ERROR LOADING HTML FROM SOURCE (http://ncf.sobek.ufl.edu//design/skins/UFDC/html/header_item.html)

Towards a Synthetic Model for RuBisCo

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004821/00001

Material Information

Title: Towards a Synthetic Model for RuBisCo Synthesis of a Magnesium Complex of Bis(3,5-di-tert-butylpyrazol-1-yl) Acetate
Physical Description: Book
Language: English
Creator: McCamant, Samuel
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: RuBisCo
Inorganic
Chemistry
Scorpionate
Botbpza
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ligand bis-(3,5-ditertbutylpyrazol-1-yl) acetic acid was prepared in 37% yield using a three step procedure, and characterized using NMR, NMR, and IR spectrosopies. A complex of this ligand and magnesium was prepared in 51% yield and characterized using NMR, NMR, and IR spectroscopies. The changes in chemical shifts between the unbound ligand and the complex support the conclusion that a complex formed, but were not sufficient to determine the formula of the complex. A decrease in the C=O stretch from 1754 cm to 1657 cm also supports the conclusion that a complex formed. The suitability of the complex for modeling the active site of RuBisCo is also addressed.
Statement of Responsibility: by Samuel McCamant
Thesis: Thesis (B.A.) -- New College of Florida, 2013
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 Libraries, 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. 2013 M12
System ID: NCFE004821:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004821/00001

Material Information

Title: Towards a Synthetic Model for RuBisCo Synthesis of a Magnesium Complex of Bis(3,5-di-tert-butylpyrazol-1-yl) Acetate
Physical Description: Book
Language: English
Creator: McCamant, Samuel
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: RuBisCo
Inorganic
Chemistry
Scorpionate
Botbpza
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ligand bis-(3,5-ditertbutylpyrazol-1-yl) acetic acid was prepared in 37% yield using a three step procedure, and characterized using NMR, NMR, and IR spectrosopies. A complex of this ligand and magnesium was prepared in 51% yield and characterized using NMR, NMR, and IR spectroscopies. The changes in chemical shifts between the unbound ligand and the complex support the conclusion that a complex formed, but were not sufficient to determine the formula of the complex. A decrease in the C=O stretch from 1754 cm to 1657 cm also supports the conclusion that a complex formed. The suitability of the complex for modeling the active site of RuBisCo is also addressed.
Statement of Responsibility: by Samuel McCamant
Thesis: Thesis (B.A.) -- New College of Florida, 2013
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 Libraries, 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. 2013 M12
System ID: NCFE004821:00001


This item is only available as the following downloads:


Full Text

PAGE 1

TowardsaSyntheticModelforRuBisCo:Synthesisofa MagnesiumComplexofBis,5-di-tert-butylpyrazol-1-ylAcetate BySamuelE.McCamant AThesis SubmittedtotheDivisionofNaturalSciences NewCollegeofFlorida InpartialfulllmentoftherequirementsforthedegreeofBachelorofArtsinChemistry Sarasota,Florida May,2013

PAGE 2

Acknowledgements I'despeciallyliketothankprofessorShermanfortakingmeontoherresearch team,andherguidanceandassistance.Thanksgoesouttoallmyprofessors,chemistryandotherwise,forimpartingknowledgeandhelpingmeunderstandtheworld better.Additionally,I'dliketothankSethHearldforassistingwithlabworkduringfallsemester,andforperformingthesynthesisofdtbpz.Thanksarealsoowed totheotherShermanlabmembers:LauraCunningham,JaclynnWindsor,Melissa Campbell,StephanieBamberger,andNealLacey,foroccasionaladvice,support, andcompany. Iwouldalsoliketothankthosewhogavemesupportduringthethesisprocessoutsidethelab.Naturally,Iwouldliketothankmyparentsforalltheircontributionsovertheyears.SpecialthanksgoestoSusannaPayne-Passmore,forher endlesssupportandencouragement,aswellasherfrequenteortstokeepmeon track.I'dalsoliketothankJamesCarterandDylanGygaxforaccompanyingme inthelabwhenIneededtostaylate,andotherwisekeepingmecompanywhenI neededit.Also,myneighborsinB-dorm:RobertWardwhokindlyprintedoutmy committeecopiesandlibrarycopyofmythesisattheprintshop,EthanFenner, ChelseaLucas,AnnieCarter,ShaneDonglasan,FebeRamirez,andColtonDodd, forencouragingmetopersistinmywork,andbeingavailablewhenIneededa break.

PAGE 3

TowardsaSyntheticModelforRubisco:SynthesisofaMagnesium ComplexofBis-3,5-di-tert-butylpyrazol-1-yl-acetate SamuelMcCamant NewCollegeofFlorida,2013 ABSTRACT Theligandbis-,5-ditertbutylpyrazol-1-ylaceticacidwaspreparedin37% yieldusingathreestepprocedure,andcharacterizedusing 1 H NMR, 13 C NMR, andIRspectrosopies.Acomplexofthisligandandmagnesiumwaspreparedin 51%yieldandcharacterizedusing 1 H NMR, 13 C NMR,andIRspectroscopies.The changesinchemicalshiftsbetweentheunboundligandandthecomplexsupport theconclusionthatacomplexformed,butwerenotsucienttodeterminetheformulaofthecomplex.AdecreaseintheC=Ostretchfrom1754cm )]TJ/F41 7.9701 Tf 6.586 0 Td [(1 to1657cm )]TJ/F41 7.9701 Tf 6.586 0 Td [(1 alsosupportstheconclusionthatacomplexformed.Thesuitabilityofthecomplex formodelingtheactivesiteofRuBisCoisalsoaddressed. Dr.SuzanneE.Sherman DivisionofNaturalSciences/Chemistry

PAGE 4

Contents Abstract3 1.Introduction:5 1.1.RuBisCo..................................5 1.2.OptimizingRuBisCo...........................19 1.3.ActiveSiteModeling...........................25 2.Experimental33 2.1.General..................................33 2.2.Synthesisof3,5-Ditertbutylpyrazole...................33 2.3.SynthesisofBis-,5-ditertbutylpyrazol-1-ylmethane.........34 2.4.SynthesisofBis-,5-ditertbutylpyrazol-1-ylaceticAcid........35 2.5.Synthesisof[Magnesiumbis-,5-ditertbutylpyrazol-1-ylacetateChloride]....................................37 3.ResultsandDiscussion38 3.1.LigandSynthesis.............................38 3.1.1.3,5-Ditertbutylpyrazole......................38 i

PAGE 5

Contents 3.1.2.Bis-,5-ditertbutylpyrazol-1-ylmethane............39 3.1.3.Bis-,5-ditertbutylpyrazol-1-ylaceticacid...........43 3.2.MetalComplexSynthesis.........................45 3.2.1.[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ].......................45 3.3.FutureWork:...............................51 A.SelectedNMRandIRSpectra53 Bibliography64 ii

PAGE 6

ListofFigures 1.1.AdiagramoftheCalvinCyclereactions.Notethatthenumberofarrowsindicatesthequantityofeachmoleculethatundergoeseachspecicstep,andtheoverallreactionsarestoichiometricallybalanced.Three RuBPformsix3PGreferredtoasPGAinthegure,andveofthese areusedtoregeneratethreeRuBPwiththeinputof9ATPinthephosphorylationof3PG,andthreeinthephosphorylationofRu5P,andsix NADPHusedtoreducetheDPGA.Thesixth3PGisremovedfrom thecycletothegeneralcycoplasmafterconversiontoGAL3Pandused forsugarbuilding.Takenfromref.9.................7 1.2.ExamplesofthefourmainformsofRuBisCo:formI,formII,formIII, andformIVorRuBisCo-likeprotein.Takenfromref.44.....13 1.3.ThestructureofformIRuBisCo.Thelargesubunitsareshowninblue andaqua,andthesmallsubunitsinyellow.Theredmoleculeisatransitionstatemimic.AisthetetramersubunitofformI,composedoftwo LSdimers.BandCillustratehowfouroftheseunitsarearrangedinform IRuBisCo'sstructure.Takenfromref.6...............14 1

PAGE 7

ListofFigures 1.4.OneproposedmechanismforthexationofcarbondioxidebyRuBisCo. Takenfromref.47...........................16 1.5.ArecentlyproposedmechanismforcarbondioxidexationbyRuBisCo, withanemphasisonprotontransfer.Protonsfromsolventarecircled, andtheprotonsfromH3andO3arenotedbyboldtextandaz-shaped arrow,respectively.Takenfromref.48...............18 1.6.Thesimilaritiesanddierencesofthephotorespiratorycycleinplants andcyanobacteria.Thepathwayforphotorespirationinplantsishighlightedingreen,andthereactionsspecictocyanobacteriaareshown inorange.Takenfromref.10.....................21 1.7.X-raycrystalstructureofaMgTCMAcomplex.Takenfromref.5626 1.8.bis,5-diisopropylpyrazole-1-ylacetate,orbdippza.Takenfromref.6228 1.9.Schemeforsynthesisof3,5-diisopropylpyrazolefrom1,6-dimethyl-3,5heptanedione.Takenfromref.62...................30 1.10.Schemeforsynthesisofbdippzmfrom3,5-diisopropylpyrazoleanddichloromethane. TEBA,ortriethylbenzylammoniumchloride,wasusedasaphasetransfercatalyst.Takenfromref.62....................30 1.11.Schemeforthesynthesisofbdippzafrombdippzm.Takenfromref.6230 1.12.Schemeforthesynthesisof[ZnbdippzaCl] 2 fromZnCl 2 andbdippza. Takenfromref.62...........................31 1.13.Schemeforsynthesisof[Mgbdippza 2 ]frombdippzaandMgX 2 .Taken fromref.62................................32 2.1.Schemeforthesynthesisofbdtbpzm.TEBA,ortetrabutylammonium chloride,wasusedasaphasetransfercatalyst.Takenfromref.66.34 2

PAGE 8

ListofFigures 2.2.SchemeforthesynthesisofHbdtbpza.Takenfromref.66.....36 3.1. 1 H NMRspectrumofbdtbpzm,showingassignmentsofpeaks.....40 3.2. 13 C NMRspectrumofbdtbpzm,showingassignmentsofpeaks....42 3.3. 1 H NMRspectraofHbdtbpzatopand[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]bottom, alignedonthesameaxisfordirectcomparison.Notethatupperpeak at2.18ppmandlowerpeakat3.65ppmareduetoacetoneandmethanol, respectively.................................47 3.4. 13 C NMRspectraofHbdtbpzatopand[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]bottom, alignedonthesameaxisfordirectcomparison.Thepeakaround210ppm isduetoacetone.............................49 A.1.IRSpectrumofbdtbpzm.........................54 A.2. 1 H NMRSpectrumofbdtbpzm.....................55 A.3. 13 C NMRSpectrumofbdtbpzm.....................56 A.4.IRSpectrumofHbdtbpza........................57 A.5. 1 H NMRSpectrumofHbdtbpza....................58 A.6. 13 C NMRSpectrumofHbdtbpza....................59 A.7.IRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]..................60 A.8. 1 H NMRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]..............61 A.9. 13 C NMRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]..............62 3

PAGE 9

ListofTables 3.1.Changesof 1 H NMRshiftsoftheligandsbdtbpza,bdippza,andbdmpzauponforming1:1andbiscomplexeswithzincandmagnesiumions. 1,2 Onlyincludespeakscommontoallthreeligands............50 3.2.Changesof 13 C NMRshiftsoftheligandsbdtbpza,bdippza,andbdmpza,uponforming1:1andbiscomplexeswithzincandmagnesiumions. 1,2 Thetert-butylcarbonandpyrazolylcarbonshiftsarelistedfromlowesttohighestchemicalshift........................51 4

PAGE 10

1.Introduction: 1.1.RuBisCo Anoverview ItissafetosaythatwithoutRuBisCo,lifeonearthasweknowitwouldnot bepossible.RuBisCoisaubiquitousenzymeresponsibleforthexationofatleast 10 11 tonsofcarbondioxideperyear. 3 Asbettingforanenzymethatisresponsible forsuchamonumentalandessentialtask,RuBisCoisalsoestimatedtobeoneof themostplentifulenzymes,ifnotthemostplentifulenzyme,bymassonthe planet,accountingforasmuchas50%ofthesolubleproteininsomeplantsand bacteria. 4,5 Thecarbondioxidethusxedisconvertedintosugarsand carbohydrates,tostoreenergyforlateruseorasinthecaseofcellulosecreate rigidstructures.Inthisway,RuBisCoisresponsibleforliterallypullingthe immenseamountsofbiomassthatcomposetheworld'sforestsoutofthinair. Whetherweconsumethemdirectlyorindirectly,byeatinganimalsthatfeed onthem,werelyonplantstogeneratethesesugarsthatsustainourlivesand civilization.Fewotherpathwaysforcarbondioxidexationexist,andnoneofthem areperformedonanywherenearaslargeascaleasthatcatalyzedbyRuBisCo. 3,6 5

PAGE 11

1.1RuBisCo ThebiologicalusefulnessofRuBisCoisattestedtobyitsubiquity:itisfoundina broadspectrumoforganismsrangingfromhigherplants,toeukaryoticalgae, photosyntheticbacteria,andevenarchae. 7 Despitethewidevarietyoforganisms thatcontainit,thesecondarystructureandactivesiteofRuBisCoarehighly conservedthroughoutitsdierentforms. 8,9 WhileRuBisCoistheenzymedirectlyresponsibleformostcarbondioxide xation,itisproperlyconsideredtobejustonestepofacycleofreactionscalled theCalvincycle,illustratedinFigure1.1. 10,11 TheCalvincycledescribesthesetof reactionsbywhichcarbondioxideisultimatelyxed.ATPandNADHaresupplied bythelightreactionsofphotosynthesis,socalledbecausetheyrequireand harnesstheenergyofincomingphotons.TheCalvincyclereactionsareusually referredtoasthedarkreactionsofphotosynthesis,sincetheyrequirenodirect inputoflight. IntheportionoftheCalvincycleinvolvingRuBisCo, ribulose-1,5-bisphosphateRuBPbindstoRuBisCoandsubsequentlyreactswith carbondioxidetoformtwo3-phosphoglyceratePGmolecules.Atthispoint,a few3PGareextractedforuseinothermetabolicpathways,butveoutofeverysix 3PGarerecycledbackintoRuBPtocontinuethecycle.Thegenerationofone3PG moleculeforuseincomplexsugarbuildingultimatelyrequiresthehydrolysisof9 moleculesofATP,andtheoxidationof6moleculesofNADPH. 10 6

PAGE 12

1.1RuBisCo Figure1.1.: AdiagramoftheCalvinCyclereactions.Notethatthenumberof arrowsindicatesthequantityofeachmoleculethatundergoeseachspecicstep, andtheoverallreactionsarestoichiometricallybalanced.ThreeRuBPformsix 3PGreferredtoasPGAinthegure,andveoftheseareusedtoregeneratethreeRuBPwiththeinputof9ATPinthephosphorylationof3PG,and threeinthephosphorylationofRu5P,andsixNADPHusedtoreducethe DPGA.Thesixth3PGisremovedfromthecycletothegeneralcycoplasmafter conversiontoGAL3Pandusedforsugarbuilding.Takenfromref.9 Forthepurposeofthisdiscussion,letusconsidertherststepoftheCalvin cycletobethexationofcarbondioxideandtheproductionofsix3PGfromthree 7

PAGE 13

1.1RuBisCo RuBPshowninthebottomrightofFigure1.1.Each3PGisphosphorylatedwith anATP,producingsix1,3-bisphosphoglycerateordiphosphoglycerate,DPGA molecules,whicharethenreducedwithNADPHinordertoformglyceraldehyde 3-phosphateGAL3P. 11 Atthispoint,theCalvincyclebeginstobecomemorecomplex.Oneofthe sixGAL3Pmoleculesleavesthecycletobeusedinbuildingsugars. 10,11 Two GAL3PareisomerizedintodihydroxyacetonephosphateDHAP,oneofwhich subsequentlyiscombinedwiththefourthGAL3Pinordertoformfructose 1,6-bisphosphateFDP,whichissubsequentlydephosphorylatedintofructose 6-phosphateF6P. TheF6Pthusformedhastwoofitscarbonstransferredontothefth GAL3P,formingerythrose-4-phosphateE4Pandxylulose-5-phosphate Xu5P. 10,11 E4PandthesecondDHAParecombinedintoasingle sedoheptulose-1,7-bisphosphateSDP,whichisdephosphorylatedinto sedoheptulose-7-phosphateS7P.ThisiscombinedwiththesixthGAL3Pinorder toformasecondXu5Pandribose-5-phosphateR5P. BothoftheXu5PandtheR5Parethenisomerizedintoribulose-5-phosphate Ru5P,whichafterphosphorylationbyATPyieldsthreemoleculesofRuBPready forusebyRuBisCo. 10,11 Alloftheabovestepsarecatalyzedbyanensembleoften enzymes. 10 Inthepresenceofoxygen,RuBisCowithboundRuBPcanincorporate oxygenratherthancarbondioxide,whichresultsintheproductionofone3PGand one2-phosphoglycolate. 12 The2-phosphoglycolateisrecoveredinto3PGviathe photorespiratorycycle,whichinvolvesthelossofcarbondioxideandammoniaand 8

PAGE 14

1.1RuBisCo isconsideredtobedetrimental. 8 BesidestheCalvincycle,therearealsocurrentlyveotherknowncarbon xationpathways.ThesearethereductivetricarboxylicacidrTCAcycle,the reductiveacetyl-CoAWL,forWood-Ljundgahlcycle,the3-hydroxypropionate -HPbicycle,the3-hydroxypropionate/4-hydroxybutyrate-HP/4-HBcycle, andthedicarboxylate/4-hydroxybutyrateDC/4-HBcycle. 6 Organismsthatutilize themtendtobefoundarounddeepseaventsorinotherdeepseaenvironments. TheyaregenerallymoreenergyecientthantheCalvincycle,butalllacka mechanismsuchasthephotorespiratorycyclefordealingwiththepresenceof oxygen,andsocanonlybeutilizedinanaerobicenvironments. 6 AbriefhistoryofRuBisCoresearch RuBisCowasrstisolatedin1947fromspinachleaves. 13 Itwasnot characterizedatthattime,however,andwasreferredtoasFraction1.RuBisCo's activitywasdiscoveredasaresultofresearchintotheCalvinCycle.Startingin 1948,Calvin'sresearchgroupused 14 C labeledcarbondioxidetotraceits integrationintosugars. 14 Theyinitiallyreferredtotheenzymeresponsiblefor thexationstepascarboxydismutase.Anotherresearchgrouppuriedtheenzyme in1956,againfromspinachleaves,anddeterminedthatitwastheenzyme responsibleforthisstep. 17 Ayearlater,thefactthatthisenzymewaslikely identicaltoFraction1wasrealized, 18 thoughthiswasnotproveduntil1965. Duringthe60's,investigationusingelectronmicroscopesdeterminedthatthe enzymecomplexeswereoblatespheres,100angstromshighand200widewitha centraldepression,thoughinitiallythecomplexeswerethoughtoconsistof24 9

PAGE 15

1.1RuBisCo subunitsinsteadof16. 19 In1962,researchersnoticedthatoxygenseemedtoinhibit photosynthesis,interpretedcorrectlytomeanthatoneofthereactionintermediates thatwouldnormallybecarboxylatedwascapableofbeingoxygenated,whichat thetimewasacontroversialconclusion. 20 In1963itwasdeterminedthatthe puriedenzymeonlybecameactivatedbythepresenceofmagnesiumand carbonate, 21,22 andresearchin1966indicatedfurtherthatRuBisCoseemedtobe activatedbyincreasedlight invivo 23 In1968therstindicationsthatthe enzyme'scatalyticrate,notcarbondioxidediusion,wasoftenthelimitingfactor incarbondioxidexationbegancomingtolight. 24 Otherresearchersalsofound in1969thatthesubstratewasnotcarbonateionsashadbeenthought,butcarbon dioxide. 27 In1970,atightlybindinginhibitorforRuBisCowasdiscovered -carboxy-D-ribitol1,5-bisphosphate,andfromstudyingitsbindingwith RuBisCoitwassuggestedthatRuBisCoconsistedof8largeand8smallsubunits theacceptedstructureforTypeIRuBisCoinsteadofthepreviouslyposited24 subunits. 28 Studiesofphotosynthesisin1971foundthatinhibitionof photosynthesisbyoxygenindicatedthatRuBisCowaslikelythesourceof 2-phosphoglycolateusedinthephotorespiratorycycle,whichatthetimewas consideredtobeacontroversialconclusionandtookseveralyearstogaingeneral acceptance. 29,30 1973laterbroughtaboutthediscoveryofRuBisCoin cyanobacteriaandotherchemautrophicbacteria,containedinpreviouslyobserved polyhedralstructurescalledcarboxysomes. 31 The70'salsobroughtaboutanumberofothernotableeventsinthestudyof RuBisCo:therstpuricationofthelargesubunitofRuBisCooccuredin1974, 32,33 10

PAGE 16

1.1RuBisCo alongwithananalysisofthecompetitivekineticsbetweencarbondioxideand oxygen.RuBisCo'slargesubunithappenedtobetherstplantproteintranslated invitro frommRNAin1975. 34 Improvingx-raycrystallographytechniquesalso broughtaboutanimprovedknowledgeofthestructure,whichwasdeterminedtobe a2-layeredstructurewith4-2-2symmetryposessingonefour-foldrotationalaxis paralleltothecentralchannel,andapairoftwo-foldrotationaxesperpendicularto thechannelandeachotherandacentralchannelnotadepressionaspreviously thought. 35 Themechanismbywhichmagnesiumionsandcarbondioxideactivated RuBisCowasdeterminedthefollowingyear. 36 However,itwasnotuntil1979,when theenzyme,previouslysaddledwiththenameribulose-1,5-bisphosphate carboxylaseoxygenase,wasrstreferredtoasRuBisCo. 37 RuBisCowasfullysequencedin1980,thoughthesequencesforthelargeand smallsubunitwererstdeterminedfromseparatespeciescornandpeas, respectively. 38,39 Duringthefollowingyear,itwasdiscoveredthatRuBisCofrom dierentspeciesdieredintheirrelativeratesofcarboxylationandoxygenation, withanespeciallylargedierencebetween C 3 plantsand C 4 plantsthedistinction betweenthetwoisdiscussedinthesectionStrategiesforOptimizing RuBisCo. 40,41 During1982,studyoftheactivityofRuBisCointheabsenceof carbondioxideprovidedthemechanisticinsightthatenolizationofthesubstrate RuBPisoneoftheintermediatestepsduringcarbonxation,whichwaslater supportedbyNMRevidence. 42,43 Ahigh-resolutionX-raystructureofformII RuBisCofrom Rhodospirrillumrubrum wasobtainedin1986,demonstratingitsL2 dimericstructurewithnosmallsubunits. 44 Similaritybetweensmallchloroplast proteinsthatareinvolvedintheassemblyofRuBisCoandaproteincalled gro EL 11

PAGE 17

1.1RuBisCo from Escherichiacoli leadtothecreationofthetermchaperoninstodescribea classofproteinsinvolvedintheassemblyofotherproteinsin1988. 45 Sincethe90's,understandingofRuBisCo'sstructureandpropertieshas progressedsignicantly.However,researchfrom1990untilthepresentdayisherein presentedasmodernratherthanhistoricalinformation,andthuscoveredinallthe otherportionsofthisthesis. 12

PAGE 18

1.1RuBisCo DierentFormsofRuBisCo Figure1.2.: ExamplesofthefourmainformsofRuBisCo:formI,formII,form III,andformIVorRuBisCo-likeprotein.Takenfromref.44 Rubiscoisfoundinfourdistinctlydierentforms,andwhiletheysharean activesiteandmuchsecondaryandtertiarystructure,theydierinquaternary structure. 7 InformIRuBisCoIllustratedinFigure1.2,whichprovidesatop view,themostwidespreadandstudiedtype,consistsofeightdimers,each 13

PAGE 19

1.1RuBisCo consistingofalargeandsmallsubunit.Thelargesubunitscontaintheactivesite, andwhenseparatedfromthesmallsubunitstillretainsomecatalyticability. 7 Each dimerispairedwithanotherdimer,asillustratedinFigure1.3A. 8 Fourofthese tetramerunitsarearrangedinacircle,sothatthereisafour-foldrotationalaxis goingdownthecenterascanbeseenclearlyinFigure1.3C. 8 Thisformof RuBisCoisthemostcommon,foundinallhigherplants,aswellasmanybacteria andalgae. 46 Thesmallsubunitisthoughttocarryoutaregulatoryroleandis encodedbyaseparategene. 8 Figure1.3.: ThestructureofformIRuBisCo.Thelargesubunitsareshownin blueandaqua,andthesmallsubunitsinyellow.Theredmoleculeisatransition statemimic.AisthetetramersubunitofformI,composedoftwoLSdimers. BandCillustratehowfouroftheseunitsarearrangedinformIRuBisCo's structure.Takenfromref.6 TheformIRuBisCoenzymesareclassiedintoseveralsubgroupsbasedupon dierencesinaminoacidsequences.Thisclassicationalsoreectsthegeneral classicationsofRuBisCocontainingorganisms. 46 FormsIAandBarethemost common,foundinallhigherplants,greeneukaryoticalgae,andcyanobacteria. 46 Whileitmightseemcounterintuitivefortwogroupsthatareunrelatedtoeach othertohavesuchsimilarenzymes,thechloroplastsofeukaryoticphotosynthetic 14

PAGE 20

1.1RuBisCo organismsarederivedfromendosymbioticcyanobacteria,thusalleukaryotic RuBisCoaredescendedfromcyanobacterialRuBisCo.FormsICandD,however, arerestrictedtoredeukaryoticalgaeandsomephototropicbacteria. 46 FormIIRuBisCodiersfromformIbyconsistingofdimers,whichare composednotofalargeandsmallsubunit,butofidenticalunitsanalogoustothe largesubunitofTypeIRuBisCo. 46 ThisversionofRuBisCoisfoundinpurple non-sulphurbacteria,somechemautrophicbacteria,anddinoagellates. 8 Thereare severalspeciesofbacteriathatactuallycontaingenesforbothformIandformII RuBisCo. 47,48 Becauseofthis,andtheresultsofanalysisofthealignmentofalarge numberofdierentRuBisCovariants,formIIisthoughttobederivedfromform I. 8 FormIIIRuBisCoisonlyfoundinarchaea,andcanconsistofdimers, octamers,ordecamersofsubunitsanalagoustothelargesubunitofformI. 46 While theycouldlikelybefurtherclassiedbasedonquaternarystructure,littleresearch hasbeenmadeintoclassifyingformIIIRuBisCo,duetotheirrelativelyrecent discovery. FormIVRuBisCoisoftenreferredtoasRuBisCo-LikeProteinorRLP,since itdoesnotactuallycatalyzethesamereaction,despitestrongstructural similarity. 8,46 Theseproteinsarelikelyusedforsulfurmetabolism,astheyoccurin sulfurmetabolizingbacteria.AllthedierentformsofRuBisCoincludingRLP appeartohavebeenderivedfromasinglelineage,insteadofbeingtheresultof convergentevolution,baseduponphylogeneticanalysis. 8,46 15

PAGE 21

1.1RuBisCo RuBisCoactivesiteandcatalysis TheactivesiteofRuBisCoisamagnesiumion,whichiscoordinatedfacially toanasparticacid,aglutamicacid,andacarbamylatedlysine.Inspinach,the residuesinvolvedarelys201,asp203,andglu204. 49 Thecarbamylationisnecessary forRuBisCo'scatalyticactivity,andprovidesamechanismforregulatingthe activityoftheenzymeasRuBPandthenon-carbamylatedenzymeformaninactive complex. 7 Intheenzyme'srestingstate,themagnesiumioniscoordinatedto3 aqualigands,andposessesadistortedoctahedralcoordinationenvironment. 49 Figure1.4.: OneproposedmechanismforthexationofcarbondioxidebyRuBisCo.Takenfromref.47 OnemechanismforthexationofcarbondioxidebyRuBisCoisillustratedin 16

PAGE 22

1.1RuBisCo Figure1.4:First,RuBPbindstotheactivesiteoftheligand,withO2andO3 coordinatingtothemagnesiumionanddisplacingtwoaqualigands. 49 Thiscauses theprotonsonC3andO3tobecomemoreacidic.C3isdeprotonatedbythe uncoordinatedoxygenofthecarbamylatedlys201,andthentheprotonis transferredtoO2.O2isthendeprotonatedbylys175,andthecarbamylatedlys201 transfersaprotonfromO3toO2.Theresultoftheseprotontransfersistoconvert theRuBPintoa cis -2,3-enediolate.Theelectron-rich bondthenattacksacarbon dioxidemolecule,whichcoordinateswiththemagnesium,displacingtheremaining aqualigandandstabilizingitsnegativecharge.Awatermoleculefromthesolvent isdeprotonatedbyhis294andhydratestheC3-C2doublebond,either simultaneouslywithorshortlyaftertheattackofcarbondioxide.Next,theC2-C3 bondiscleaved,andafterthestereospecicprotonationofC2andtheprotonation ofO2,resultsinapairof3PGmolecules,whicharethenreleased. 17

PAGE 23

1.1RuBisCo Figure1.5.: ArecentlyproposedmechanismforcarbondioxidexationbyRuBisCo,withanemphasisonprotontransfer.Protonsfromsolventarecircled, andtheprotonsfromH3andO3arenotedbyboldtextandaz-shapedarrow, respectively.Takenfromref.48 18

PAGE 24

1.2OptimizingRuBisCo Asomewhatdierent,recentlyproposedmechanism,derivedfromisotope eectdataandcomputationalanalysis,isillustratedinFigure1.5. 50 Whilelargely similar,itdoesdierinafewpoints.Theinitialprotontransfersequenceis somewhatdierent,withprotonationofO2bylys175insteadofbythe carbamylatedlys201.His294isimplicatedasbeingresponsiblefordeprotonationof O3,insteadofthecarbamylatedlys201.Whetherthesourceofwaterforhydration istheaqualigandorthegeneralsolventisnotcurrentlyclear.Anotherdierenceis thatthenegativelychargedcaroxylatecreateduponbindingofthecarbondioxide isonlyshownascoordinatingwiththemagnesiumionaftercleavageoftheC2-C3 carbonbond,asopposedtoimmediatelyafteritsbinding. 1.2.OptimizingRuBisCo IssueswithRuBisCo RuBisCoisaparticularlyinecientenzyme. 8,51 Themostobviousproblem withRuBisCoisthattherateatwhichitcatalyzescarbondioxidexationis abysmallyslow.Whilemanyenzymescatalyzethousandsofreactionspersecond, undertypicalconditionsRuBisCocatalyzesaboutonexationpersecond. 52 This servestoexplainwhysuchimmensevolumesofRuBisCoaremaintainedbymany organisms,butalsoimpliesthepossibilityofimprovement. AnotherissuewithRuBisCoisthat,asmentionedpreviously,undermany conditionsitispronetoincorporatingoxygeninsteadofcarbondioxide,which 19

PAGE 25

1.2OptimizingRuBisCo resultsintheproductionofamoleculeof2-phosphoglycolateandamoleculeof 3PG,insteadoftwomoleculesof3PG. 7 The2-phosphoglycolatecannotbe regeneratedviatheCalvinCycleorutilizedfortheconstructionofsugars,andis insteadrecoveredinthephotorespiratorycycle. Thephotorespiratorycycleisnotgenerallyconsideredtoprovideanyuseful functionotherthanrecovering2-phosphoglycolate. 12 Itconvertstwomoleculesof 2-phosphoglycolateintoone3PG,withthelossofacarbondioxidemoleculeand inhigherplantsanammoniamolecule. 12 BecauseofRuBisCo'sreactivitywith oxygen,almostallorganismsthatuseitforcarbonxationhaveaversionofthe photorespiratorycycle,andorganismslackingitandmutantswithnon-functional cyclescanonlysurviveintheabsenceofoxygen. 12 20

PAGE 26

1.2OptimizingRuBisCo Figure1.6.: Thesimilaritiesanddierencesofthephotorespiratorycycleinplants andcyanobacteria.Thepathwayforphotorespirationinplantsishighlightedin green,andthereactionsspecictocyanobacteriaareshowninorange.Taken fromref.10 TheprocessofphotorespirationisdetailedinFigure1.6.Photorespiration beginswithhydrolysisofthe2-phosphoglycolateintoglycolatetoprightcornerof 21

PAGE 27

1.2OptimizingRuBisCo Figure1.6. 12 Inhigherplants,thisissubsequentlyoxidizedintoglyoxylate,which produceshydrogenperoxidethatissubsequentlybrokendownbycatalase.The glyoxylateisthenconvertedtoglycine,andtwomoleculesofglycinearethen convertedintoonemoleculeofserine,withthelossofcarbondioxideandammonia viaamulti-enzymesystem.ThisstepalsoreducesanNAD + moleculeintoNADH. Theserinethusgeneratedisusedtoconverttheglyoxylateintoglycine,keepingthe secondaminegroupinthecycle,andthisconvertstheserineintohydroxypyruvate. ThehydroxypyruvateisreducedwithNADHintoglycerate,whichafter phosphorylationwithATPcanbere-usedbytheCalvincycleas 3-phosphoglycerate. Incyanobacteria,photorespirationproceedssimilarlytohigherplantsuntil theproductionofglycolate,afterwhichglyoxylateisproducedwiththereduction ofNAD + insteadofoxygen. 12 Followingthis,twoglyoxylateareusedtoformone tartronicsemialdehydemolecule,withthelossofcarbondioxide.Thisisthen reducedtoglycerate,whichisphosphorylatedinto3PGasinhigherplants.The cyanobacterialpathwayavoidsthelossofammoniaandproductionofhydrogen peroxide,butstillinvolvesthelossofcarbondioxide. RuBisCofromdierentorganismsalsovariesinspecicity.Thepreferencefor carbondioxideoveroxygen,oftenreferredtoas ,isdenedas V C K O V O K C ,where V C and V O arethemaximumratesofcarboxylationandoxygenation,and K C and K O arethevaluesof K M forcarboxylationandoxygenation. 53 Mosthigherplantshave a of60-100,butvariationinotherorganismsismuchwider,withsome photosyntheticbacteriaposessing inthe5-40range,andredalgaehaving ofup to180-240. 8 Theratiobetweencarboxylationandoxygenationisafunctionof 22

PAGE 28

1.2OptimizingRuBisCo andtherelativeconcentrationsofcarbondioxideandoxygenintheenvironment inhabitedbyRuBisCo, carboxylation oxygenation = [ CO 2 ] [ O 2 ] Limitingfactorsofphotosynthesis WhileRuBisCo'scarbonxationstepisoftenthoughtofastheratelimiting stepinphotosynthesis,thisisactuallyonlysometimesthecase. 53 Inconditions wheretherearehighlevelsofsunlight,theregenerationofRuBPisspedupbythe highavailabilityofenergyfromtheLightreactions.Thiscausestheconventional casewhereRuBisColimitsthespeedofcarbonxation,andundertheseconditions anyincreasesinboth and V C wouldincreasetheoverallrateofcarbonxation. However,inlowenoughlevelsoflight,theregenerationofRuBPoftenslowsdown tothepointthatitbecomesthelimitingfactorincarbonxation.Underthese conditions,itisonlyhelpfultoincrease ,sincethiswoulddecreasetherateof photorespiration. StrategiesforoptimizingRuBisCo Interestingly,speedandspecicityinRuBisCoseemtobeinverselylinked.In general,avariantofRuBisCowithahigher willhavealower V C .This relationshiphasbeenexplored,andcanbeapproximatedbythefunction V C = e 5 : 16 1 0 : 69 ,with r 2 = : 89 53 Inlightofthis,onealternativestrategyfor improvingRuBisCoreliesnotonincreasingboth andthecatalyticrateof RuBisCo,butonguringouttheoptimalcombinationforagivensituation. Accordingtoonemodel,takingintoaccountwhichportionsofthedayanaverage C 3 plantwouldbeRuBPorRuBisColimited,adecreaseof andacorresponding 23

PAGE 29

1.2OptimizingRuBisCo increasein V C couldactuallyprovideaslightimprovement,ofaround3%,incrop yields. 53 Thelogicbehindthisisthatcurrent and V C balanceinplantsis optimizedforhistoricalcarbondioxidelevels,andwithrisinglevelslower and higher V C isoptimal. Anotherstrategy,currentlyoneofthemostpromising,involvesidentifying organismswithRuBisCothatexhibithigherthanexpected fortheir V C 53 Itis predictedthat,forinstance,RuBisCofrom GrithsiaMonilis wouldbeableto improvecropyieldsofplantswithmoretypicalRuBisCoby27%. 51 Also,RuBisCo fromoneorganismdoesnotneedtobecompletelyreplaced: Thermococcus kodakaraensis, athermophilicarchaea,hasbeenshowntogrow31%fasterwhenits relativelyinecientformIIIRuBisCoismodiedtohavetheactivesiteofamore ecientformIRuBisCofromspinach,requiringthealterationofonly11 residues. 54 Manyplants,referredtoas C 4 plants,havedevelopedavarietyofmechanisms forconcentratingcarbondioxideinthevicinityoftheirRuBisCo,reducingthe incorporationofoxygen. 55 Severaldierentmechanismshaveevolvedapparently independently,butmostfollowthesamegeneralstrategy.First,carbondioxideis convertedtobicarbonatebycarbonicanhydrase,andthenxedintheformof oxaloacetatebyanenzymecalledPEPcarboxylaseviareactionwith phosphoenolpyruvate.Then,thisisconvertedintomalateoraspartateand transportedtointernalleaflayersprotectedbyspecializedsheathcellstoprevent penetrationbygas,andconvertedbackintocarbondioxide,creatingacarbon dioxiderichenvironmentforxation. C 4 plantstendtobefoundprimarilyinhot areas,asathighertemperaturesphotorespirationbecomesmoreofaproblemdue 24

PAGE 30

1.3ActiveSiteModeling toahigherrelativesolubilityofoxygenathighertemperatures,andahigher activationbarrierforoxygenationwhichmakeshighertemperaturesincreasethe rateofreactionwithRuBisCo.Someresearchhasbeendoneintryingtointroduce the C 4 carbonconcentratingmechanismintoplantsthatlackit C 3 plants,but therehasyetbeenlittlesuccessinthiseld. 51 Anotherstrategyusedbyplantstoimprovephotosynthesiseciencyis crassulaceanacidmetabolismCAM,inwhichcarbondioxideisxedasin C 4 plants,butprimarilyatnight. 55 Malateisstoreduntildaytime,whenitis convertedbackintocarbondioxideandxednormally.Storingcarbondioxidein thisfashionallowsCAMplantstoclosetheirstomataduringthedayandminimize waterloss. 1.3.ActiveSiteModeling Abriefoverviewofactivesitemodeling DirectlystudyingRuBisCoisnottheonlywaytogaininsightintohowto improveitsfunctionality.Itcanbeinsightfultopreparesmallmetalcomplexesthat havesimilaritiestotheactivesiteofanenzyme. 56,57 Thesecompoundscannever replicatethecompexitiesoftheactivesiteenvironmentspresentinenzymes,butby determiningthepropertiesoftherawcomplex,itispossibletogaininsightintothe eectsofthecomplexenzymeenvironmentontheactivesite.Smallcomplexesare mucheasiertostudythanenzymes,sincethereismuchlessworryabout denaturation,andtheyarespectroscopicallymuchsimpler. 25

PAGE 31

1.3ActiveSiteModeling PreviousworkintheShermanlab:TCMA WorkintheShermanlabonsynthesizingasyntheticmodelforRuBisCo startedwiththeligand1,4,7-triazacyclononane-monoacetateTCMA. 58 TCMA coordinatesatthe3aminenitrogens,andthenegativelychargedcarboxyloxygen, makingitatetradentate[ N 3 O ]ligand.TCMAwaschosenbecauseofseveral factors.TCMAenforcesanoctahedralcoordinationgeometryonthemagnesium ion,asdoesRuBisCo.Also,TCMAhasacarboxyldonor.InRuBisCo,the complexedmagnesiumisboundtotwocarboxylsandacarbamylateinan[ O 3 ] bindingscheme,buthavingonlyonecarboxyldonorallowedthecomplextoavoid havinganoverallnegativecharge,whichwouldcomplicatecomplexformationwith negativesugars.TCMAisalsosolubleinpolarsolvents,whichisadvantageous sinceRuBPandsimilarcompoundsarereadilysolubleinpolarsolvents. Figure1.7.: X-raycrystalstructureofaMgTCMAcomplex.Takenfromref.56 26

PAGE 32

1.3ActiveSiteModeling ThemagnesiumcomplexofTCMAwasprepared,andisshowninFigure 1.7. 58 Thetwocoordinationsitesnotboundtotheligandcoordinatewithapairof labileaqualigands. 58 TotestifitcouldcatalyzeasimilarreactiontoRuBisCo, acetolwasusedasasubstrateanalogofRuBP,asunlikemanyothersimilarsmall carbohydratesitcanonlybindinoneconguration,andonlyformsdimersin solutionafterextendedperiodsoftime.A1:1solutionofMgTCMAindeuterated methanolwaspreparedinanNMRtube,andobservedviaNMRspectroscopy. 59 TheMgTCMAwasfoundtocatalyzetheslowdeuterationoftheacetolsubstrate, whichwashypothesizedtooccurviaanenolateintermediatesimilartothe intermediateformedintheRuBisComechanismpriortotheattackofcarbon dioxide. Unfortunately,furtherexaminationsofMgTCMA'sactivitydeterminedthat simplemagnesiumsaltsinthepresenceofaddedbasecouldcatalyzethe deuterationofacetolathigherrates,andthatMgTCMAwasincapableofxing carbondioxidetotheacetolsubstratemimic. 60 Additionally,sinceTCMAisa tetradentateligand,itcannotprovideacompletesimulationoftheactivesiteof RuBisCo,asthesixthcoordinationsitehasbeensuggestedtohavearolein catalysis. 49,50,63 Itwasdecided,inlightoftheseresults,topursuethedevelopment ofacomplextomodelRuBisCowithanew,tridentateligandposessingsimilar propertiestoTCMA. Anewavenue:bdippza Thenextligandselectedforresearchwas bis,5-diisopropylpyrazole-1-ylacetatebdippza,amemberofaclassofligands 27

PAGE 33

1.3ActiveSiteModeling calledscorpionates. 1 Scorpionateligandsshareabasicstructure,withtwoclaws andonestingerattachedtoacommonatom.Theclawsandstingereachcontain adonoratom,andtheycoordinatefaciallywithmetalions.Intherstscorpionate ligands,theclawsandstingeroftheligandwerethesame,andtheatomaround whichallthreewerecenteredwasaboron,makingitaboroncentered homoscorpionate. 1 Laterligandsinthisclassintroduceddierentcenteratomssuch ascarboncarboncenteredscorpionates,whichweremorestablethantheoriginal boroncenteredligands,andligandswithdierentclawsandstings,referredtoas heteroscorpionates,werequicklydeveloped,whichallowedforligandssimulating morediversecoordinationenvironmentstobedeveloped. 1 Figure1.8.: bis,5-diisopropylpyrazole-1-ylacetate,orbdippza.Takenfromref. 62 Manyscorpionateligands,suchasbdippza,usepyrazolenitrogensforsomeor allofthedonoratoms.Becauseitispossibletosynthesizepyrazoleswithdierent substituentsattachedtothe3and5positionsofthering,itispossibletointroduce bulkygroupsandadjustthestericsoftheligandfairlynely.Manyscorpionate ligandsarepronetoformingunreactivebiscomplexes,butwithlargeenough groupstheformationofthesecomplexescanbeprevented. 28

PAGE 34

1.3ActiveSiteModeling Theparticularligandselectedisdescendedfromaligandsynthesizedin 1999,bis,5-dimethylpyrazole-1-ylacetate,orbdmpza. 64 Itwaspreparedina fairlystraightforwardsynthesis,inwhich3,5-dimethanepyrazolereactedwith dichloromethaneinthepresenceofbase,followingwhichthecentralcarbonwas deprotonatedandthelonepairusedtoattackcarbondioxide,thusfunctionalizing thecomplexwithacarboxylgroup. Anotherresearchgrouppreparedzinccomplexesofthisligand,andfound thattheligandformedbiscomplexesduetolowsterichindrance. 2 Theyquickly adaptedthesynthesisforusewith3,5-ditertbutylpyrazoleordtbpz,resultingin bis,5-ditertbutylpyrazole-1-ylacetatebdtbpza,fromwhichazinccomplexwas preparedandcharacterized. 2 Thisligandwasfoundtobehinderedenoughthatit formeda1:1tetrahedralzinccomplex. Forinitialresearch,theShermangroupdecidedtofocusonthethen-novel bis,5-diisopropylpyrazole-1-ylacetatebdippza,sincethelowersterichindrance oftheisopropylgroupsrelativetothetert-butylgroupswashopedtomakeitmore likelythatanoctahedralcoordinationgeometrywouldbefavored,whilestill preventingtheformationofbis-complexesasinthecaseofbdmpza. 1 Theligand wasprepared,usingaprocedureadaptedfromtheliterature. 2 First, 1,6-dimethyl-3,5-heptanedionestartingmaterialreactedwithhydrazineinan ethanolsolutiontoproduce3,5-diispropylpyrazole,asshowninFigure1.9.This wasthenusedtopreparebdippzausingthesamebasicprocedureasforbdtbpza, asshowninFigures1.10and1.11. 29

PAGE 35

1.3ActiveSiteModeling Figure1.9.: Schemeforsynthesisof3,5-diisopropylpyrazolefrom1,6-dimethyl-3,5heptanedione.Takenfromref.62 Figure1.10.: Schemeforsynthesisofbdippzmfrom3,5-diisopropylpyrazoleand dichloromethane.TEBA,ortriethylbenzylammoniumchloride,wasusedasa phasetransfercatalyst.Takenfromref.62 Figure1.11.: Schemeforthesynthesisofbdippzafrombdippzm.Takenfromref. 62 Bothzincandmagnesiumcomplexeswereprepared.Therstcomplex preparedwasazinccomplex.Bdippzawasdissolvedinmethanolandneutralized usingsodiumhydroxide.Aseparatesolutionofzinctriateinmethanolwasalso prepared,andtheligandsolutionaddedtothezincsolution.Initially,thezinc 30

PAGE 36

1.3ActiveSiteModeling complexesformedwereonlybiscomplexes,asdeterminedbyx-raycrystallography. Byusingzincchlorideinsteadofzinctriate,a1:1complexwasobtained,asshown inFigure1.12.AnX-raycrystalstructurerevealedadimeric[ZnbdippzaCl] 2 complex,thoughitisthoughtthatthe[ZnbdippzaCl]complexexistedinsolution. Figure1.12.: Schemeforthesynthesisof[ZnbdippzaCl] 2 fromZnCl 2 andbdippza.Takenfromref.62 Preparationofmagnesiumcomplexeswithmagnesiumtriateandmagnesium chlorideresultedinbothcasesintheformationofabiscomplex,asillustratedin Figure1.13.Eventheuseofdi-n-butylmagnesiumasthesourceofmagnesium resultedexclusivelyinbiscomplexes.NMRdatasupporttheconclusionthatonly biscomplexeswereformed,asthespectrawerealmostidenticaltothoseofthebis complexofzinc. 31

PAGE 37

1.3ActiveSiteModeling Figure1.13.: Schemeforsynthesisof[Mgbdippza 2 ]frombdippzaandMgX 2 Takenfromref.62 Fromtheseresults,itwasconcludedthatbdippzaisnotsucientlysterially hindered,anditwasdecidedtocontinueresearchusingbdtbpza.Whilethe synthesisoftheliganditselfcanbefoundintheliterature,nomagnesiumcomplex ofbdtbpzahasyetbeenprepared.Baseduponthe1:1zinccomplexthatwas synthesizedfromit,itislikelythatamagnesiumcomplexofbdtbpzawillalsobe 1:1.Itisunclearifthemagnesiumcomplexofbdtbpzawillbeabletosupport octahedralcoordination,duetothepossibilitythatitistoohindered. 32

PAGE 38

2.Experimental 2.1.General ReagentswerepurchasedfromSigma-Aldrichandusedasdeliveredunless otherwisenoted.CompoundswereweighedusingaDenverInstrumentsAPX-203 balance.THFwasdistilledtodrynessoverpotassiuminthepresenceofabenzophenoneindicator.DryicewaspurchasedfromPublix.Reactionaskswere heatedusingaPEGbath,whennecessary.NMRspectraweretakenusingaBruker AC250MHzspectrometerforbothprotonandcarbonspectra,andTMSasastandard.AMel-Tempapparatusandathermocouplewereusedtodeterminethemeltingpoints.InfraredspectrawereobtainedusingaNicoletAvatar320FT-IRspectrometer,usingsolidsamples. 2.2.Synthesisof3,5-Ditertbutylpyrazole Thissynthesiswasadaptedfromliteratureprocedures. 1,65 Thissynthesiswas performedbySethHearld.To30mlethanolinathree-neckedroundbottomask, 9.0ml.1mmol2,2,6,6-tetramethyl-3,5-heptanedionewasadded.Underani33

PAGE 39

2.3SynthesisofBis-,5-ditertbutylpyrazol-1-ylmethane trogenatmosphere,2.3mlmmolhydrazinemonohydratewasaddeddropwise tothesolutionwhilestirring.Thesolutionwasthenreuxedforonehour,washed withanaqueoussodiumchloridesolution,andextractedwithdiethylether.The aqueousphase,whichcontainedunreactedhydrazinemonohydrate,wasdestroyed withbleach.Theorganiclayerwasdriedovermagnesiumsulfate,andthesolvent wasremovedinvacuotoyield5.514g.6mmol3,5-ditertbutylpyrazoleasa whitepowder,a71%yield. HazardNote:Thisprocedureinvolvestheuseofhydrazinemonohydrate,which ishighlytoxic,corrosive,combustible,andcarcinogenic. 1 H NMRMHZ, CDCl 3 =1.32s,18H, CH 3 ,5.90s,1H, H pz 2.3.Synthesisof Bis-,5-ditertbutylpyrazol-1-ylmethane Figure2.1.: Schemeforthesynthesisofbdtbpzm.TEBA,ortetrabutylammoniumchloride,wasusedasaphasetransfercatalyst.Takenfromref.66 Theprocedureusedforthepreparationofbdtbpzmwasadaptedfromlit34

PAGE 40

2.4SynthesisofBis-,5-ditertbutylpyrazol-1-ylaceticAcid eratureprocedures,andisillustratedinFigure2.1. 1,2,66 Approximately110ml dichloromethanewasaddedtoaaskcontaining3.194g.7mmolbdtbpz,9.590 g.45mmolanhydrouspotassiumcarbonate,0.391g.71mmolbenzyltriethylammoniumchloride,and3.844g.5mmolpotassiumhydroxide.Thissolution wasreuxedwhilestirringunderanitrogenatmospherefor24hours.Thesalts werethenremovedviavacuumltration,andthesolventwasremovedinvacuo. Thecrudebdtbpzmwasthenwashedwithwatertoremoveresidualbenzyltriethylammoniumchloride,extractedwithpentane,anddriedovermagnesiumsulfate. Thepentanewasremovedinvacuotoyield2.562g.88mmolo-whitebdtbpzm crystals,a78%yield.Themeltingrangewas135-136 Celcius,ascomparedtothe literaturevalueof132 C. 2 1 H NMRMHZ, CDCl 3 =1.21s,18H, CH 3 ,1.25s,18H, CH 3 5.91s,2H, CH 2 ,6.52s,2H, H pz 13 C NMRMHZ, CDCl 3 =29.9 CH 3 ,30.6 CH 3 ,32.1 C )]TJ/F27 11.9552 Tf 11.027 0 Td [(t bu ,67.0 CH 2 ,102.2 HC pz ,153.2 C pz ,159.3 C pz IR: = 1538 cm )]TJ/F41 7.9701 Tf 6.586 0 Td [(1 C=Nstretch 2.4.Synthesisof Bis-,5-ditertbutylpyrazol-1-ylaceticAcid TheprocedureusedforthepreparationofHbdtbpzawasadaptedfromliteratureprocedures,andisillustratedinFigure2.2. 1,2,66 ToapurgedSchlenkask, 1.464g.93mmolbdtbpzmwasadded,anddissolvedinaround50mldryTHF. Theaskwasthenttedwithaseptumandcooledinanacetone/dryicebath 35

PAGE 41

2.4SynthesisofBis-,5-ditertbutylpyrazol-1-ylaceticAcid Figure2.2.: SchemeforthesynthesisofHbdtbpza.Takenfromref.66 whilestirringunderanitrogenatmosphere.Tothissolution,4.4ml1.6M.03 mmoln-butyllithiumwasaddeddropwise,causingthesolutiontoturnlightyellow.Thesolutionwasthenstirredforanhour.Carbondioxideproducedfromthe sublimationofdryicewasbubbledthroughthesolution,andtheSchlenkaskwas allowedtoventintotheatmosphere,allowingcarbondioxidetobubblefreely.The solutionslowlylostitsyellowcoloration.Thebubblingcontinuedfor2hoursasthe reactionaskwasallowedtoslowlyheattoroomtemperature.Thesolventwas subsequentlyremovedinvacuo.Thecrudeproductwasthenpartiallydissolvedin watertoformacloudywhitesuspension,andacidiedtoapHof1.Hbdtbpzawas thenextractedwithdiethylether,using3washesof100,50,and50ml.Theorganicphasewasdriedovermagnesiumsulfate,andthentheetherwasremovedin vacuoyieldingathick,lightyellowuidwhichcrystallizeduponexposuretoatmosphereoverthecourseof5days.Thiscrudeproductwasthenrinsedwithpentane, yielding1.093g.62mmolofwhitebdtbpzacrystals,a67%yield.Themelting rangewas74-77 C,ascomparedtoaliteraturevalueof92 C. 2 1 H NMRMHZ, CDCl 3 =1.26s,36H, CH 3 ,2.18 Acetone ,6.02 s,2H, H pz ,7.27s,1H, CH 36

PAGE 42

2.5Synthesisof[Magnesiumbis-,5-ditertbutylpyrazol-1-ylacetateChloride] 13 C NMRMHZ, CDCl 3 =30.3 CH 3 ,32.1 C )]TJ/F27 11.9552 Tf 12.391 0 Td [(t bu ,32.4 C )]TJ/F27 11.9552 Tf 12.391 0 Td [(t bu 73.0 CH ,103.3 HC pz ,154.1 C pz ,159.8 C pz ,166.7 CO 2 H ,208.0 Acetone IR: = 1754 cm )]TJ/F41 7.9701 Tf 6.586 0 Td [(1 C=Ostretch, = 1552 cm )]TJ/F41 7.9701 Tf 6.587 0 Td [(1 C=Nstretch 2.5.Synthesisof [Magnesiumbis-,5-ditertbutylpyrazol-1-yl acetateChloride] Theprocedureusedforthepreparationof[MgbdtbpzaCl]wasadaptedfrom literatureprocedures. 1 To1mlofmethanol,0.041g.098mmolHbdtbpzawas added.Upontheadditionof0.005g.125mmolsodiumhydroxide,theHbdtbpza wasneutralizedanddissolved,formingacloudysolution.In1mlofmethanol, 0.011g.115mmolanhydrousmagnesiumchloridewasdissolved,andthenthe bdtbpzasolutionwasaddedtothis.Transparent,colorlesscrystalsof[MgbdtbpzaCl] slowlyformedoverthecourseofaweek,witha51%yield.Themeltingrangewas 156-165 C. 1 H NMRMHZ, CDCl 3 =1.15s,18H, CH 3 ,1.22s,18H, CH 3 3.65 MeOH ,5.87s,2H, H pz ,6.89s,1H, CH 13 C NMRMHZ, CDCl 3 =30.7 CH 3 ,32.0 C )]TJ/F27 11.9552 Tf 12.391 0 Td [(t bu ,32.1 C )]TJ/F27 11.9552 Tf 12.391 0 Td [(t bu 75.8 CH ,101.7 HC pz ,152.9 C pz 159.8 C pz ,170.4 CO 2 H ,211.1 Acetone IR: = 1657 cm )]TJ/F41 7.9701 Tf 6.586 0 Td [(1 C=Ostretch, = 1536 cm )]TJ/F41 7.9701 Tf 6.587 0 Td [(1 C=Nstretch 37

PAGE 43

3.ResultsandDiscussion 3.1.LigandSynthesis 3.1.1.3,5-Ditertbutylpyrazole Since3,5-ditertbutylpyrazoleisnotcurrentlyavailablecommercially,itwas synthesizedfrom2,2,6,6-tetramethyl-3,5-heptanedione,usingaprocedureadapted fromtheliterature. 65 ThisreactedwithhydrazinemonohydrateSeehazardwarninginexperimentalsection,whichattackedthecarbonylcarbonsoftheheptanedioneinordertoformapyrazolering.Likely,thereactionproceededrstwiththe nucleophilicattackofeitherC3orC5byoneofthehydrazinenitrogens.Thenegativelychargedoxygenwasthenprotonated,andthenitrogendeprotonated,andthe othernitrogenwasthenkineticallyfavoredtoattackthesecondcarbonylcarbon withthesameprotonationanddeprotonatingsteps,forminga5-memberedring. Oneofthehydroxylgroupscouldthenbeprotonatedandeliminatedalongwitha nitrogenboundhydrogentoformadoublebond.Thepyrazoleringcouldthenbe completedbyasimilarprotonationandeliminationoftheremaininghydroxyl,this timeaccompaniedwithahydrogenfromC4. Oncethedtbpzhadbeensynthesized,aworkupofrstawashwithanaque38

PAGE 44

3.1LigandSynthesis oussodiumchloridesolutionfollowedbyextractionwithdiethyletherwasused, andafterdryingtheorganicphaseandremovingthesolventinvacuo,dtbpzwas isolatedasawhitepowderwitha71%yield,signiantlyhigherthanthe41%yield reportedintheliterature. 65 Thepurposeofthesodiumchloridesolutionwashwas toremovetheunreactedhydrazine,andthiswassubsequentlytreatedwithbleach todestroyitpriortodisposal. TheNMRspectrumofdtbpzisfairlysimple,withthe 1 H spectrumhaving onlytwopeaks.Therst,at =1.32,correspondstothe18 CH 3 hydrogensinthe twotert-butylgroups,whichareallchemicallyequivalentduetobeinglargelysymmetricalandrotationallyunhindered.Thesecondpeak, =5.90,correspondsto thecarbonboundpyrazolehydrogen.Thefactthatthetwotert-butylgroupsare chemicallyequivalentalsosuggeststhatthenitrogen-boundhydrogenexchangesbetweenthetwoadjacentnitrogensrapidlyontheNMRtimescale,whichwouldalso accountfortheabsenceofasignalfromthishydrogen. 3.1.2.Bis-,5-ditertbutylpyrazol-1-ylmethane Thenextstepintheligandsynthesiswasbdtbpzm.Thiswaspreparedbyreactingdichloromethanewith3,5-ditertbutylpyrazoleinthepresenceofabase.Since dichloromethaneboilsat40degreescelcius,itwasusedasasolventforthisreaction,andheatedtoreux.Thebaseusedinthisreactionwaspotassiumhydroxide, andsincethisisinsolubleinthedichloromethanesolvent,triethylbenzylammonium chloridewasusedasaphasetransfercatalyst.Additionally,sincethereactionis sensitivetothepresenceofwater,potassiumcarbonatewasaddedinordertoremoveanytraceamountsofwaterpresent. 39

PAGE 45

3.1LigandSynthesis Figure3.1.: 1 H NMRspectrumofbdtbpzm,showingassignmentsofpeaks. Thereactionlikelyproceedsasfollows:Thelonepaironthenon-protonated pyrazolylnitrogenofthedtbpzattacksadichloromethane,withthelossofachlorineion.Theotherpyrazolylnitrogenisthendeprotonated,andthenowmorereactivepyrazolylchloromethylisattackedbyaseconddtbpz,whichisalsodeprotonated.Thisstepisvulnerabletothepresenceofwater,aswatercouldeasilyattack thereactivechloromethyl,resultinginahydroxylgroupbeingsubstitutedtothe 40

PAGE 46

3.1LigandSynthesis methaneinsteadofthepyrazoleasdesired. Themeltingrangeof135-136 Celciuswasreasonablyclosetothatreported intheliterature,132 Celcius. 2 Theyieldof78%alsocomparedfavorablywiththe 77%literatureyield. 2 The 1 H NMRspectrumofbdtbpzm,showninFigure3.1,ismorecomplicatedthanthesimpledtbpzspectrumlargelybecauseoftheloweredsymmetry. Insteadofshowingupasonepeak,thetert-butyl CH 3 hydrogensshowupattwo separateshifts, =1.21and =1.25,sincetheyarenolongerinterchangeable. Thenewlyadded CH 2 hydrogensshowupat =5.91,duetotheiradjacencyto twoaromaticrings,andthepyrazolylhydrogensbothappearat =6.52dueto theirsymmetry.Alloftheseshiftswerewithin0.01ppmofthevaluesreportedin theliterature. 2 41

PAGE 47

3.1LigandSynthesis Figure3.2.: 13 C NMRspectrumofbdtbpzm,showingassignmentsofpeaks. The 13 C NMRspectrum,showninFigure3.2,wassimilarlymorecomplex. Thetert-butyl CH 3 sappearedat =29.9and =30.6,duetotheirchemicalinequivalence.Thequaternarytert-butylcarbons,however,bothappearedat = 32.1.The CH 2 carbonappearedat =67.0,duetoproximitytothetwoaromatic pyrazolerings.Thepyrazolylcarbonsappearedatthreedierentchemicalshiftsinsteadoftwo, =102.2, =153.2,and =159.3,duetothenon-interchangeability 42

PAGE 48

3.1LigandSynthesis ofC1andC3.Alloftheseshiftswerewithin0.3ppmofthevaluesreportedinthe literature. 2 3.1.3.Bis-,5-ditertbutylpyrazol-1-ylaceticacid First,bdtbpzmwasdissolvedindryTHF.Thisclearsolutionwasthenchilled, usingadryice/acetonebath,whilestirringandunderaneutralnitrogenatmosphere.ExcessN-butyllithiuminhexaneswasaddeddropwisethroughaseptum, andthismixturewasstirredforanhour.Thencarbondioxideproducedbysublimatingdryicewasbubbledthroughthereactionmixture,asthemixturewas allowedtowarmupto0deg.C.Theworkupthenfollowed,consistingofremoving theTHFinvacuo,washingtheproductinwaterandacidifyingittoapHof1,and extractingitwithether.Theetherwasremovedinvacuo,thoughitcouldnotall beremovedinitially,sothecrudeproductwasallowedtositfor5daysuntilitwas largelydry.Thiswasthenrinsedwithpentanetoyieldbdtbpza. TherstattemptedsynthesisofHbdtbpzawasconductedonasmallerscale thantheproceduregivenintheexperimentalsection,about 1 3 thescale.NoHbdtbpzawasisolated,instead,smallamountsofanasyetunelucidatedproductwere produced.Thereasonforthiswasthoughttobeeitherrelatedtowatercontamination,ortothesharptemperatureramprate,asfortherstrunthereactionask wasdirectlytransferredtoanicewaterbathfromthedryice/acetonebath.NMR spectraoftheresultantcompoundappeareddissimilartotheliteraturespectraof Hdtbpza. 2 Thesecondattemptwasconductedonasimilarscale.However,insteadof thesharptemperatureramp,theacetonebathwasallowedtoslowlywarmtofreez43

PAGE 49

3.1LigandSynthesis ingoverthecourseof2hours,byceasingtheadditionofdryicetothebath.Pure bdtbpzawasobtainedatabouta50%yield,anditwasanticipatedthattheyield wouldimproveifthereactionwasscaledup.Thenalattempt,detailedintheexperimentalsection,producedbdtbpzawitha67%yield,signicantlypoorerthan theliterature88%yield. 2 Themeltingrangewas74-77 C,somewhatlowerthanthe valuereportedintheliteraturewhichwas92 C. 2 Themechanismforthisreactionwaslikelyfairlystraightforward.First,the centralmethylgroupwasdeprotonatedbytheN-butyllithium.Thelonepaircould thenattackthecentralcarbonofcarbondioxide.Duringtheacidicationstep,the carboxylatethusformedisprotonated. The 1 H NMRspectrumseeFigure3.3ofHbdtbpzaisfairlysimilartothat ofbdtbpzm,thoughtheshiftsaresomewhataltered.Theformerlyseparatetertbutyl CH 3 resonancesbothappearat =1.26,andcouldnotbedistinguished usingtheavailableinstrument.Theremainingpyrazolylhydrogenappearedat =6.02,andthecentral CH hydrogenhad =7.27,overlappingwiththe CHCl 3 peak.Athighersampleconcentrations,theratiobetweenthemethylandpyrazolyl hydrogenpeaksapproached1:2,supportingthisassignment.Thecarboxylhydrogenwasnotvisible.Theindistinguishibilityofthetert-butylmethylresonances possiblyduetoshimmingissuesorlowerresolutionthantheinstrumentusedin theliteraturewastheonlyvariationfromthereportedvalues,whichotherwise werewithin0.01ppm. 2 Inthe 13 C spectrumseeFigure3.4,the CH 3 carbonsonthetert-butylgroups allappearedat =30.3.Thetertiarytert-butylcarbonshadshiftsof =32.1and 32.4.Thecentralcarbonappearedat =73.0,andthethreepyrazolylcarbonsat 44

PAGE 50

3.2MetalComplexSynthesis =103.3, =154.1,and =159.8,thedeshieldingrelativetobdtbpzmdueto thecarboxylgroupsupportingtheconclusionthatthecentralcarbonhadbeen functionalized.Thenalandmostconclusiveevidenceinfavorofthiswastheappearanceofanewpeakduetothecarboxylcarbonat =166.7.Allpeakswere within1ppmofthevaluesreportedintheliterature. 2 3.2.MetalComplexSynthesis 3.2.1.[Mgbdtbpza n Cl 2 )]TJ/F28 9.9626 Tf 7.748 0 Td [(n ] Inordertopreparethemagnesiumcomplex,theligandandanequimolar amountofsodiumhydroxideweredissolvedinmethanol.Aseparatesolutionofa slightexcessofmagnesiumchlorideinmethanolwasprepared,andtherstsolution mixedintothesecond.Asthemethanolevaporated,[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]crystallizedoutofsolutionafter5daysasthesolventslowlyevaporated. Asevidenceforthebindingofmagnesiumtotheligand,the 1 H and 13 C chemicalshiftsarealteredrelativetothoseoftheunboundligand,asillustratedinFigures3.3and3.4.Allofthe 1 H peaksareshieldedvaryingamountsrelativetounboundHdtbpza.Thetert-butyl CH 3 hydrogensappearattwoseparateshifts, = 1.15and =1.22,duetothedierentchemicalenvironmentstheynowinhabit, themoreshieldedofthetwoprobablybeingclosertotheboundmagnesiumasit experiencedalargerchangeinchemicalshiftuponbinding.Thisisasignicantalterationfromthesingle =1.25peakintheunboundligand.The CH 3 hydrogens at1.22ppmprobablycorrespondtothosemostdistantfromtheboundmagnesium, astheyhavetheleastalteredchemicalshiftofanyhydrogenspresentinthecom45

PAGE 51

3.2MetalComplexSynthesis plex.Thepyrazolylhydrogens,consistentwiththeirpositionnearthemagnesium, arealsomoreshieldedthantheunboundligand,at =5.87insteadof =6.02. Thecentral CH hydrogencomesinat =6.89,anupeldshiftfrom7.27ppm.A peakat3.65ppmwasdeterminedtobelongtoresidualmethanol,andintensied upontheadditionofmethanol. 46

PAGE 52

3.2MetalComplexSynthesis Figure3.3.: 1 H NMRspectraofHbdtbpzatopand[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]bottom,alignedonthesameaxisfordirectcomparison.Notethatupperpeak at2.18ppmandlowerpeakat3.65ppmareduetoacetoneandmethanol, respectively. 47

PAGE 53

3.2MetalComplexSynthesis Thecarbonspectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ]isalsoshiftedfromthatof theunboundligand,butinalessstraightforwardfashionthanthehydrogenspectrum,ascanbeviewedinFigure3.1CandD.The CH 3 carbonsappearedasasinglepeak,at =30.7asopposedto30.3ppm,andthetert-butylcarbonsastwo closelyspacedpeaksat =32.0and =32.1insteadof =32.1and32.4forthe unboundligand.Thecentral CH carboncameinat =75.8,shifteddowneld 2.8ppm.Thethreepyrazolylcarbonswereshiftedto =101.7, =152.9,and = 159.8alteredby1.6,-1.2,and0ppm.Thecarboxylcarbonwasnotablyshiftedto =170.4,downeldby3.7ppm.Apeakat211.0ppmwasdeterminedtobedue toresidualacetone. 48

PAGE 54

3.2MetalComplexSynthesis Figure3.4.: 13 C NMRspectraofHbdtbpzatopand[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ]bottom,alignedonthesameaxisfordirectcomparison.Thepeakaround210ppm isduetoacetone. 49

PAGE 55

3.2MetalComplexSynthesis Severalrelatedheteroscorpionatezinc/magnesiumcomplexeshavebeenpreparedandcharacterizedintheliterature:[Znbdmpza 2 ],[Znbdippza 2 ],[Mgbdippza 2 ], [ZnbdippzaCl],and[ZnbdtbpzaCl]. 1,2 Itwashopedthatbyexaminingtherelativechemicalshiftsoftheresonancesofportionsoftheligandthatwerecommonto alltheligands,andnotingwhichcomplexeswere1:1orbiscomplexes,itcouldbe determinedwhether[MgBdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ]wasa1:1orabiscomplex.Thechanges inthechemicalshiftsofthesharedportionsoftheseligandsuponformationofvariouscomplexesistabulatedinTables3.1 1 H and3.2 13 C .Thequaternarytertbutyl,tertiaryisopropyl,andmethylcarbonsofthethreeligandswerecompared sincetheyoccupythesamepositionrelativetotheion,anddespitedieringchemicalenvironmentswouldbeexpectedtoexperiencesimilareects. Table3.1.: Changesof 1 H NMRshiftsoftheligandsbdtbpza,bdippza,andbdmpzauponforming1:1andbiscomplexeswithzincandmagnesiumions. 1,2 Only includespeakscommontoallthreeligands. Complex Central CH Pyrazolyl H BisComplexes [Znbdmpza 2 ] 2 -0.19 -0.03 [Znbdippza 2 ] 1 -0.05 -0.35 [Mgbdippza 2 ] 1 -0.07 -0.36 1:1Complexes [ZnbdippzaCl] 1 0.06 -0.37 [ZnbdtbpzaCl] 2 0.15 0.15 Indeterminant [Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ] -0.14 -0.38 Unfortunately,thesecomparisonsyieldambiguousresults.Theshared 1 H peaksseemtosupportthepossibilitythat[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ]isabiscomplex. Theshieldingofoneofthepyrazolylcarbonsalsoseemstosupportthisconclusion. However,thestrongdeshieldingofthecentral CH carbonandthecarboxylcarbon 50

PAGE 56

3.3FutureWork: Table3.2.: Changesof 13 C NMRshiftsoftheligandsbdtbpza,bdippza,andbdmpza,uponforming1:1andbiscomplexeswithzincandmagnesiumions. 1,2 The tert-butylcarbonandpyrazolylcarbonshiftsarelistedfromlowesttohighest chemicalshift. Complex T-bu/iPr/M C Central CH pzC pzC pzC COO )]TJETq1 0 0 1 547.929 600.413 cm[]0 d 0 J 0.398 w 0 0 m 0 14.446 l SQq1 0 0 1 108 600.214 cm[]0 d 0 J 0.398 w 0 0 m 439.929 0 l SQq1 0 0 1 108 598.222 cm[]0 d 0 J 0.398 w 0 0 m 439.929 0 l SQq1 0 0 1 108 583.577 cm[]0 d 0 J 0.398 w 0 0 m 0 14.446 l SQBT/F17 11.9552 Tf 290.425 587.91 Td [(BisComplexes [Znbdmpza 2 ] 2 -0.1 -0.6 -2.8 -1.1 -2.0 1.3 1.2 [Znbdippza 2 ] 1 -0.37 0 -3.0 -2.1 -1.7 2.1 1.0 [Mgbdippza 2 ] 1 -0.35 0 -2.5 -2.1 -1.3 2.8 1.1 1:1Complexes [ZnbdippzaCl] 1 0.5 -1.0 -3.6 -1.1 2.1 5.2 1.3 [ZnbdtbpzaCl] 2 0.3 0.4 -0.4 0.7 2.4 4.8 -0.9 Indeterminant [Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ] -0.1 -0.3 3.1 -1.4 -1.1 0.2 4.2 lendsupporttothepossibilitythatthecomplexis1:1. Intheinfraredspectrum,thecarbonylstretchat = 1750 cm )]TJ/F41 7.9701 Tf 6.587 0 Td [(1 wasshifted to = 1654 cm )]TJ/F41 7.9701 Tf 6.587 0 Td [(1 ,consistentwiththeweakenedbondfoundinmagnesiumandzinc complexesofbdippza. 1 Thisinformationdoesnotcontributetodeterminingthe natureofthecomplexformedbeyondthatitisametalcomplex,asboth1:1and biscomplexesexperiencedasimilarshiftinIRresonancefortheC=Ostretch. 3.3.FutureWork: Amagnesiumcomplexoftheligandbdtbpzahasbeenprepared.Thecomplex hasbeencharacterizedusingNMRandIRspectroscopy,butfurtherworkisneeded todetermineifthiscomplexis1:1.Inordertodothis,highqualitycrystalsmust beprepared,andanX-raycrystalstructureobtained.Anelementalanalysiswould alsobehelpfulincompletelycharacterizingthiscomplex.Ifthecomplexprepared 51

PAGE 57

3.3FutureWork: isfoundtoactuallybe[MgbdtbpzaCl],itssuitabilityasanactivesitemodelof RuBisCocanthenbeevaluated. Inordertoevaluatethis,themoststraightforwardmethodwouldbetoconductsimilarexperimentsaswereconductedonMgTCMApreviously. 59 First,an equimolarmixtureofacetolandthecomplexcouldbedissolvedindeuteratedmethanol alongwithaprotonsponge,tomaintainaconstantbasicpDandavoidcomplex dissociationandaddedtoanNMRtube.Ifthecomplexisabletocatalyzethe deuterationoftheacetolsubstratemimic,thehydrogenpeaksfromacetolwillgraduallydisappear,asbindingdecreasesthepKa'softheacetolevenifthesubsequent complex'slifetimeisshortontheNMRtimescale.Ifthecomplexisnotabletocatalyzethedeuterationofacetol,itislikelythatbdtbpzaistoohinderedtoorderto supportcoordinationoftheacetol. Thepossibleabilityofthecomplextoxcarbondioxidecouldalsobeprobed usingasimilarmethod. 59 AnNMRtubewithasolutionofthemagnesiumcomplexindeuteratedmethanolcouldbeprepared,andaddedtoanNMRtube.The tubecouldthenbepressurized,purged,andagitatedwithdrycarbondioxideseveraltimes.Followingthis,itcouldbeinjectedwithacetolthroughaseptum.This samplecouldthenbemonitoredviaNMRfortheappearanceofcarbonxation products. 52

PAGE 58

A.SelectedNMRandIRSpectra 53

PAGE 59

SelectedNMRandIRSpectra FigureA.1.: IRSpectrumofbdtbpzm 54

PAGE 60

SelectedNMRandIRSpectra FigureA.2.: 1 H NMRSpectrumofbdtbpzm 55

PAGE 61

SelectedNMRandIRSpectra FigureA.3.: 13 C NMRSpectrumofbdtbpzm 56

PAGE 62

SelectedNMRandIRSpectra FigureA.4.: IRSpectrumofHbdtbpza 57

PAGE 63

SelectedNMRandIRSpectra FigureA.5.: 1 H NMRSpectrumofHbdtbpza 58

PAGE 64

SelectedNMRandIRSpectra FigureA.6.: 13 C NMRSpectrumofHbdtbpza 59

PAGE 65

SelectedNMRandIRSpectra FigureA.7.: IRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ] 60

PAGE 66

SelectedNMRandIRSpectra FigureA.8.: 1 H NMRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.586 0 Td [(n ] 61

PAGE 67

SelectedNMRandIRSpectra FigureA.9.: 13 C NMRSpectrumof[Mgbdtbpza n Cl 2 )]TJ/F43 7.9701 Tf 6.587 0 Td [(n ] 62

PAGE 68

SelectedNMRandIRSpectra 63

PAGE 69

Bibliography [1]Kriegel,B.Bdippza:SynthesisandMetalComplexesofanewMonoanionic [N2O]HeteroscorpionateLigand.UndergraduateThesis,NewCollegeof Florida,DivisionofNaturalSciences,2010. [2]Beck,A.;Weibert,B.;Burzla,N. EuropeanJournalofInorganicChemistry 2001 ,521. [3]Field,C.;Behrenfeld,M.;Randerson,J.;Falkowski,P. Science 1998 281 237. [4]Kung,S. Sciene 1976 191 ,429. [5]Ellis,R. TrendsinBiochemicalSciences 1979 4 ,241. [6]Huegler,M.;Sievert,S.M.In AnnualReviewofMarineScience,VOL3 ; Carlson,CAandGiovannoni,SJEd.;AnnualReviewofMarineScience; AnnualReviews:4139ElCaminoWay,POBOX10139,PaloAlto,CA 94303-0897USA,2011;Vol.3;pp261. [7]Andersson,I. JournalofExperimentalBotany 2008 59 ,1555. 64

PAGE 70

Bibliography [8]Andersson,I.;Backlund,A. PlantPhysiologyandBiochemistry 2008 46 275. [9]Karkehabadi,S.;Satagopan,S.;Taylor,T.C.;Spreitzer,R.J.;Andersson,I. Biochemistry 2007 46 ,11080. [10]Kelly,G.In ProgressinBotany ;Esser,K.,Luttge,U.,Beyschlag,W., Murata,J.,Eds.;ProgressinBotany;SpringerBerlinHeidelberg,2005; Vol.66;pp218. [11]Bassham,J. PhotosynthesisResearch 2003 76 ,35. [12]Bauwe,H.;Hagemann,M.;Kern,R.;Timm,S. CurrentOpinioninPlant Biology 2012 15 ,269. [13]Wildman,S.;Bonner,J. ArchivesofBiochemistry 1947 14 ,381. [14]Benson,A.;Calvin,M. AnnualReviewofPlantPhysiologyandPlant MolecularBiology 1950 1 ,25. [15]Jorgensen,E.;Bassham,J.;Calvin,M.;Tolbert,B. JournaloftheAmerican ChemicalSociety 1952 74 ,2418. [16]Benson,A.;Bassham,J.;Calvin,M.;Hall,A.;Hirsch,H.;Kawaguchi,S.; Lymch,V.;Tolbert,N. JournalofBiologicalChemistry 1952 196 ,703. [17]Hurwitz,J.;Weissbach,A.;Horecker,B.;Smyrniotis,P. JournalofBiological Chemistry 1956 218 ,769. [18]Dorner,R.;Kahn,A.;Wildman,S. JournalofBiologicalChemistry 1957 229 945. 65

PAGE 71

Bibliography [19]Park,R.;Pon,N. JournalofMolecularBiology 1961 3 ,1. [20]Bassham,J.;Kirk,M. BiochemicalandBiophysicalResearchCommunications 1962 9 ,376. [21]Pon,N.;Rabin,B.;Calvin,M. BiochemischeZeitschrift 1963 338 ,7. [22]Akoyunoglou,G.;Calvin,M. BiochemischeZeitschrift 1963 338 ,20. [23]Jensen,R.;Bassham,J. BiochimicaetBiophysicaActa 1968 153 ,227. [24]Bjorkman,O. PhysiologiaPlantarum 1968 21 ,1. [25]Bjorkman,O. PhysiologiaPlantarum 1968 21 ,84. [26]Wareing,P.;Khalifa,M.;Treharne,K. Nature 1968 220 ,453. [27]Cooper,T.;Filmer,D. JournalofBiologicalChemistry 1969 244 ,1081. [28]Wishnick,M.;Lanes,M.;Scrutton,M. JournalofBiologicalChemistry 1970 245 ,4939. [29]Ogren,W.;Bowes,G. Nature-NewBiology 1971 230 ,159. [30]Bowes,G.;Ogren,W.;Hageman,R. BiochemicalandBiophysicalResearch Communications 1971 45 ,716. [31]Shively,J.;Ball,F.;Brown,D.;Saunders,R. Science 1973 182 ,584. [32]Tabita,F.;McFadden,B. JournalofBiologicalChemistry 1974 249 3453. 66

PAGE 72

Bibliography [33]Tabita,F.;McFadden,B. JournalofBiologicalChemistry 1974 249 3459. [34]Hartley,M.;Wheeler,A.;Ellis,R. JournalofMolecularBiology 1975 91 67. [35]Baker,T.;Eisenberg,D.;Eiserling,F.;Weissman,L. JournalofMolecular Biology 1975 91 ,391. [36]Lorimer,G.;Badger,M.;Andrews,T. Biochemistry 1976 15 ,529. [37]Wildman,S. PhotosynthesisResearch 2002 73 ,243. [38]McIntosh,L.;Poulsen,C.;Bogorad,L. Nature 1980 288 ,556. [39]Bedbrook,J.;Smith,S.;Ellis,R. Nature 1980 287 ,692. [40]Jordan,D.;Ogren,W. Nature 1981 291 ,513. [41]Yeoh,H.;Badger,M.;Watson,L. PlantPhysiology 1981 67 ,1151. [42]Saver,B.;Knowles,J. Biochemistry 1982 21 ,5398. [43]Gutteridge,S.;Parry,M.;Schmidt,C.;Feeney,J. FebsLetters 1984 170 355. [44]Schneider,G.;Lindqvist,Y.;Branden,C.;Lorimer,G. EmboJournal 1986 5 3409. [45]Hemmingsen,S.;Woolford,C.;Vandervies,S.;Tilly,K.;Dennis,D.; Georgopoulos,C.;Hendrix,R.;Ellis,R. Nature 1988 333 ,330. 67

PAGE 73

Bibliography [46]Tabita,F.R.;Satagopan,S.;Hanson,T.E.;Kreel,N.E.;Scott,S.S. Journal ofExperimentalBotany 2008 59 ,1515. [47]Gibson,J.;Tabita,F. JournalofBiologicalChemistry 1977 252 ,943. [48]Hayashi,N.;Oguni,A.;Yaguchi,T.;Chung,S.;Nishihara,H.;Kodama,T.; Igarashi,Y. JournalofFermentationandBioengineering 1998 85 ,150. [49]Cleland,W.;Andrews,T.;Gutteridge,S.;Hartman,F.;Lorimer,G. Chemical Reviews 1998 98 ,549. [50]Tcherkez,G.G.B.;Bathellier,C.;Stuart-Williams,H.;Whitney,S.;Gout,E.; Bligny,R.;Badger,M.;Farquhar,G.D. Biochemistry 2013 52 ,869. [51]Parry,M.A.J.;Madgwick,P.J.;Carvalho,J.F.C.;Andralojc,P.J. Journal ofAgriculturalScience 2007 145 ,31. [52]vonCaemmerer,S. PlantCellandEnvironment 2003 26 ,1191. [53]Zhu,X.-G.;Long,S.In Photosynthesisinsilico ;Laisk,A.,Nedbal,L., GovindjeeEds.;AdvancesinPhotosynthesisandRespiration;Springer Netherlands,2009;Vol.29;pp401. [54]Yoshida,S.;Atomi,H.;Imanaka,T. AppliedandEnvironmentalMicrobiology 2007 73 ,6254. [55]Zabaleta,E.;VictoriaMartin,M.;Braun,H.-P. PlantScience 2012 187 97. [56]Ibers,J.;Holm,R. Science 1980 209 ,223. 68

PAGE 74

Bibliography [57]Karlin,K. Science 1993 261 ,701. [58]Odom,D.;Gramer,C.;Young,V.;Hilderbrand,S.;Sherman,S. Inorganica ChimicaActa 2000 297 ,404. [59]Hilderbrand,S.InvestigationofInteractionsBetween [MGTCMAH2O]O3SCF3andRibulose-1,5-BisphosphateMimicsasa ModelSystemfortheActiveSiteoftheEnzymeRuBisCo.Undergraduate Thesis,NewCollegeofFlorida,DivisionofNaturalSciences,1998. [60]Wheeler,K.IdenticationandKineticStudiesoftheReactionBetween MgO3SCF3andAcetol:AModelfortheFirstStepintheCatalytic PathwayofRuBisCo.UndergraduateThesis,NewCollegeofFlorida,Division ofNaturalSciences,2001. [61]Horowitz,J.ReactionsofMagnesiumandZincSaltswithAcetolandCarbon DioxideasModelsforCatalysisbyRuBisCo.UndergraduateThesis,New CollegeofFlorida,DivisionofNaturalSciences,2008. [62]Liang,A. Unpublishedresults [63]Kannappan,B.;Gready,J.E. JournaloftheAmericanChemicalSociety 2008 130 ,15063. [64]Otero,A.;Fernandez-Baeza,J.;Tejeda,J.;Antinolo,A.; Carrillo-Hermosilla,F.;Diez-Barra,E.;Lara-Sanchez,A.; Fernandez-Lopez,M.;Lanfranchi,M.;Pellinghelli,M. JournaloftheChemical Society-DaltonTransactions 1999 ,3537. 69

PAGE 75

Bibliography [65]Kitajima,N.;Fujisawa,K.;Fujimoto,C.;Morooka,Y.;Hashimoto,S.; Kitagawa,T.;Toriumi,K.;Tatsumi,K.;Nakamura,A. Journalofthe AmericanChemicalSociety 1992 114 ,1277. [66]Burzla,N.In AdvancesinInorganicChemistry,Vol.60 ;vanEldikREd.; AdvancesinInorganicChemistry;ElsevierAcademicPressINC:525BStreet, Suite1900,SanDiego,CA92101-4495USA,2008;Vol.60;pp101. 70


ERROR LOADING HTML FROM SOURCE (http://ncf.sobek.ufl.edu//design/skins/UFDC/html/footer_item.html)