determinefreehost

ARTICLEReceived27May2013|Accepted10Sep2013|Published8Oct2013Macromolecularsemi-rigidnanocavitiesforcooperativerecognitionofspeciclargemolecularshapesTakaneImaoka1,YukiKawana1,TakutoKurokawa1&KimihisaYamamoto1Molecularshaperecognitionforlargerguestmolecules(typicallyover1nm)isadifculttaskbecauseitrequirescooperativitywithinawidethree-dimensionalnanospacecoincidentallyprobingeverymolecularaspect(size,outlineshape,exibilityandspecicgroups).
Althoughtheintelligentfunctionsofproteinshavefascinatedmanyresearchers,thereproductionbyarticialmoleculesremainsasignicantchallenge.
Herewereporttheconstructionoflarge,well-denedcavitiesinmacromolecularhosts.
Throughtheuseofsemi-rigiddendriticphe-nylazomethinebackbones,evensubtledifferencesintheshapesoflargeguestmolecules(uptoB2nm)maybediscriminatedbythecooperativemechanism.
Aconformationallyxedcomplexwiththebest-ttingguestissupportedbyathree-dimensionalmodelbasedonamolecularsimulation.
Interestingly,thesimulatedcavitystructurealsopredictscatalyticselectivitybyarutheniumporphyrincentre,demonstratingthehighshapepersistenceandwideapplicabilityofthecavity.
DOI:10.
1038/ncomms35811ChemicalResourcesLaboratory,TokyoInstituteofTechnology,Yokohama226-8503,Japan.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoK.
Y.
(email:yamamoto@res.
titech.
ac.
jp).
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Theultimatechallengeinhost–guestchemistryisthedesignofsynthetichoststhatcanuniversallyrecognizeguestmoleculeswithvariousstructuralcharacteristicsincludingsize,shapeanddetailssuchasthepositionsoffunctionalgroups.
Ofparticularscienticinterestistheconceptofthearticialenzyme,whichcanactasacatalystforaspecicsubstrateviacooperativerecognition1.
Theimportanceofpracticalmaterialscanbeseeninsolution-phaserecognition,whichallowsefcientchemicalseparationdespiteverysmallstructuraldifferences2.
Theessentialrequirementforrecognitionisashape-persistentcavitandgenerallymadeofcovalentmacrocycles3–5orsupra-molecularcages6–9.
However,recognitionhasbeenlimitedtorelativelysmallguestmoleculesofsubnanometresize(uptotwoaromaticrings).
Apartfromparticularexamples10,11,attemptsatsimpleenlargementofthecavitandswithoutastrategyaregenerallyfruitlessbecausethelargerhostmoleculesarenolongershapepersistent.
Onetypicalexampleisthecaseofdendrimers.
Themolecularweightandchemicalstructureofthedendrimersareunity.
Inaddition,thecore-shellarchitecturegenerallyprovidesauniqueencapsulationeffect12.
Atthedawnofdendrimerchemistry,itwasbelievedthatthegiantinnercavitywouldbeapplicabletovarioustypesofmolecularrecognition,similartonaturalenzymes13,andthatdendrimerswerepromisingcandidatesforlarge-scalerecognition14.
Theideawasthattheywouldactaslargecontainers,allowingencapsulationofmanyguestmoleculessimultaneously15–17.
However,thepreviousdendrimerswerefoundtobesuitableonlyforidenti-cationofsimpleandsmallguests18–22.
Althoughdendrimersarepotentialcontainersforalargerguestormanyguestmolecules,theycanbecomecompactthroughback-foldingoftheirterminalmonomerstowardstheinsideofthecavity,leadingtolossofthecavity23–25.
Meanwhile,dendriticstructureswithextra-rigidbackbonesdonotparticipatetoasignicantextentinhost–guestbinding26,27.
Exceptforsomeintriguingapproachessuchasmolecularimprinting28,molecules41nmarestilldifculttorecognize.
Evennow,itisimpossibleachievingthisonlybythemolecularshapebecausethisrequiresdetaileddesignofthenanospacethroughasemi-rigidarchitecture.
Inthisstudy,werevisithost–guestchemistryusingamoderndendrimerdesignforthehostmolecules.
Phenylazomethinedendrimerscomposedofrigidp-conjugatedbackbonesexhibitexceptionallynecoordinationprocesses,suchasstep-by-stepassembly29–31,allowingone-atomcontrolledsynthesisofmetalclusters32.
Inaddition,previousstudieshaveshownthatthestablequadrupolecharacterofsuchdendrimersallowsanefcientphotoelectricprocessbasedontheirrecticationproperties33.
Byusingthisuniquedendrimer,wesuccessfullyachieveverynerecognitionoflargeguestmolecules.
ResultsScreeningandrenementofguests.
Thehostmoleculesemployedinthisstudyaredendrimerswithametalloporphyrincoreasabindingcentreforguestmoleculessuchaspyridinederivatives.
Fourdendronunitswerehorizontallyspreadtotheoutsidefromtheporphyrincore,whereasthetwoaxialspaceswereretained(Fig.
1).
Forexample,theexperimentalhydro-dynamicdiameterofZnG4aisover4nm.
Thepercentageoffreevolumeinthedendrimer,denedasvacantspaceoftheatom-icallyoccupiedvanderWaalsvolumeinthemolecularhydro-dynamicspace,isca.
80%(ref.
34).
Tostudythecharacteristicsofthesesyntheticcavities,weinitiallyinvestigatedbindingbetweenaguestmoleculeandthezincporphyrincoreburiedinthecavity.
Apyridinederivativewasusedasaguestmolecule,because1:1complexformationbetweenzincporphyrinandthisderivativehasbeenestablishedandiswidelyaccepted35,36.
ZnG4awassynthesizedaccordingtoaliteraturemethod37.
BycomparingthebindingconstantsofZnG4aandthecorrespondingrst-generationmodel(ZnG1a),wewereabletoestimatethestericcontributionofthedendrongroupstohost–guestcomplexation.
Thereafter,theratioKG4a/KG1awasestimatedforeachguestmolecule(pyridinederivatives,Fig.
2)asanindexparameterforadaptabilitywiththesyntheticcavity.
Initialscreeningwascarriedoutusingpyridinederivativeswithseveralaromaticcomponentswithvariousconnections.
Anultraviolet–visibletitrationanalysiswascarriedoutinatoluene/acetonitrile(v/v1:1)solvent,followedbytheoreticalcurvettingoftheexperimentalresultstoobtainbindingconstantswiththesynthetichostsat20°C(SupplementaryTableS1).
Thebindingstoichiometryofthepyridinetothezincporphyrincore(axialposition)is1:1,asdeterminedbyultraviolet–visibleabsorption,isothermaltitrationcalorimetry(ITC)andNMRspectroscopy(atypicalexampleisshowninFig.
3).
Thebindingconstantswereindependentlyobtainedusingallofthesemeasurements,andeachresultwasfoundtobeingoodagreementwiththeothers.
ItisnotablethatthebindingafnitywiththedendrimerhostZnG4avariedinasensitivemannerdependingontheshapeoftheguestmolecule.
Pyridinederivativescontainingtwophenylgroupsatdifferentpositions(5,6,7,8,9and10)gaveverydifferentbindingconstants(KG4a),althoughtheirformulasandmolecularweightswereidentical.
Oneofthederivatives,whichwasY-shaped(4-(3,5-diaryl)phenylpyridine,11)wasfoundtocoordinatetothedendrimerhostwithalargerbindingconstant,whereasguestswithothershapes(15,16,17and18)didnotcoordinatesostrongly(Fig.
3f).
Eventheshapesofverylargemolecules(ca.
19width)withthesamechemicalformula(12,13and14)werecorrectlyrecognized.
Incontrast,amongtheselectedguestmolecules,therewaslittlevariationinbindingafnitywiththenon-dendritichost(ZnG1a).
Theseobservationsareevidenceofshaperecognition,butnotofanelectroniceffectcausedbytheelectron-donating/withdrawingcharacterofthesubstitutedgroups.
ThedendrimershellexhibitedapositiveeffectonthebindingofY-shapedguests,ascanbeobservedfromthevalueofthebindingconstantratioKG4a/KG1a(upto4.
2for11).
Previousreportsonhost–guestchemistryNNNNBDNHHnp-Phm-Php-BPhBDBDBDBDDPAGnMBDOOPhPhBzEGnnMZnG1ap-PhDPAG1ZnZnG2ap-PhDPAG2ZnZnG3ap-PhDPAG3ZnZnG4ap-PhDPAG4ZnZnG4bm-PhDPAG4ZnZnG4cp-BPhDPAG4ZnZnG4d–BzEG4ZnRuG1ap-PhDPAG4Ru(CO)RuG4ap-PhDPAG4Ru(CO)H2G4ap-PhDPAG4H2Figure1|Structuresofdendrimers.
Thedendrimersarecomposedofthreedifferentparts.
Thecoreunitisazincorrutheniumcarbonylporphyrin,whichcanbindonepyridinederivativeattheaxialposition.
Thebridges(B)arelinearorbentphenylenegroups,adjustingthepositionanddirectionofthedendronsubunits.
Thedendron(D),whichconsistsofdendriticphenylazomethineorbenzylethergroups,createsananospacethroughtheformationofarigidorexibleshell,respectively.
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involvingdendrimerarchitecturehaveshownthatanincreaseinthegenerationnumberresultsinaseveredecreaseinbindingafnitybecauseoftheshelleffect18–22.
Infact,thedendrimerhostunderdiscussionexhibitsunusualenhancementonhost–guestcomplexation.
Toseekthebest-ttingmolecule,wecarriedoutfurthertuningoftheY-shapedguests.
Chemicalmodicationofthetipsof11,giving19,20and21,increasedthebindingconstant(KG4a).
Ofparticularinterestwasthendingthatchemicalmodicationofthe'dip'oftheY-shapedpyridine25signicantlyenhancedthebindingstrength,resultinginahighbindingconstantratio(KG4a/KG1a)ofupto19for25.
Insharpcontrast,suchanenhancementwasnotobservedforanI-shapedderivative(24).
Thedendrimershelldidnotactasapositivefactorforotherguestssuchasunsubstitutedpyridine(1)andrigidI-shapedpyridines(4and10).
Further,thekinkedmolecules5,6,7and8,andoversizedderivativesthatdeviatedfromtheY-shape(12,22and23),wereexcludedfromthehost.
Thus,thecommonY-shapedstructureactedasananchorthatspecicallyboundtothedendrimerhost.
Modulationofinteractions.
Thedependenceofcomplementaryhost–guestbindingonthehoststructurewasstudiedbytestingzinc-porphyrin-coredphenylazomethinedendrimers(ZnGna:n1B4)withgenerationnumbersof1–4.
Thisexaminationrevealedthatadendriticstructurewithatleastfourgenerationsisessentialfortheformationofacavitythatiscapableofmoleculardiscrimination.
Indeed,noshape-selectivebindingtothehostwasobservedwhenthegenerationnumberwaso4(Fig.
4a).
Aroughestimateofthebindingstructureusingamolecularspace-llingmodelsuggestedthatthecavitiesindendrimersofgeneration3orlessweretooshallowtotightlyanchortheguest.
Thestructureofthedendrimersignicantlyaffectedhost–guestcomplexationthroughshapemodicationofthecavity.
Wesynthesizeddendrimerswithalternativestructures:onehadfourdendronsatthemeta-positionsonthephenylgroupsofthezincporphyrincore(ZnG4b),whereastheotherhadfourdendronsatthepara-positionswith1,4-phenylenespacers(ZnG4c).
Onthebasisofspace-llingmodelsobtainedbymolecularmodelling,itbecameapparentthatZnG4chadanextendedcavity,whereasthatofZnG4bwasshrunken.
Thesemolecularimageswereconrmedbyexploringthefreevolumepercentageswithinthehydrodynamicvolume(ZnG4a,78%;ZnG4b,71%;ZnG4c,77%)34.
TheZnG4bhost,whichhadanarrowcavity,didnotallowbindingoftheguestmolecule20,buttheZnG4chost,whichhadanextendedcavity,alsoshowedalowerbindingafnitywith20.
ZnG4cpreferredlargerguestmoleculessuchas14and22.
Wealsoexaminedaexibledendrimercomposedofabenzyletherstructure(ZnG4d)withthesametopologicalarchitecturearoundthezincporphyrincore,butwithaverysmallfreevolumepercentage(45%)38.
Asshowninpreviousstudies,exibledendronsexhibitexclusivehost–guestbind-ing18,20–22.
ThehostZnG4dallowedbindingonlyofsmallguestmolecules(1and4)withoneornophenylgroup(Fig.
4b).
Thisobservationwasconsistentwithpreviousndingsthatexibledendrimerssimplydecreaseguestmolecularbinding(thewell-recognized'shelleffect').
Conversely,exchangingthemetalcomplexdidnotaffecttheselectivityofthehostmoleculetothesameextent.
Theruthenium-porphyrin-coredphenylazomethinedendrimerRuG4a,acarbonyl-cappedcomplex,showedalmostthesametrendinitsshapeselectivityasZnG4a(SupplementaryFig.
S1).
Thisfactindicatedthatmetalcomplexationintheporphyrincoredidnotaffecttheshapeofthecavity.
Structuralanalysis.
Todeterminethebindingstate,a1HNMRanalysiswascarriedoutusingtheY-shapedguestmolecule20withtert-butyl(tBu)groups.
ThechemicalshiftcorrespondingtotheprotonsofthetBuunitin20wasshiftedupeldonadditionof20toaZnG4asolutionat20°C(Fig.
5a).
Thisobservationimpliedthatchemicalexchangebetweenthetrappedandfree-guestmoleculeswasfasterthantheNMRtimescale(afewmil-liseconds).
Atalowtemperature(20°C),wecouldobservetheprotonsignalscorrespondingtothetrappedandfree-guestmolecules.
Whentwoequivalentsof20wereadded,thesignalNR3R2R1R1R2R11:R1=R2=R3=H2:R1=Ph,R2=R3=H3:R2=Ph,R1=R3=H4:R3=Ph,R1=R2=H17:R2=TPP,R1=R3=H5:R1=Ph,R2=R3=H6:R2=Ph,R1=R3=H7:R3=Ph,R1=R2=HNR1R1R2R2R38:R1=Ph,R2=R3=H9:R2=Ph,R1=R3=H10:R3=Ph,R1=R2=HNR3NR1R2R3R3R2R111:R1=R2=R3=H12:R1=Ph,R2=R3=H13:R2=Ph,R1=R3=H14:R3=Ph,R1=R2=H19:R3=iPr,R1=R2=H20:R3=tBu,R1=R2=H21:R3=cHex,R1=R2=HNR215:R1=R2=H16:R1=Ph,R2=HTPPNRRRR22:R=Ph23:R=tBuN24:R1=HR2=C18H3725:R1=PhR2=C18H37R1R1OR218:R3=TPP,R1=R2=H26:R1=HR2=C6H1327:R1=PhR2=C6H13Figure2|Structuresofguestmolecules.
Shape-persistentpyridinederivativeswereselectedasmolecular'keys'forthehostmoleculesbasedonthedendrimerarchitecture.
Eachkeywascomposedofapyridineandseveralphenylgroupswithdifferentconnections.
Thepyridinegroupactedastheconnectingtiptotheaxialcoordinationsiteofthezincporphyrincorexedinthedendrimerhost.
Thephenylgroupactedasanidentierforthemolecules,recognizedbythecavityaroundtheaxialligationsiteofthehost.
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integrationvaluesofthesetrappedandfree-guestmoleculeswerealmostequivalent,supporting1:1host–guestcomplexation.
Oneofthesesignalsappearingdowneldhadachemicalshift(d)of1.
31p.
p.
m.
andaspin-latticerelaxationtime(T1)of684ms,whichwereidenticaltothoseofthefree-guestmolecule(d1.
31p.
p.
m.
,T1665msfor20),whereastheothersignalexhibitedaverydifferentchemicalshift(d1.
02p.
p.
m.
)andrelaxationtime(T1874ms).
ThelongerT1forthesignalat1.
02p.
p.
m.
canbeunderstoodbasedoneffectiveencapsulationbythedendrimershell,aspreviouslyreported16,18,37,39,40.
Thisbehaviouristypicalfortheenvironmentinrigidphenyl-azomethinedendrimers,whereT1andT1/T2increasewereobservedfortheencapsulatedprotonsasthegenerationnumberincreases37,41.
Thisincreaseisinterrelatedasaslowerdynamicmotionofthedendrimer(longerrotationalcorrelationtime)42.
1Hdiffusion-orderedspectroscopy(DOSY)-NMRmeasure-mentssupportedourcontentionthatthesignalsat1.
31and1.
02p.
p.
m.
correspondedtothefreeandbindingguest,respectively.
Thediffusioncoefcient(D)ofZnG4a(2.
31010m2s1)determinedbyDOSY-NMRwasmuchsmallerthanthatofzinctetraphenylporphyrin(9.
21010m2s1)becauseofthelargerhydrodynamicradius(SupplementaryFig.
S2).
ThesevaluesarecomparabletothediffusioncoefcientsofZnG4a(1.
11010m2s1)andzinctetraphenylporphyrin(8.
01010m2s1)intetrahydrofuran,calculatedfromthehydrody-namicradiiusingtheStokes–Einsteinequation34.
IftheguestmoleculebindstoZnG4a,theD-valuecorrespondingtothebindingguestmoleculeshouldbeidenticaltothatofthedendrimerhostmoleculebecausetheywoulddiffusetogetherinthesolution.
However,DofthetBuunitcorrespondingtoZnG4a–20wasslightlydifferentonthediffusionaxisevenatalowtemperature(20°C).
Thisresultindicatedthatthetimescaleofchemicalexchangebetweenthecomplexandthefreebasewasclosetothediffusiondelay(40ms).
Forquantitativeanalysis,wenextemployedRuG4a,inwhichbindingwasstrongerthanthatinZnG4a.
ItwasfoundthatthevalueofDforthetBuunitcorrespondingtoRuG4a–20agreedexactlywiththevalueofDforthedendronmoieties(Fig.
5c).
1HnuclearO¨verhauserenhancementspectroscopy-NMRmeasurementoftheRuG4a–20systemwascarriedoutforthestablecomplexovera100-mstimescale.
Thistechniquehasbeenextensivelyusedtoelucidatedendrimer-basedhost–guestsys-tems43.
Inthetwo-dimensionalspectrum,manycross-peakswereobservedamongthearomaticprotonsofthedendronsubunitsasintramolecularnuclearO¨verhausereffect(NOE)couplings.
Inaddition,aclearcross-peakbetweenthetBuprotonsofthebindingguestmolecule(20)andaspecicaromaticprotonsignal01234512345678910111213141516171819202122232425GuestmoleculeLogK(M–1)ZnG4aZnG1aNo'Y'motifContaining'Y'motifDeviated'Y'motif26270.
80.
60.
40.
20.
0450440430420λ(nm)Abs.
432nm442nm02460–15–10–50020406080100Heatflow(μJs–1)Heat(kJmol–1)Time(min)[Guest]/[Host]K=6.
0*104(M–1)–ΔH=55(kJmol–1)TΔS=–28(kJmol–1)135–0.
2–0.
10.
00.
13020100ExperimentalCurvefitting1.
01.
240[Guest](μM)ΔAbs.
442nm432nm–40–30–20–10NNNNZnNNNNNZnNCooperativerecognitionBindingcentreHostGuestKFigure3|Examinationofhost–guestbinding.
(a)Schematicrepresentationofcooperativebinding.
Dockingtestsforguestmoleculeswithvariousshapesallowedprobingoftheshapeofthecavity.
(b)Ultraviolet–visibleabsorptionspectra(20°C)ofZnG4a(2.
5mM)onadditionoftheguestmolecule20intoluene/acetonitrile(1:1).
(c)Plotsofthechangeinabsorbanceattwodifferentwavelengthsforvariousconcentrationsof20.
Atheoreticalttingcurve(solidline)wasappliedtotheabsorbancedata.
(d)Isothermaltitrationcalorimetricdatafortheadditionof20toasolutionofZnG4a(51mM)intoluene/acetonitrile(1:1).
(e)Aplotofthemolarreactionheatforeachtitrationpoint.
Theoreticalttingoftheexperimentaldatagavethethermodynamicparametersforhost–guestbinding.
(f)Host–guest-bindingconstants(K)betweenzincporphyrin-containingdendrimers(ZnG1aandZnG4a)andpyridinederivativesintoluene/acetonitrile(1:1).
Overall,Y-shapedguestmoleculesshowedhighbindingconstants.
112131418K(103M–1)K(103M–1)Guestmolecule14201422Guestmolecule23n=1n=2n=3n=4G4aG4bG4cG4d010203040500102030405060FitWideNarrowShrunkenGenerationnumberCavityshapeFigure4|Bindingconstantsforeachhost–guestpair.
(a)Dependenceonthegenerationnumber(n)ofthedendrimerZnG4n.
Thefourth-generationdendrimer(ZnG4a)showedastrongafnityforY-shapedguestmolecules.
Nosignicantdependencewasfoundfordendrimerswithagenerationnumbero3.
(b)Dependenceoncavitysizeandexibilityofthedendrimers.
ThespecicrecognitionobservedforZnG4awaslostwhenthecavitysizewasextendedorshrunk.
Theexiblebackbonealsoexcludedthelargerguestmolecules.
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ofthedendronsubunitwasalsoobservedasintermolecularNOEcoupling(Fig.
5d).
Asthephenylazomethinemonomerhadtwonon-equivalentaromaticringsatsyn-andanti-positions,theNMRprotonsignalsforeveryaromaticringinonedendronsubunitcouldnotbereconciled.
Onthebasisofthis,thetBuprotonsofthebindingguestmolecule(20)werethoughttobeclosetoaspecicareneinthedendrimercavitywithoutanytiltorrotation.
Itisnoteworthythatthegeneration-3dendrimerRuG3adidnotshowanyNOEcross-peakwith20,suggestingthatthefourthlayerofthedendrimercontributedtogueststabilization,asnotedabove.
Thisresultfeasiblyexplainsthemolecularpictureofthecomplexinathree-dimensional(3D)space(Fig.
5b)inwhichthemonomerunitonthefourthlayerofadendronapproachesthetBuunitoftheguestmolecule(20),probablybecauseofweakCH/pinteractions.
Theupeldshiftofthealiphaticprotonsignalobservedinthe1HNMRspectrumoftheZnG4a–20system(Fig.
5a)isatypicalbehaviourforaCH/pinteraction44,45.
Thermodynamicanalysis.
ThermodynamicdatafromITCandultraviolet–visibleanalysis(van0tHoffplot)providedinformationaboutthemechanismofmolecularrecognition.
ThevaluesofDH°(enthalpychange)andDS°(entropychange)obtainedfromITCanalysiswereingoodagreementwiththosefromthevan0tHoffanalysisandwereverydifferentforeachhost–guestpair.
Inthecaseof20(agoodt),DH°hadaverylargenegativevalue,whereasTDS°(T293K)hadalargepositivevalue(SupplementaryTableS1).
Incontrast,theabsolutevaluesofDH°andTDS°forthemorebadlyttingmoleculesweresmaller.
Thesefactscanbeinterpretedtomeanthatmaximalcontactbetweenthehostandguestresultingreaterstabilizationintermsoffreeenergy(DG°DH°TDS°).
Thisstabilizationismainlyduetothelargeintermolecularforceassociatedwiththeenthalpyfactor(DH°).
Surprisingly,thevalueofDH°for20wasalmosttwicethatfor4,whichimpliesthatthecontributionsfromtheupper'anchor'structureandfrompyridinecoordinationwerenearlyequivalent.
ThisincreaseinDH°canbeunderstoodasasummationofweakintermolecularinteractionsincludingp/p,CH/N,CH/p,vanderWaalsattractionandhydrophobicinter-action.
Thenon-dendritichost–guestsystemgaveamuchsmallerDH°variationforguestmoleculeswithdifferentshapes.
There-fore,theprimaryfactorinthehighmolecularrecognitionabilityofthedendrimerZnG4aisthoughttobetheenthalpycon-tribution,basedonthelargecontactareabetweenthehostandguestmolecules.
Animpressivedifferencewasfoundbetweenguestmolecules24and25,althoughtheonlystructuraldifferencewasthepresenceoftwophenylringsin25.
Althoughthealkylchainin25contributesthetightbindingverymuch,thesameDendrontButBuChemicalshift(p.
p.
m.
)8.
06.
04.
02.
08.
06.
04.
04.
06.
08.
02.
0–9.
0log[D(m2s–1)]1.
41.
31.
21.
11.
00.
940°C20°C0°C–20°CGuestonly(20°C)Chemicalshift(p.
p.
m.
)tButButButButButBuCoalescencePorphyrin(β-proton)SolventDendronDendronPorphyrin(β-proton)SolventF1(p.
p.
m.
)F2(p.
p.
m.
)–9.
5IntermolecularNOEIntramolecularNOEacdbCross-sectionTopNNNNZnNNNNRuCONEncapsulated(T1=874ms)Freebase(T1=665ms)EncapsulatedFreebaseHostGuestCH-ππ-πNNtButButButBuFigure5|Structuralanalysisofthehost–guest-bindingcomplex.
(a)Variable-temperature1HNMRspectraofaZnG4awith20atamixingratioof1:2(H:G)intoluene-d8/acetonitrile-d3(2:1).
ThesignalsoftBugroupsintheguestmolecule(20)areshownforboundandfreestates.
(b)Acomputer-generatedspace-llingmodelofZnG4aandacross-sectionalimageshownasasolvent-accessiblesurface(proberadius:1.
4)with20.
Theporphyrincoreishighlightedinmagenta.
This3Dcoordinatewasobtainedfromsemi-empiricalmolecularorbitalcalculations(MOPAC-AM1).
(c)1HDOSY-NMRspectrumofaRuG4awith20atamixingratioof1:2(H:G)indichloromethane-d2.
(d)1HnuclearO¨verhauserenhancementspectroscopy-NMRspectrumofRuG4awith20atamixingratioof1:1(H:G)indichloromethane-d2.
Theinsetgureisa3Dmodelofthehost–guestcomplex.
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structurein24didnotcontributeanyimprovementinhost–guestbinding.
Asimilardifferencewasfoundbetween26and27.
ThetitrationexperimentsdemonstratedthatbindingbetweenthehostandtheY-shapedpyridineinpuretoluenewasnotasstrongasinamixedsolventoftoluene/acetonitrile(1:1).
Ahighermixingratioofalesssolubilizingmedium(acetonitrile)allowedstrongerbindingbasedonanenthalpicdrivingforce(SupplementaryTableS2).
Inparticular,theuseofaspecicallypolararomaticsolvent(forexample,dimethylphthalate)asagoodmediumsignicantlyenhancedthebindingafnity(KG45.
4105M–1)andselectivity(KG4a/KG1a142)ofthereaction,asshowninSupplementaryTableS3.
Thisresultindicatesthedominanceofanon-classicalhydrophobiceffectasdiscussedbyDiederichandco-workers46,whichenablesstrongerhost–guestbindinginadendrimersystem.
Thethermodynamicbehaviourisdifferentfromthatoftheclassicalhydrophobicinteraction,inwhichentropicfavourabilityduetodesolvationisaprincipaldrivingforce47.
Ingeneral,hydrophobicinteractionsdonothaveacrucialroleinmolecularrecognition.
However,itisinterestingtonotethatthesolventeffectenhancesselectivityinthepresentsystem.
Thisenthalpicfavourabilitywasfoundonlyintheshape-matchingguestmoleculesandnotintheotherpyridinederivatives.
Selectivityincatalysis.
Thesyntheticnanocavitycanselectfavourableguestmoleculesbasedontheirshapes.
Inanaturalenzyme,thisprincipleisutilizedtoachieveexcellentselectivitybetweensubstratesandproducts.
OnthebasisofaMichaelis–Mentenmodel,thestabilityofanintermediateenzyme–substratecomplexisimportantinthedeterminationoftheentirekinetics.
Catalyticepoxidationofaromaticolensbyarutheniumpor-phyrin(RuPor)wasdemonstrated48–50.
Themechanismofcatalysis,usinga2,6-dichloropyridine-N-oxideastheoxidativereagent,iswellestablished,asfollows:(1)thereagentoxidizestheRuPortoaffordanoxo-complex(RuOPor)49.
(2)AnolenderivativeapproachestheoxygenatomonRuandformsanadductastheintermediatestate51.
(3)TheRuOPorrevertstoRuPorbydesorptionoftheepoxideproduct.
WereasonedthatifaRuPorinadendrimer-baseddeepcavitywasusedasacatalyst,molecularrecognitionoftheintermediatestatemightsignicantlyaffectthekineticsaspredictedbytheMichaelis–Mentenmodel.
Beforetheexperiment,wesimulatedthestructureofinter-mediatespecieswithvariousolens.
Forexample,methoxy-styrenehasthreestructuralisomers(o-,m-andpMeO-Sty).
Thesesubstratesweretestedwiththe3Dstructuralmodelofthedendrimerveriedfromtheabove-mentionedhost–guestchemistry.
Inthispredictionanalysis,mMeO-Stytthecavityverywell,whereasthepMeO-Styconictedwiththesidewall,asshowninFig.
6a,b.
Thekineticsweredetermined(SupplementaryTableS4)foreachpairoftwodendrimers(RuG1aandRuG4a)andtwosubstrates(mMeO-StyandpMeO-Sty).
Thereactionswerecarriedoutusing100mmoll1ofthesubstrates,anequimolaramountofoxidativereagent(2,6-dichloropyridine-N-oxide)and0.
05mol%ofthecatalyst(RuG1aandRuG4a)atroomtemperature(25°C)inchloroform.
Thereactionprogresswasmonitoredusing1HNMRandgaschromatography.
AlthoughtheentirekineticsofthereactionusingRuG4a(VG4)wereslowerthanthoseforRuG1a(VG1)becauseofhighsterichindrance,theratioVG4/VG1clearlydemonstratedtheeffectofthecavityonsubstrateselectivity.
AsshowninFig.
6c,theratioVG4/VG1formMeO-StywastwicethatforpMeO-Sty.
Toachieveagreaterunderstandingofthecatalyticactivity,thedependenceonsubstrateconcentrationwasexaminedbasedonaMichaelis–Mentenmodel.
Inthecaseofarutheniumtetraphenylporphyrinmonocarbonylcatalyst,theapparentproductformationkineticsshowedalmostlineardependenceonthesubstrateconcentrationbetween100and1,000mmoll1(SupplementaryFig.
S3).
TheMichaelis–Mentenconstantwasdeterminedtobe45moll1formMeO-Sty.
Incontrast,amuchsmallerconcentrationdependencewasobservedwhenRuG4awasusedasacatalyst,suggestingahigherafnitybetweenmMeO-StyandRuG4a.
TheestimatedMichaelis–MentenconstantforRuG4awas90mmoll1.
AsimilarkineticinvestigationwasalsoappliedtomPh-StyandpPh-Sty,whichexhibitedalmostthesametrend.
Thissuggestedthatselectivityinthepresentexperimentalconditions([S]100mM)wasthermodynamicallycontrolledbytheenzyme–substratecomplexequilibrium,butnotkineticallybycomplexformation.
Ofparticularimportanceistheagreementbetweentheexperimentalresultsandthesimplesimulationbasedonastaticmolecular3Dmodel.
Theseresultsareincontrasttopreviouslyreportedselectivitybydendrimersintermsofpredictability52.
DiscussionAlthoughtheentropyfactor(TDS°)actsasanegativedrivingforceforbinding,thisissmallerthanthepositivedrivingforcebecauseofenthalpy(DH°).
Similartotheliterature5,13,53,54,thehost–guestsystemunderdiscussionshowedagoodlinearcorrelation(correlationcoefcient:r0.
98)betweenDH°andTDS°(SupplementaryFig.
S4).
Thisbehaviour,whichiscalledenthalpy-entropycompensation,hasbeenwidelyobservedinnaturalandsynthetichost–guestsystems.
AsitisknownthatanapparentcorrelationbetweenDH°andTDS°mayalsobe0.
000.
050.
100.
150.
00.
10.
20.
3o–m–p–m–p–VG4/VG1VG4/VG1PhMeORROCat.
:RuG4aorRuG1aNClClOOMeOMeox:Cat.
,oxChloroform25°CFigure6|Catalyticepoxidationofolensbythedendrimercatalyst.
(a)Reactionscheme.
(b)3DsimulationmodelofmMeO-StyintheRuG4acavity.
Thissubstratetsinthecavityinaside-onpositioninrelationtotheoxygenatomontheRuPorcore.
(c)3DsimulationmodelofpMeO-StyintheRuG4acavity.
Themethoxygroupconictswiththesidewall.
(d)RatioofthereactionratecatalysedbyRuG4aandRuG1aforeachmethoxystyrenesubstrate.
(e)RatioofthereactionratecatalysedbyRuG4aandRuG1aforeachphenylstyrenesubstrate.
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observedbecauseofastatisticalartifact,weanalysedthesedatawithcaution13.
Indeed,weexaminedthereproducibilityofhost–guestbindingbetweenZnG4aand20vetimes,andtheresultingexperimentalvalues(DH°andTDS°byITC)werefoundtobewithinatypicalexperimentalerrorof±10%,stemmingfromthesummationofmeasurements(weightorvolume)anddataprocessing(curvetting).
TheexperimentalvaluesofK(DG°)andDH°foralloftheguestmoleculesweredistributedoverasubstantialrangeexceedingtheerrorrange;therefore,theobservedrelationshipmayberegardednotasanapparentcorrelationbasedonstatisticalerrorbutasaphysicalphenom-enon.
Hereanunexpectedlylowproportionalcoefcienta(TDDS°/DDH°0.
75at293K)wasobservedfortheZnG4asystem;thiswassubstantiallylowerthanthatofthenon-dendriticzincporphyrin(zinctetraphenylporphyrin:a0.
91).
Theobservedrelationshipsuggeststhat25%(1–a)oftheenthalpygaincanbeusedforfree-energygain(DG°)onmolecularrecognition.
Thisslopeissimilartothatofa-cyclodextrinsandrathersmallerthanthatofg-cyclodextrins5.
Theinterceptoftheenthalpy-entropycompensationplotgaveavalueofTDS0°13.
9kJmol–1,closetothevalueforg-cyclodextrins5.
Thisinterceptvalueisreferredtoasanentropygainaccompanyingdesolvationfromthebindingcentreinthecavityofthefreehostmolecule.
Therelativelysmalla(comparabletoa-cyclodextrins)andlargeTDS0°(comparabletog-cyclodextrins)indicategoodshapepersistenceofthelargecavityinthedendrimer.
Thesignicantcontributionofthelongalkylchainon25canbeinterpretedasthehydrophobiceffectorCH/pinteraction,whichenhanceshost–guestafnity.
AnupeldshiftoftheNMRprotonsignalscorrespondingtothealkylchainwasobservedoncomplexationwithZnG4a.
Thisresultindicatedacooperativerecognitionmechanism55withinterlockingateachrecognitionsiteincludingthepyridine(tip),Y-shapedaromaticrings(anchor)andlongalkylchainmoieties(thread)asshowninFig.
7.
Inspiteofthesignicantcontributionoftheanchorandthreadin25(anadditional40kJmol1DH°),itisnotablethat25didnotshowanyinteractionwiththefree-baseporphyrinformofthedendrimer(H2G4a),asmonitoredbyultravioilet–visibletitrationandITC.
Thisresultdemonstratesthatprexingoftheguestmoleculesthroughpyridinecoordinationisthekeytothesubsequentmolecularshaperecognition.
Inaddition,furtherinterlocking,whichcanbeseenasthesecondkey,allowsthelongalkylchaintofunctionasapositivedrivingforce.
Signicantamplicationofrecognitionwasachievedthroughsequentialcooperativitybetweendifferentpartsoftheguestmolecule,whichmayberelatedtoaninduced-tmechanism13,47.
Suchamechanismmaybethekeytounderstandingprotein-basedrecognitionsystems.
Inconclusion,wehavecreatedasynthetickeyholethatnelydiscriminatestheshapeofaspecicmolecularkey.
Theessentialelementinprobingthemultidimensionalstructureofguestmoleculesistheshapepersistenceofthemacromolecularhostintermsofconformationandcavitygeometry.
Whereastheshapeofthecavityinconventionaldendrimersisundened,anappro-priatedesignwithsufcientrigidityallowedustoprovidealargecavitywithaprogrammedfunctionformolecularrecognitionandcatalysis.
Further,thereasonablysemi-rigidkeyhole,inwhichshapepersistenceandadaptabilitygotogether,providescoopera-tiverecognition,allowingamplicationofrecognitionthroughcomplexation-inducedxationofthecavitystructure,whichmayberelatedtoallostericnatureofthiskindofhostmolecule56.
MethodsChemicals.
Phenylazomethinedendrimerswithazincporphyrincore(ZnGna:n1,2,3and4)usedinthisstudyweresynthesizedbyaconvergentmethodasdescribedintheliterature37.
Othertypesofphenylazomethinedendrimer(RuG4a,ZnG4bandZnG4c)werenewlysynthesizedasdescribedintheSupplementaryMethods.
Abenzylether(Frechettype)dendrimerwithazincporphyrincorewassynthesizedasdescribedintheliterature57.
AllpyridinederivativesusedasguestmoleculeswerepurchasedorsynthesizedasdescribedintheSupplementaryMethods.
NMR.
1Hand13CNMRspectrawereobtainedusingaBrukerAvanceIII-400spectrometerequippedwitha5-mmBBFOz-gradientprobe(gradientstrength:57Gcm1).
Thechemicalshiftvalueswerecalibratedtoaninternaltetra-methoxysilanestandard.
TheDOSYexperimentwascarriedoutwiththefollowingsettings.
Thediffusiondelaywasintherangeof40–60msdependingonthesample.
Thegradientpulselengthandthegradientstrengthwerevariedfrom1.
5to3.
0msandfrom2to100%,respectively.
The1H–1HnuclearO¨verhauserenhancementspectroscopyexperimentwascarriedoutusingstandardpulsesequencesonthesamespectrometerwitha120-msmixingtime.
Determinationofbindingparameters.
Coordinationconstantsandthermo-dynamicparametersforallhost–guestpairsweredeterminedbytitrationexperi-mentsbasedonultraviolet–visibleabsorptionmeasurementsandisothermalcalorimetryat20°C(seeSupplementaryFigsS5–S30).
Ultraviolet–visibleabsorp-tionspectraweremeasuredonaShimadzuUV-3150PCspectrometer.
Curve-ttinganalysesbasedonaleast-squaresmethodattwoindependentwavelengthsweredoneusinghomemadesoftwaretodeterminethebindingconstants.
ITCwascarriedoutusingaMicrocalVP-ITCinstrument.
Analysiswascarriedoutonbuilt-insoftwaretodeterminethebindingconstantsandenthalpyandentropydiffer-ences(DHandDS).
Determinationofthekineticparameters.
CatalyticepoxidationofstyrenederivativesbytheRuPorcatalystwasinitiallyconrmedby1HNMRmeasure-mentsinCDCl3.
DetailedkineticdatainCHCl3wereobtainedbymonitoringtheepoxideproductusingagaschromatograph(Shimadzu,GC-14B)withaTCDdetectorandHecarriergas.
Turnoverfrequencyvaluesweredeterminedfromthetimecourseofproductformation.
AllthereactionswerecarriedoutunderaN2atmosphereat25°C.
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AcknowledgementsThisworkwassupportedinpartbytheCRESTprogramoftheJapanScienceandTechnology(JST)AgencyandGrants-in-AidforScienticResearchonInnovativeAreas'CoordinationProgramming'(area2107,Number21108009)fromtheJapanSocietyforthePromotionofScience(JSPS).
ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms35818NATURECOMMUNICATIONS|4:2581|DOI:10.
1038/ncomms3581|www.
nature.
com/naturecommunications&2013MacmillanPublishersLimited.
Allrightsreserved.
AuthorcontributionsT.
I.
,Y.
K.
andT.
K.
preparedthedendrimersandpyridinederivatives.
T.
I.
andY.
K.
carriedoutthetitrationexperiments.
T.
K.
carriedoutthecatalyticepoxidationexperiment.
T.
I.
andK.
Y.
conceivedexperimentsandco-wrotethemanuscript.
AdditionalinformationSupplementaryInformationaccompaniesthispaperathttp://www.
nature.
com/naturecommunicationsCompetingnancialinterests:Theauthorsdeclarenocompetingnancialinterests.
Reprintsandpermissioninformationisavailableonlineathttp://npg.
nature.
com/reprintsandpermissions/Howtocitethisarticle:Imaoka,T.
etal.
Macromolecularsemi-rigidnanocavitiesforcooperativerecognitionofspeciclargemolecularshapes.
Nat.
Commun.
4:2581doi:10.
1038/ncomms3581(2013).
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms3581ARTICLENATURECOMMUNICATIONS|4:2581|DOI:10.
1038/ncomms3581|www.
nature.
com/naturecommunications9&2013MacmillanPublishersLimited.
Allrightsreserved.