mLofacetonitrile.Thebenzylbromidesolutionwasaddedviasyringeovervehours.Oncethe additionwascomplete,thereactionwasallowedtoproceedfortwohours.Afterthistime, thesolutionwashotltered,andtheltratewasrotaryevaporatedtoadeepgoldenoil.By NMR,theproductappearedtobeimpureandnotcompletelyreacted.Thecrudeyieldwas 99.5%.Asilicacolumnwasattemptedwithnosuccess. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.60d,2H, 7.50-7.20m,integrationcomplicatedbyCDCl 3 ,7.05d,2H,4.85s,1.5H,H 2 O,4.50 s,0.2H,PhCH 2 Cl,4.20s,1.4H,3.60s,0.5H,3.45s,2H,3.25s,1.3H,2.80s, 0.7H,2.40s,1.7H,2.05s,2.3H,CH 3 CN,1.85s,3.1H,H 2 O.SeegureA.7. ExperimentalNotes: Thereactionwaslikelynotconductedforalongenoughperiodoftime. Theamountofsodiumcarbonatewaslikelynothighenough. ConditionsclosertothoseusedforthebenzylationofTACNtsseebelowwould likelybeideal. 2.2.5RouteTwoB TACNtsSynthesis:HydrogenBromidewithPhenol ThisprocedurewastakenfromSessleretal.[40].Warning:ametalstirringrodshould notbeusedinHBrsolutions.Additionally,syringesusedwithHBrshouldbewashedwith

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48 Figure2.2. Awaterhosewasattachedtotheexitofthebubbler.Aglassfunnelwastted totheotherendofthehose.Afunnelwithanopeningroughlythesizeofabeakerwas used.Thisfunnelwassetontherimofabeakerofwaterandthejunctionwaswrapped withparalm.Atinyopeningwasleftoverthelipofthebeakertopreventthepressure frombuildingtoohigh.TheHBrwillbedissolvedinthewaterratherthanllingthehood withgas,pollutingtheenvironment. waternotacetone.TheHBrwillturnpurpleinacetone.Althoughitisn'tclearwhatthis colorisfrom,itprobablyisn'tbenecialtothereaction. Important:Thisreactionwillcoatthetygontubingtotheschlenklineandturnthe mineraloilinthebubblergrey/black.Itisimportantthatwhateverisinthetygontubing doesnotfallintothereaction.Inordertoassurethis,thetubingshouldbeloopedwith thehighestpointatthenitrogenadaptorattachedtothecondenserofthereactionask.A dedicatedtygontubeshouldbeused,soasnottoruinmultiplelines.Additionally,more precautionswerenecessarytotraptheHBr.Thisisshowningure2.2.Note:thefunnel shouldnottouchthetopofthewater!BecausetheHBris highly solubleinwater,thewater willbepulledupintothewaterhoseandpossiblyintothebubbler.Thiscouldpotential resultinwaterenteringthereactionvesselandthuscontaminationofreactionsusedinthe future. ImpureTACNts 3 .487g;56.59mmol;591.77g/molandphenolroughly40g; 425mmol;94.11g/molwereaddedtoatwoneckedroundbottomask,equippedwitha thermometerwithanadaptorandacondenser.Then450mlofHBr%byweightin glacialaceticacidwereaddedtothereactionaskatroomtemperature.Themixturewas

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49 thenplacedunderanitrogenatmosphereandstirredat90 Cfor36hours.Afterthistime, thereactionwasremovedfromheat.Onceithadreachedroomtemperature,thereaction wasltered.TheTACNts 2HBrwaswashedwithdiethylether.Thecolorofthesolid variedfromwhite/tantotan/purple. Thetannishsolidwasdissolvedin1MNaOHtopHgreaterthan12.Apinksolution resulted.Oncedissolved,theliquidwasextractedwithchloroform8x15ml.ThecombinedorganicphasesweredriedoverMgSO 4 formorethan30minutes.Afterltration,the liquidwasrotaryevaporatedtoyieldaclearoil.Thisoilwasextremelycleanandwasused withoutfurtherpurication.Theyieldofthisrepresentativereactionwas70%.218g. Theaverageyieldwas73%witharangefrom52%-89%.Thescalesofthereactionsvaried from7.5mmolto56.59mmol. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.69d,2H,Ar,7.35d,integration complicatedbyCHCl 3 ,Ar,3.3-3.2m,4H,TsNCH 2 ,3.2-3.05m,4H,CH 2 NH,2.90 s,4H,CH 2 NH,2.42s,3H,ArCH 3 ,1.70*s,4H,H 2 Oppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 : 143.444,135.791,129.868,127.425,54.239,49.873,49.702,21.705ppm.Seegure3.5. Experimentalnotes: Anicebathwasusedforthereuxcondenserofthereaction. Thecolorofthereactionuctuatedfromareddish-orangetoalmostblack.However, colordidnotseemtobeanindicatorforsuccessorfailure. Thereactionseemedstableduringsomeruns,butHBrgaswouldpouroutofthe schlenklineandtheglassjointsduringotherruns.Surprisingly,thisresultalsowas notanindicatorofsuccessorfailure. LargerscalereactionsseemedlessdesirablebecauseoftheamountofHBrthatwould bereleased. BecauseoftheHBrgasthatrunsthroughouttheschlenkline,otherreactionswere neverrunconcurrentlyinthesamehood.Thiswasimmenselyfrustrating,butno satisfactorysolutionwasestablishedduetothehazardofthegas.

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50 Synthesisof1,4-Dibenzyl-7-tosyl-1,4,7-triazacyclononane[ Bn 2 TACNts ] Theproductofthisreactionwastherstnovelcompound.However,asimilarprocedure,fromMahapatraetal.,wasmodiedforthisreaction[41].Theprocedurewas completed7times. Asingle-neckedroundbottomaskwasequippedwithareuxcondenserandanitrogen inlet.Intheroundbottom,TACNts.1mmol,1.162g,283.3915g/molwassuspended with10mloffreshlydistilledacetonitrile.Thesolutionwasplacedunderanitrogenatmosphere.Sodiumcarbonate.8mmol,3.476gwasnelygroundandmicrowavedbriey toremovewater.Thewhitesolidwasthenaddedtothestirredsolution.Rapidly,benzylchloride.61mmol,0.99mlwasaddedviasyringebeneaththeliquid.Thereaction wasbroughttoreux.TheexternaltemperatureofthePEGbathwasmeasuredbetween 70-80 C.Thereactionwasheldatreuxfor18hours.Afterthistime,themixturewas lteredandwashedwithacetonitrile.Theyellowltratewasrotaryevaporatedtoayellowoil,consistentlycontainingBn 2 TACNtsandBnCl.Theoilwasdissolvedinasmall amountofethanolandpouredintoroughly50mLofwater.Uponadditiontowater,a whitecrystallineproductprecipitated.ThisproductwasfreeofBnCl,butwasverywet. Thewhitesolidwasdriedinvacuoovernight.Afterdryingtheproductstillcontainedsome water,butwasotherwisepure.Theyieldforthisrepresentativereactionwas80.5%.524 g.Thereactionsrangedinscalefrom2.68mmolto5mmol.Theaverageyieldofthese reactionswas78%withavariationfrom65%to90.9%. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCD 3 CN:7.62d,2H, Ar,7.50-7.15m,12H,Ar,3.65s,4H,ArCH 2 NR,3.3-3.19m,4H,NCH 2 CH 2 NTs, 3.19-2.95m,4H,NCH 2 CH 2 NTs,2.62s,4H,BnNCH 2 CH 2 NBn,2.40s,3H,ArCH 3 2.19*s,13H,H 2 O,1.60*s,3.6H,H 2 Oppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCD 3 CN:144.463,141.283, 137.247,130.760,130.060,129.236,128.059,127.880,62.993,56.431,56.153,41.457, 21.529ppm.SeeguresA.8andA.9. Experimentalnotes: AlargeexcessofNa 2 CO 3 wasrequired,andthereactiontimewasincreasedto18 hours.Thesechangesweretheonlymodicationstotheliteratureprocedure.

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51 Synthesisof1,4-Dibenzyl-1,4,7-triazacyclononane[ Bn 2 TACN ]fromBn 2 TACNts Thisprocedurewasmodiedfromtwoprocedures[42,43].Important:Careshouldbe takennottotouchthequartzofthemercurylamp.Thelampshouldbecarefullywiped withaKimwipebeforeuse. Inasingle-neckask,Bn 2 TACNts.197g,2.58mmol,463.6378g/molwasaddedto 150mLof95%ethanoland22mLofdeionizedwater.p-Methoxybenzene.426g,10.3 mmolandNaBH 4 .992g,51.6mmolwereaddedtothesolution.Astirbarwasadded andtheroundbottomwasplacedundernitrogen. Thesingle-neckroundbottomwasplacedadjacenttothewatersleeveofthe450W mercurylamp.Ablastshieldwascoveredinaluminumfoilandplacedaroundthelampand roundbottom,sothattheroundbottomwasatthefocalpointoftheshield.Alargecardboard box,supportedbythreeringstandswasplacedovertheblastshield,lampandroundbottom. Heavyblackfeltwaswrappedaroundthebottomofthecardboardtocompletelyobstruct anylight.The`out'hoseofthewatersleevewasconnectedtoaninterlockwaterregulator whichturnsoffthelampifwaterstopsowing. Oncethewaterwasrunningrapidly,thelampwasturnedon.Thereactionwasleftto stirnexttothelampforthreedays.Themixturebecamenoticeablywarmduetotheheat fromthestirplate.Attheendofthethreedays,thereactionwasacidiedwithconcentrated hydrochloricacidtopH1-2.Thesolutionwasrotaryevaporatedtoayellowresidue.This residuewastakenupinwaterandwashedwithtoluenex10ml.Theaqueouslayer wasthenbasiedwith1MNaOHtopH12.Thebasicsolutionwasthenextractedinto chloroformx15ml.Thechloroformlayerwasrotaryevaporatedtoabrownoil,which wasthendissolvedin10mloftolueneand10mlofmethanol.Thesolutionwasmade acidicpH2withthesmallestvolumeofconcentratedhydrochloricacid.Thenliquid waslightlyrotaryevaporatedtoremoveonlyasmallamountofsolvent.Theroundbottom wasthenplacedintherefrigeratorfor2hourstoprecipitatethetanproduct.Thetan solidwaslteredtoyieldBn 2 TACNH HCl.Thesolidwasthendissolvedin1MNaOH topH12,andthebasicsolutionwasextractedwithchloroformx8ml.Thecombined chloroformlayerswererotaryevaporatedtoaclearyellowoil.Theyieldwas0.102gof unidentiedoil.Becausetheproducthasnotbeenfullycharacterized,nopercentyield

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52 wasdetermined. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCD 3 CN:7.60*s,0.2H,possiblyH 3 O + ,7.35-7.15m,10H, ArofBn,3.72s,2H,CH 2 ofBn,3.45s,2H,CH 2 ofBn,2.60t,4H,CH 2 ,2.422.30m,tripletoversingle,8H,CH 2 ,2.15s,20H,H 2 O,1.95p,11H,CH 3 CNppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCD 3 CN:206.854*,142.471,139.946,130.123,129.328,129.151,128.028, 127.727,63.644,58.789,54.492,54.242,46.787ppm.Seegures3.6and3.8. ExperimentalNotes: TheBn 2 TACNtswasnotcompletelysolubleinthemethanolandwaterreactionmixture.Forbetterresults,1-butanolshouldbetried. Thechambershouldcompletelyshieldeveryonefromlookingatthelamp.Thelamp isverybrightandwilldodamagetoeyesifvieweddirectly. Therotaryevaporationoftheacidiedreactionsolutionshouldbecomplete.Otherwise,theproductwillpartitionbetweenthetwophases. Dichloromethanewasnotused,asindicatedbytheprocedure,becauseitdegrades theTACNring. Thesaltissolubleinchloroform,sochloroformshouldnotbeusedfortherst extraction. Itwasnecessarytoisolatethesaltforpurity.Additionally,TACNsaltsarevery stable,soisolationofthesaltisidealifoneintendstostoretheproduct. Extractionswithbaseandchloroformshouldalwaysbedonequicklytoavoidcarbeneformation.CarbeneswilldegradetheTACNring. AttemptedSynthesisof1,4-Dibenzyl-1,4,7-triazacyclononane-7-monoacetate Inascrew-capNMRtube,0.75mlofCD 3 ODwereaddedtotheproductfromthe previousreaction.070g,0.23mmol.Lithiumhydroxidemonohydrate.030g,0.71 mmolandchloroaceticacid.033g,0.35mmolwereaddedtotheNMRtube.The NMRtubewasplacedundernitrogenthroughaneedle.Thereactionwasheatedtoreux

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53 for21hours.AcrudeNMRwastakentodeterminewhenthereactionwascomplete.Once thiswasdone,thereactionwasrotaryevaporatedtoayellowoil.Theoilwastakenupin 1mlofwaterandwashedwithchloroformx1ml.Thecombinedchloroformlayers weredriedoveranhydrousmagnesiumsulfatefor30minutes.Thesolidwasltered,and theltratewasrotaryevaporatedtoayellowoilmg.Theyieldcontainedanimpurity: chloroaceticacid.Becausetheproducthasnotbeenfullycharacterized,nopercentyield wasdetermined. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :7.25ppmm,integrationcomplicatedbyCHCl 3 Bn,4.70s,integrationcomplicatedbyunidentiedbroadpeak,ClCH 2 COOH,4.80-3.90 s,vbroad,possiblywater,3.80s,2H,CH 2 ofBn,3.52s,2H,CH 2 ofBn,3.35s,2H, CH 2 ofacetatearminligand,2.80-2.30m,12H,CH 2 ofTACNring,2.25*m,1.5H and1.12-1.00*m,1.5Hppm. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRCDCl 3 :176.164,138.274,137.729,129.331, 129.211,128.748,128.604,127.904,127.578,62.644,62.062,58.476,55.063,52.236, 51.066and50.557ppm.Seegure3.9.

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54 Chapter3 ResultsandDiscussion Bn 2 TCMAisanovelligand.Thusitssynthesisrequiresexploringmanymethodsbecauseasyntheticschemehasnotbeenpublished.Someaspectsofthesynthesis,however, maybesimilartootherligandscontainingaTACNring.Thusexaminingmethodsused tosynthesizesimilarligandscanbeavaluablerststeptowardsdevelopingapotential syntheticroute. C 3 symmetrictrisubstitutedTACNcompoundsareverycommonintheliterature[44 46].Synthesisofthesecompoundsisoftenstraightforwardbecauseeachnitrogenatomis bondedtothesamesubstituent.MonosubstitutedTACNligandsarealsopresentinliteraturemethods[47,48].SynthesesofthesecompoundsaretrickierthantheC 3 symmetric trisubstitutedligandsduetotheneedforselectivity.Morerecently,synthesesofTACN compoundscontainingdifferentsubstituentsontwoofthethreenitrogenatomshavebecomemorewidelyestablished[30,41,49].Thesemethodsrelyonsub-stoichiometricadditionsofsubstituentgroups,sub-stoichiometricadditionsofprotectinggroupsand/orclever deprotectionmethods. Inrecentyears,previousstudentsintheShermanlabhaveencounteredobstaclestowardsgeneratingcleansynthesesofamonosubstitutedTACNligand,1,4,7-triazacyclononanemonoacetate[TCMA].Becausetheproposedligand,1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate[Bn 2 TCMA],sharesmanysyntheticstepswithTCMA,onegoal ofthisworkwastodeterminethemostreliablemethodforselectivefunctionalizationof TACN.Therefore,thedecisionsregardingwhichexperimentalrouteswereandwerenot

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55 attemptedarepertinenttothecontinuationofresearchwithsimilarcompounds. Scheme3.1. AboveisthesynthesisemployedbyDuncanStewardandmostsubsequent studentsintheShermanlabtosynthesizeTCMA. In1994,DuncanStewardwastherststudentintheShermanlabtosynthesizeTCMA[50]. TCMAandtheTCMAcomplexwithCu 2+ hadbeenpreviouslycharacterizedbyStuderet al.in1989[51].Scheme3.1showsthesyntheticstepsthatStewardtooktopreparethe ligand.Themethodshownisrelevanttothecurrentgoalbecausethesyntheticschemeis similartothatusedinthiswork.Theimportantdistinctionisthelackofbenzylationin Steward'smethod.Inscheme3.1,therststepistoprotectthethreenitrogenatomsofdiethylenetriaminewithtosylgroups.Thetosylgroupsarealsoaddedtotheethyleneglycol toformleavinggroups.Thesetwocompoundsarethencombinedtoformtritosyltriazacyclononane.Thetosylgroupsarethenremoved.Stewardusedtwomethodsforthisstep: thesulfuricacidmethodandtheaqueoushydrogenbromidemethod.BothofthesemethodswereexploredinthisworkseeresultsanddiscussionofroutetwoAbelow.From thecompletedetosylation,thefreeTACNisthenallowedtoreactwithchloroaceticacidto formTCMA.MostotherstudentsintheShermanlabfollowedthisroute[36,52,53]. In2002,EllenWolfgangexploredaseparateroutethatsoughttoproduce1,4,7-triazacyclononane-7-methylpropionate[TCMP]investeps[53].Thissyntheticrouteisshown inscheme3.2.Inthismethod,ethylenediamineisprotectedwithtosylgroups.Thetosylateddiethylaminewasreactedwithethylenecarbonate.Theresultingtenmemberchain istosylatedagaintoproducegoodleavinggroups.Subsequently,thetosylatedproductis reactedwithbeta-alaninemethylesterhydrochloridetoproduce1,4-tosyl-1,4,7-triazacyclononane-N-methylpropionate.Unfortunately,thedetosylationmethodsmostcommonly

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56 Scheme3.2. Inthissyntheticroute,exploredbyEllenWolfgang,ethylenediamineistosylated,andthenreactedwithethylenecarbonate.Theresultingcompoundisthentosylated toformbetterleavinggroups.Thetosylatedproductisreactedwithbeta-alaninemethyl esterhydrochloride.AsubsequentdetosylationofTCMPts 2 wasattempted.Unfortunately, thedetosylationdestroyedthependant-arm. usedinTACNsynthesisweretooharshforWolfgang'sligand.Thedetosylationdestroyed thependantarm.Throughanalysis,thependantarmwasdeterminedtoselfeliminatein thehighlyacidicsolution[53]. Fromthesetwoapproaches,insightintopossiblesyntheticmethodsforthenovelligand, 1,4-dibenzyl-1,4,7-triazacyclononane-7-monoacetate[Bn 2 TCMA],canbedetermined.Becausethependantarmisreactive,thedibenzylationoftheTACNringneedstobecompletedbeforetheadditionofthependantarm.Thusthetwobenzylgroupsmustbeattached immediatelyafterdetosylation,eitherusingsub-stoichiometricquantitiesforastatistical yieldorselectivelyprotectingoneofthethreenitrogenatoms. Severalmethodswerereviewed.TherstmethodconsideredwasbasedonWolfgang's desiredroute.Thisproposalisshowninscheme3.3.Thesynthesiswasintriguingbecause ithasonlyvesteps.Fewerstepsusuallymeanhigheroverallyields,shortertimeto product,lesschemicalwasteandlesshassle.Mostnotably,thisreactionpathwouldrequire noprotectinggroups.Thusnodetosylationwouldbenecessary.Unfortunately,thisroute wasnotexplored.Atthistime,itisnotclearwhy.Futurestudentsshouldseriouslyconsider thismethodasanalternativeapproach! Scheme3.4showsasecondroutethatwasconsidered.Thisroutewasnotinitially attempted.Coincidentally,apaperbyHuangetal.waspublishedduringthetimeof

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57 Scheme3.3. Unexploredsyntheticroute:Thisproposedroutewasnotattempted,buthas goodpotential.ItisbasedofftheworkofEllenWolfgang[53]. Scheme3.4. Unexploredsyntheticroute:Thisproposalissimilartoarecentlypublished procedurethatwasnotabletocorrectlyformtheTACNringinsteptwo. thisresearchsuggestingthatscheme3.4wouldnotyieldthedesiredproduct[35].Inthe reactionusedbyHuangetal.,theRgroupsaremethylgroups.Aproposedmechanism fortheobservedreaction,showninscheme3.5wasgiventoexplaintheproductformed. Althoughtheproposedreactionwouldusebenzylgroupsinsteadofthemethylgroups, thereisahighprobabilitythatthereactionwouldresultinthesamesixmemberedring. ThethirdsyntheticrouteconsideredwasproposedbyDr.PaulScudderfortheaddition ofphenylrings.Theproposalhasbeenadapted,showninscheme3.6,forbenzylgroups. Withthebenzylgroups,thecyclizationsteprequirestheparticipationofmoresterically hinderedsites.However,itseemsmorethanplausible.Thisroutewasconsidered,but notattempted.Thiswasprimarilyduetotiming.Iftimepermits,thisreactionwouldbe advantageoustoattempt. Aftereliminatingthethreesyntheticschemesabove,tworoutestowardstheproposed ligandwereexplored.Routeoneisshowninscheme3.7.Routetwo,showninscheme3.8, requiredthesynthesisofTACNts 3 withsubsequentdetosylation.Additionally,RouteTwo

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58 Scheme3.5. ThismechanismwasproposedbyHuangetal.in2009toexplainthesmall sixmemberedringthatwasformedinthereaction.Whilebothpathsaandbareshown, pathadominatedintheliteraturewhenRwasCH 3 .ThisgurewasmodiedfromHuang etal.[35]. Scheme3.6. ThissyntheticroutewasproposedbyDr.Scudder,NewCollegeofFlorida. TherouteshownherestopsattheformationofBn 2 TACNts.Thesubsequentstepswould involvedetosylationandpendantarmattachment. subdividesintotwoseparateroutesatthepointofdetosylation. 3.1RouteOne RouteonesuggestedthesynthesisofTACNtswithouttheformationofatritosylintermediate.Themethodforthispathisshowninscheme3.7.Thetosylationofthe diethanolamine,stepone,wasnecessarytoformbetterleavinggroupstosylategroups -OTsandtoprotectthenitrogenatom.Thetosylamideislessreactivethantheamineof thestartingmaterialbecausetosylgroupsareelectronwithdrawing.Thisrststepwasvery successful,butsomedetailstoyieldbetterresultswereabsentfromtheliterature.These

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59 Scheme3.7. TsCl,triethylamine,methylenechloride;ethylenediamine,K 2 CO 3 acetonitrile. detailsareincludedintheexperimentalsectionforthereaction. ThesecondstepofroutetwoattemptedtoformthemonotosylatedTACNring.However,thereactiondidnotsuccessfullyproducecleanmonomerofthecompoundinsuitable quantities.Polymerformationishighlypreferredinthereaction.Theliteraturesuggests thataddingallofthereactantsatoncewillresultina78%yieldofcleanmonomer.As expected,thiswasnotthecase.Althoughslowadditionswereattempted,theyieldofthe monomerwasconsistentlylowandveryimpure,seegure3.1.Attemptstorecrystallize Figure3.1. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:HuangmethodcrudeTACNts theproductfrommethanolleadtoonlymoderatelycleanerproduct.Ifthehydrochloride saltofthemonomerwereattained,thisreactionmayprovetobeadvantageous.However, dramaticchangestothepublishedprocedurearenecessaryforhigheryieldsofmonomer. Thetwochangesemployedinthisworkareslowadditionofethylenediamineandamore

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60 dilutereactionseeexperimentalfordetails.Sincethepolymerandthemonomerhave similarNMRspectra,ameltingpointisthebestwaytodistinguishthetwocompounds. Themonomerisexpectedtohavealowmeltingpoint-84 C[40],butthepolymer wouldbeexpectedtohaveahighermeltingpoint-270 C. Althoughitisnotshowninscheme3.7,thelogicalnextstepwouldbetoattachbenzylgroupstothetworeactivenitrogenatomsoftheTACNtsmonomer.Subsequently,a detosylationoftheresultingBn 2 TACNtscompoundwouldbenecessary.Thiscouldbe accomplishedbythemethodsemployedinroutetwoBseebelow. 3.2RouteTwo Scheme3.8. ProposedLigandSynthesisRouteTwo:Thechemicalsusedforeachstep areasfollows:TsCl/Ether,H 2 O;TsCl/THF,H 2 O;60%NaHinoil,DMF; H 2 SO 4 ;HBr30%inAcOH,dryphenol;benzylchloride,K 2 CO 3 ,acetonitrile; benzylchloride,sodiumcarbonate,acetonitrile;400WHglamp,MeOH/H 2 O,NaBH 4 p )]TJ/F17 11.9552 Tf 9.289 0 Td [(methoxybenzene;chloroaceticacid,lithiumhydroxide,methanolandwater.

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61 SomestepsofroutetwohavebeenconductedbypreviousstudentstowardsthesynthesisofTCMA.Thusprogressonsomeofthesestepswasaidedsubstantiallybyinformation fromthesestudents[36,50,52]. Stepsoneandtwowereeasilyaccomplished.Thetosylationofdiethylenetriamine wascompletedtoprotectthenitrogenatomsofthestartingmaterial.Ethyleneglycolwas tosylatedinordertoformbetterleavinggroups-OTs.Althoughmanyprocedurescallfor theuseofpyridineasasolventfortosylationreactions,amuchsaferprocedureusingwater asthesolventwastakenfrompreviousstudentsintheShermanlab[36]. Thesynthesisof1,4,7-tritosyl-1,4,7-triazacyclononaneTACNts 3 wasslightlymore involvedthanthetwopreviousprocedures.Aswithallringclosurereactions,polymer formationisaninitialconcern.However,polymerizationhasneverbeenobservedforthis reactionbyanystudentsintheShermanlab.Techniquesforpreventingpolymerformation include:aslowadditionofoneorbothofthereactants,eventemperaturedistribution andforintramolecularclosuresdilutesolutions.Oddly,OlaKarylowskideterminedthat a86.3:1moleratioofDMFtoDETAts 3 providedabettersynthesisthana207:1mole ratio[36].Basedonthisinformation,itislikelythatpolymerizationisnotalargeriskwith thisreaction.Unfortunately,theproductofthisreactionalwayscontainedasignicant Figure3.2. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:TACNts 3

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62 amountofDETAts 3 .The 1 H-NMRspectrumoftheproductisshowningure3.2.The multipletpeakat3.20ppmisfromDETAts 3 andthebroadsingletpeakat1.70ppmis duetowater.Becauseboththeliterature[39]andpreviousstudentsindicatedthatthis wascommonanddidnotaffectsubsequentreactions,itwasignored.Havingsaidthis,a cleanerspectrumwouldbeideal,butthetimecommitmentinvolvedinndingapurication methodwastoogreatofadeterrent. FromTACNts 3 twoseparatesub-routeswereexplored.ThesearenamedroutetwoA androutetwoB.Becausebothofthesepathsleadtointerestingdiscoveries,athorough reviewofeachispertinenttobothcurrentandfutureresearch. 3.2.1RouteTwoA RoutetwoAbeginswithacompletedetosylationofTACNts 3 .Scheme3.9showsthe Scheme3.9. ProposedSynthesisforRouteTwoA:thenumbersusedoverthearrows correspondtothenumbersinscheme3.8forconsistency.H 2 SO 4 ;benzylchloride, K 2 CO 3 ,acetonitrile;chloroaceticacid,lithiumhydroxidemonohydrate,methanoland water. stepsinvolvedinthescheme.ThissyntheticmethodissimilartothatusedbyDuncan Stewardseescheme3.1[50]. TherstreactionofroutetwoA,stepfour,hasseenmixedresultsinthepast.Dueto difcultiesencounteredbypreviousstudents,severaldetosylationmethodswereattempted forstepfour.Table3.1isasimplecomparisonofthereactionconditionsused. Stewardhadsuccesswithtwoformsofdetosylation:thesulfuricacidmethodandthe aqueoushydrobromicacidinaceticacidmethod.Inhisthesis,itisclearthatheultimately preferredtheaqueousHBrmethodfromKoyamaandYoshino[28]duetothestability

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63 Literature Reactantsmmol Runs Yield Product Yang[31] HBrinAcOH TACNts 3 1 N/A astatisticalmixoftosylation states Koyama[28] HBrinH 2 O AcOH TACNts 3 2 NA astatisticalmixoftosylation states Searleand Geue[39] ConH 2 SO 4 TACNts 3 1 47% pureTACN Table3.1. DetosylationMethods:Thersttwomethodsaresimilar:oneusesHBrinacetic acidandtheotherusesHBrinbothwaterandaceticacid.Thesulfuricacidmethodisthe onlymethodthatresultedincompletedetosylation. ofthesaltformed[50].In1998and1999,respectively,MichelleGeorgeandOlaKarylowskisynthesizedfreeTACN[36,52].Georgeutilizedonlythesulfuricacidmethod,and KarylowskiutilizedonlytheHBrdetosylationmethodfromKoyamaandYoshino[36]. However,subsequentstudentsintheShermanlabattemptedtheaqueousHBrmethodwith puzzlinglylittlesuccess.Theresultsofthedetosylationmethodsintable3.1arediscussed here.TheaqueousHBrmethodandtheHBrinaceticacidmethodarecombinedbecause ofthesimilarityintheresultingproduct. HydrogenBromideDetosylation AllofthepreviousstudentsintheShermanlabhavereliedonthemethodfromKoyama andYoshino[28],whichinvolvesaddingaqueousHBrandglacialaceticacidtoTACNts 3 Intheexperimentsofstudentsfromthemoredistantpast,thissynthesisresultedincompletedetosylation[36,50].However,theexperimentsofmorerecentstudentshaveyielded amixtureoftosylationstates[37,38].Onestudentproposedthatthisisduetoachange inthecommercialpurityofHBrovertime[37].However,bothStewardandKarylowski indicatedthatpartialdetosylationalsooccurredineachoftheirexperimentswhentheHBr bottlewasolder.DespitealwaysusingfreshlyopenedHBr,thismethodwasneversuccessfulintheexperimentsconductedforthiswork.Additionally,theYangandZompa procedurewasalsoattemptedwithfreshHBr33%inAcOH.However,thisexperiment

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64 wasalsounsuccessful. Becausethereactionisnotsealed,thehydrobromicacidevaporatesfromthesolution veryrapidly.ItislikelythatthelossofHBrcontributestothemixtureofTACN,TACNts, TACNts 2 andTACNts 3 inthenalproduct.Inordertopreventthisproblem,theGraham andWeatherburnprocedurecallsforasealedreactionvessel[54].However,becauseof safetyconcerns,thisreactionhasnotbeenexplored.InordertopreventthelossofHBr, anitrogenballoonwasusedinonereaction,butthehydrobromicacidistoocorrosivefor thistechnique.Whileacolumncouldbeusedtoseparatetheproducts,thisprocesswould betimeconsumingandresultinaverylowyield.Additionally,dichloromethaneisoften usedinsuchcolumns,whichposestheriskofTACNringdegradation. Duetothefailureofthesereactions,thesulfuricacidreactionwasattempted. SulfuricAcidDetosylation BothDuncanStewardandMichelleGeorgeusedasimilarreactiontothesulfuricacid detosylationemployedhereseetheexperimentalsection.Bothstudentshadsuccess cleavingallthreeofthetosylgroupsofTACNts 3 [50,52].However,inSteward'sresearch, theworkupyieldedahygroscopicproduct.Duetothisdifculty,herecommendedcontinuingwiththeHBrsynthesis.However,theSearleandGeuemethod[39],describedinthe experimentalsectionofthiswork,circumventstheisolationofthehygroscopicproductby usinganionexchangecolumntoeluteTACN 3HCl.TACN 3HClisanon-hygroscopic productthatisstableandeasytohandle. Additionally,thespectrumwasverycleandespiteusingimpureTACNts 3 .The 1 HNMR,takeninD 2 Owith0.5%TSP,isshowningure3.3.TheTACN 3HClpeakappears at3.4ppm.TheTACN 3HClderivedfromtheSearleandGeuemethodcanbereadily convertedtofreeTACN. Analternativeworkupofthesulfuricacidreactionwasattempted.Thereactionmixture wasslowlybasiedwithaqueoussodiumhydroxidetoroughlypH12.Thebasicmixture wasextractedwithchloroform,driedoveranhydrousmagnesiumsulfate,lteredandrotary evaporated.Theproductfromthealternativeworkupisshowningure3.4.Thisalternative methodwasclearlynotassuccessful.Thereiswhatappearstobeasmallamountofthefree

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65 Figure3.3. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:TACN 3HCl.Thesmallpeaksat0.6and2.9ppmare duetotheindicatorTSP. TACNinthespectrum:themediumheightsingletpeakat2.67ppmthepeakispredicted toappearmoreupeldthantheTACN 3HCl.Evendeviationsfromtheliteraturethat seemreasonablearenotworththeriskofattempting.Followingtheexactprocedurefrom theexperimentalandtheliterature[39]iscriticaltosuccess. BenzylationofTACN Thesubsequentbenzylationreactionreliesonastatisticaladditionofbenzylchloride toTACN.Thisreactionwasonlyattemptedonceandunsuccessfully.Thereuxtimewas eighthours,andtheratioofanhydrouspotassiumcarbonatetoTACNwastwotoone.The timeandreagentratiowerebasedonaliteratureprocedure[41]developedfortheaddition ofisopropylgroupstoTACNts.Unfortunately,theseconditionswerelikelynotoptimal. Basedontable3.2madeforthebenzylationofTACNtsinroutetwoB,thereactiontime waslikelytooshort,andtheratioofanhydrouspotassiumcarbonatetoTACNwaslikely nothighenoughtopushthereactionforward.Thispathmaybepossibleiftheseproblems wereadjusted.Inthefuture,thereactionshouldbeallowedtocontinueatleast18hours, andthepotassiumcarbonatetoTACNratioshouldbeclosertoeighttoone.

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66 Figure3.4. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:TACNalternateworkup 3.2.2RouteTwoB RoutetwoBbeginswithapartialdetosylationofTACNts 3 toformTACNts.Benzyl groupsarethenaddedtoTACNts.ThenextstepisthedetosylationofBn 2 TACNts,followedbytheadditionoftheacetatependantarm.Theresultsfromthesesynthesesare detailedinthefollowingsections. SynthesisofTACNtsfromTACNts 3 ThereactionfromSessleretal.calledforHBr/AcOH,phenolandTACNts 3 .Although itwassimilartoareactionemployedinroutetwoAHBrinAcOHreaction,theadditionof phenolchangedtheoutcomedramatically.TheresultwascleanmonotosylatedTACN:no statisticalmixturewasproducedinanyreactioncompleted.Figure3.5showstheproduct ofthephenolbaseddetosylationreaction.Thespectrumwasconsistentlycleandespite usingimpureTACNts 3 .Theonlyimpuritiesfoundwerewaterandvacuumgreaseat1.80 ppmand0.05ppm,respectively.

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67 Scheme3.10. ProposedSynthesisforRouteTwoB:thenumbersusedoverthearrowscorrespondtothenumbersinscheme3.8forconsistency.HBr30%inAcOH, dryphenol;benzylchloride,sodiumcarbonate,acetonitrile;400WHglamp, MeOH/H 2 O,NaBH 4 ,p )]TJ/F17 11.9552 Tf 9.289 0 Td [(methoxybenzene;chloroaceticacid,lithiumhydroxidemonohydrate,methanolandwater. SynthesisofBn 2 TACNts ThecleanthoughslightlywetTACNtswasusedinthefollowingbenzylationreaction toproducetherstnovelproductofthiswork:Bn 2 TACNts.Findingasuitablesynthesis forthisreactionrequiredasignicantamountofexperimentation.Severalbenzylation reactionswereattemptedseetable3.2.Eachofthesereactionswerebasedonliterature proceduresindicatedinthetable. ThereactionfromBeisseletal.wastherstreactionattempted.Thepotassiumhydroxideneedstobenelyground.However,inthehumidity,itpicksuptoomuchwater forthisreactiontoproceed.TheQianetal.procedurewasattemptedonce.Thereaction seemedfavorablebecausethebasewasinthesamephaseastheTACNts.Unfortunately, theproductwasveryimpureanddidnotsuccessfullyresolveonasilicacolumn. ThereactionsadaptedfromStavilaetal.andMahapatraetal.wereverysimilartoone another.AhighermoleratioofbasetoTACNtswasrequiredthantheliteraturesuggested; theratiowasincreasedfrom2:1intheliteratureto8:1inthiswork.Additionally,the timeofthereactionwasincreasedfromeighthoursto18hours.Bothofthesuccessful proceduresproducedBn 2 TACNts. Inoneexperiment,aphasetransfercatalysttetra-n-butylammoniumiodidewasadded tothereactionfromMahapatraetal.Thecatalystdidnotseemtochangetheyieldand

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68 Literature* TACNts mmol Solvent Base mmol BnCl mmol ProductandYield Beissel[45] 10 Toluene 7mL KOH 0.328g 0.65 ml Startingmaterialwasrecovered.Noreactionwas found. Qian[55] 4.65 MeOH 100mL Et 3 N 6.25 9.3 Productwastooimpure tocalculateyield. Stavila[49] 2.749 CH 3 CN 17mL K 2 CO 3 3.08g 0.63 ml 80%butcontaminated withvacuumgrease. Mahapatra*[41] 4.02 CH 3 CN 10mL Na 2 CO 3 32.2 8.44 78% Table3.2. Thesemethodsweretestedtoobservetheabilityofeachreactionconditionto produceBn 2 TACNts.Whilethersttwoliteraturemethodswerenotverysuccessful,the secondtwoweresuccessfulwithonlyminorchanges.Theproductsfromeachofthese successfulreactionsweregoodcandidatesforthenextreaction.*Theliteraturecitedwas modiedspecicallyforthedesiredsynthesis.Themodicationsarenotedintheaccompanyingtext.

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69 Figure3.5. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:TACNtsproductfromHBr/AcOHandPhenoldetosylationofTACNts 3 remainedintheproductasanimpurity.Forallfurtherreactions,themodicationsin thereactiontimeandtheamountofbasewereemployedintheMahapatraetal.method becauseofthehigherpuritythanmostothermethods.WhilethemethodfromStavilaetal. wasalsosuccessful,sodiumcarbonatewasmorereadilyavailable. DetosylationofBn 2 TACNts FromBn 2 TACNts,severaldetosylationmethodswereemployed.Therstmethods exploredwerebasedonthedetosylationmethodsdescribedforTACNts 3 :thesulfuricacid method,theaqueousHBrmethodandtheHBr/AcOHmethod.Allthreewereunsuccessful. ThesulfuricaciddetosylationremovedthebenzylgroupsattachedtotheTACNringat roughlythesamerateasthetosylgroups.Reactionswithsulfuricacidwererunat70,80, 85,90,100and115 C.By 1 H-NMR,itwasdeterminedthatdetosylationwasnotachieved until85-90 C.Atthispoint,thebenzylgroupswerealsodeterioratedatonlyaslightly lowerratethanthetosylcleavage.Theproductwasnotsufcientforfurtheruse.BothHBr methodsdestroyedtheBn 2 TACNtsbeyondNMRrecognition. Aftersearchingforlessharshmethods,aphotolysisreactionwasattempted.Thepho-

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70 tolysisreaction,ultimatelyemployedtoremovethetosylgroupfromBn 2 TACNts,was designedforsyntheticaminoaciddeprotection[42].Whilethereactioninitiallyappeared toworkwell,therearesomeodditiesinthe 1 Hand 13 C-NMRspectra.Acomparisonofthe Figure3.6. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:Bn 2 TACNH experimental 1 H-NMRspectrum,showningure3.6,withthepredictedspectrum,shown ingure3.7,makesthisclearer.Intheexperimentalspectrum,singletpeaksat7.59and 2.05ppmcorrespondtoanunidentiedimpurityandalargeamountofwater,respectively. TheunusualfeatureisintheTACNboundbenzylgroups.Inthepredictedspectrum,a 4 Figure3.7. ThetwoguresshowthechemicalshiftspredictedbyChemDrawfor Bn 2 TACNH .Thepredictedspectrumisofthe 1 H )]TJ/F54 11.9552 Tf 10.949 0 Td [(NMR spectrumof Bn 2 TACNH

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71 singletpeakat3.62ppmrepresentstheCH 2 oftheboundbenzylgroups.Thepredicted singletshouldintegrateforfourprotons.Intheexperimentalspectrum,therearetwosingletpeaksat3.72and3.45ppm.Eachofthesepeaksintegratesfortwoprotonsexactly. Theappearanceoftwosingletsseparatedthisfariscongruentwithachemicaldifference betweentheCH 2 ofthetwobenzylgroups. Moreover,afterexaminingthe 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMR,itisclearthatthedifferenceinchemical shiftofthebenzyliccarbonsdecreasesfromtheCH 2 totheparapositionofthearomaticring.Figure3.8showstheexperimental 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRofthesamecompound.The Figure3.8. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:Bn 2 TACNH 13 C )]TJ/F17 11.9552 Tf 9.29 0 Td [(NMRshowsoneimpurityat206.854ppm.Thiscarbonwouldlikelybelongtoacarbonyl.Themoreinterestingaspectofthespectrumisthechemicalshiftdifferenceofthe benzylCH 2 groups.644and58.789ppm;theipsocarbons.471and139.946;and theortho,meta,andparacarbons.123,129.328,129.151,128.028and127.727.The ortho,paraandmetacarbonsaretooclosetobeaccuratelyassigned.Ofthesearomatic carbons,oneisthoughttobethesamechemicalshiftinbothbenzylgroups.Thiswould explainthepresenceofsevenaromaticpeaksratherthaneight. Observingthebenzylpeaks,itbecomesclearthatthelargestdiscrepancyinshiftis attheCH 2 positionwithaseparationof4.855ppm.Theipsocarbonshaveaslightly

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72 smallerdifferenceinshiftwithaseparationof2.525ppm.Theremainingortho,meta andparacarbonsareallwithin2.396ppmofeachother.Thissuggeststhatwhateveris differentiatingthetwobenzylgroupshasthehighesteffectneartheCH 2 anddoesnot affectthecarbonsfurtheronthearomaticringasmuch. Manyattemptsweremadetoexplainthepresenceoftwosingletpeaksinthe 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMR throughChemDrawpredictions.However,noobservedalterationofthestructuresatisfactorilyexplainedthedifferenceinthetwoCH 2 groups.Witheverymodicationmade,the patterninthe 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRfrom3.80to2.20ppmwasdrasticallychanged:severalmore tripletpeaksaroseduetothelossofsymmetry.Thusthesestructureswerenotconsidered valid.Despiteintenseeffort,nootherstructurecouldbehypothesizedforthecompound. Thoughitmaynotbechemicallyrealistic,itishopedthatthisisthedesiredproduct.Despitetheuncertainty,thenextstep,additionofthependantarm,wasattempted.Itwasfelt thattheligandcouldbechemicallyinterestingdespiteitsunusualnature. AttemptedSynthesisof1,4-Dibenzyl-1,4,7-triazacyclononane-7-monoacetate Theproductofthependantarmadditionwasdifculttopurifybasedontheexcess ofchloroaceticacidused.Duetothemicromolarscale,therewasmorechloroaceticacid addedthanintended.Additionally,thechloroformwasheswereusedtopartitiontheproductfromthechloroaceticacid.However,bothcompoundsaresolubleinwaterandchloroform.Theproductisexpectedtobeslightlymoresolubleinchloroform,whichiswhythis workupwasused. Theuncertaintyintheidentityofthe1,4-dibenzyl-7-tosyl-1,4,7-triazacyclononanestartingmaterialmeansthattheproductisnotyetcompletelyidentiable.Thetwoseparate peaksforeachofthebenzylCH 2 groupsremainintheproductofthisreaction.Despite thiscontinuedoddity,thetentativeproductisBn 2 TCMA.Itisclearthatwhateverthestartingmaterial,theacetatearmwassuccessfullyaddedtoit.Thenewpeakat3.35ppmthat appearedduringthereactionisattributedtotheacetatearm.Therefore,itspresenceinthe spectrumisindicativeofthereaction'ssuccess,seegure3.9.Thispeakat3.35ppmintegratesfortwoprotonsasexpected.Unfortunately,chloroaceticacidisstillpresentinthe crudeproduct.Afewotherimpuritiesarealsoclearinthespectrum.Asacomparison,the

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73 Figure3.9. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:nalligand predicted 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrumforBn 2 TCMAisshowningure3.10.A 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRofthe Figure3.10. ThetwoguresshowthechemicalshiftspredictedbyChemDrawfor Bn 2 TCMA .Thepredictedspectrumisofthe 1 H )]TJ/F54 11.9552 Tf 10.95 0 Td [(NMR spectrumof Bn 2 TCMA productwasalsotakenandisshowningure3.11.Thepeaksinthis 13 C-NMRspectrum correspondwellwiththeadditionofanacetatearmtothestartingmaterial. However,fromthesespectra,theidentityoftheproductisstillunclear.

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74 Figure3.11. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:nalligand

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75 Chapter4 Conclusion Twodistinctroutestotheproposedligand,4-dibenzyl-1,4,7-triazacyclononane-7monoacetatehavebeenthoroughlystudied.RouteonewasatwostepprocesstoTACNts, butthesynthesiswasunsuccessfulinproducingthemonomericTACNtsinacceptablepurityandyield.Polymerformationprovedtobetoomuchofaprobleminthesynthetic method.Ifspecicadjustmentsaremadetothesynthesis,thisroutemaybeplausiblein thefuture.Theseadjustmentsareaddressedintheresultsanddiscussionsectionofthis work. Routetwosubdividedintotwosub-routes:routestwoAandB.RoutetwoArequired thesynthesisofTACN.Inthiswork,threemethodswereattemptedtoproducefreeTACN. However,onlytheSearleandGeuemethodwassuccessful[39].Thusinthefuture,thesynthesisofTACNshouldfollowthisprocedureexactly,whichisoutlinedintheexperimental chapterandtheliterature.FromthefreeTACN,astatisticaladditionofbenzylchloride wasattempted.Unfortunately,theexperimentwasonlyattemptedonce,unsuccessfully. Intuitionsuggeststhatifoptimized,thisprocedureshouldbesuccessful. RoutetwoBrequiredthedetosylationofTACNts 3 toTACNts,whichwascompleted successfully,withoutexception.Thisreactionwasneithertimeconsumingnordifcult toworkup.Duetotherelativeease,thismethodwaspreferredovertheSearleandGeue method.Inthefollowingstep,TACNtswassuccessfullybenzylatedtoproduceBn 2 TACNts. ThisreactionwasalsopreferredoverthecorrespondingreactioninroutetwoAthebenzylationoffreeTACNbecausetherewasnoriskofproducingastatisticalmixtureofthe

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76 monobenzylated,dibenzylatedandtribenzylatedproduct. ThemostdifcultaspectofRoutetwoBwasthedetosylationofBn 2 TACNts.After severaldifferentdetosylationexperimentswereattempted,aphotolysisreactionwasemployedtoremovethetosylgroups.Becausephotolysisreactionsrelyonbombardinga solutionwithlight,sideproductsandimpuritiesareoftencommon.However,theproductofthisreactionprovedtobeunusuallydifculttocharacterize.Through 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMR and 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMR,thereappearstobeachemicaldifferencebetweenthetwobenzylgroups basedonchangesinchemicalshift.However,theCH 2 groupsoftheTACNringarenot disturbedfromtheexpectedvalues.Allattemptstoaccountforthedifferenceinshiftwere unsuccessful.Moreover,thiseffecthasnotbeenseeninanyoftheliteraturereviewedof N-arylatedTACNcompounds.Thusthecharacterizationofthephotolyticproducthasnot beencompleted. Despitethispitfall,theproductofthephotolysisreactionwasusedinasmallscale synthesistoattachtheacetatependantarm.Theadditionofthependantarmtothestarting materialwassuccessfulbasedonboth 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRand 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectra.However,puricationoftheproductneedstobecompleted.Althoughfullcharacterizationoftheresulting ligandhasnotyetbeenaccomplished,acomplexoftheligandtomanganesewouldstillbe chemicallyinteresting.Moreover,acomplexwouldallowforX-raycrystallography,which wouldidentifytheexactligandstructure. Towardstheintendedligandstructure,otherdetosylationmethodsshouldalsobesought outandattempted.Duetotherobustnatureoftosylprotectinggroups,theyareverydifculttocleave.However,thisalsomeansthatthereisalargebodyofresearchondetosylatingtosylamides.Additionally,anHBr/AcOHwithphenoldetosylationwasneverattemptedontheBn 2 TACNtscompound.ItispossiblethattheadditionofphenolcouldpromotethereactionandpreventdegradationoftheBn 2 TACNHligand.Onceanappropriate detosylationmethodisestablished,progresstowardsthedesiredligandandthecorrespondingmetalcomplexishighlyprobable.

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77 AppendixA Appendix:SpectraandPredicted Spectra ForsomeproductspredictedNMRspectraareincludedforcomparison.Forthese predictions,chemicalshiftsforsymmetricpiecesareomittedforclarity.Thesepeaksare expectedtobethesameastheirsymmetriccounterparts;thiswouldnotbetrueforsterically hinderedstructures.

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78 FigureA.1. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:DEAts 3

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79 FigureA.2. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:Huangmethodpolymer

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80 FigureA.3. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:DETAts 3

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81 FigureA.4. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:DETAts 3

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82 FigureA.5. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:EGOts 2

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83 FigureA.6. 13 C )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:EGOts 2

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84 FigureA.7. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:StatisticalbenzylationofTACNts

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85 FigureA.8. 1 H )]TJ/F17 11.9552 Tf 9.289 0 Td [(NMRspectrum:dibenzylationofTACNts

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86 FigureA.9. 13 C )]TJ/F17 11.9552 Tf 9.29 0 Td [(NMRspectrum:dibenzylationofTACNts

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