Carbohydrate multivalent systems to functionalize proteins and surfaces for lectin interaction studies
Abstract
Las interacciones entre proteínas y carbohidratos están involucradas en muchos eventos biológicos fundamentales. En estos procesos, una clase de proteínas que reconocen azúcares denominadas lectinas juegan un papel crucial. Actualmente, numerosos grupos de investigación se encuentran estudiando esta importante clase de proteínas; sin embargo, muchas de ellas están aún por explorar y no se conoce en detalle sus especificidades frente a carbohidratos y los factores moleculares que gobiernan dicha interacción. En la naturaleza, muchas lectinas se encuentran en forma de oligómeros para promover una elevada para glicanos específicos. Esto se debe al hecho de que la interacción carbohidrato-proteínas es muy débil, con una especificidad y selectividad limitadas por unidad de azúcar. Por lo tanto, para compensar esta interacción paradójicamente débil, la Naturaleza juega la carta de la multivalencia mediante una presentación multivalente del receptor, así como de los carbohidratos. En este sentido, con el objetivo de estudiar estos procesos multivalentes de reconocimiento entre carbohidratos y proteínas, los sistemas de carbohidratos multivalentes artificiales se han revelado como herramientas muy eficientes para el estudio de dichas interacciones. En este contexto, el objetivo principal de esta Tesis Doctoral es el desarrollo y la preparación de sistemas multivalentes de carbohidratos para comprender mejor los mecanismos que impulsan las interacciones de estas proteínas de unión a carbohidratos. Para lograr este objetivo general, se propusieron los siguientes objetivos específicos: • Preparación de superficies multivalentes basadas en glicodendrones y el estudio de la interacción carbohidrato-proteína sobre dichas superficies, para evaluar el efecto de la presentación del ligando en la interacción. Para alcanzar este objetivo, hemos desarrollado microarrays recubiertos con dendrones de ciclooctino, a partir de los cuales se han generado los correspondientes glicodendrones directamente sobre el chip, a través de una reacción de tipo click en ausencia de Cu (SPAAC) con diferentes tipos de carbohidratos funcionalizados con un grupo azida en su extremo reductor. Las superficies hidrófobas utilizadas permiten el uso de la detección por medio de la espectrometría de masas MALDI-TOF, permitiendo así verificar la finalización de cada reacción (>95%). Con este nuevo método se realizaron varios estudios de interacción usando lectinas marcadas fluorescentemente, fundamentalmente ConA y DC-SIGN. En particular, se observó una fuerte unión para el disacárido αMan1,2Man, en todas las valencias y concentraciones empleadas. Como se esperaba, la señal fluorescente aumenta al aumentar la valencia todas las concentraciones utilizadas. En estos estudios también se ha puesto de manifiesto que las lectinas que reconocen manos, no muestran ninguna interacción para los glicodendrones de β-D-galactosa. Todos estos resultados demuestran la eficacia y la versatilidad real de la técnica desarrollada. • Preparación de nuevas glycodendriproteinas mediante la funcionalización de la proteína BSA con carbohidratos y evaluación de su interacción con lectinas. Se ha desarrollado un método de preparación rápido, simple, económico y versátil de proteínas funcionalizadas con glicodendrones. En primer lugar, se han sintetizado varias familias de glicodendrones con un grupo azido en la posición focal. Estos grupos funcionales se han utilizado para conjugar las moléculas en la proteína BSA mediante metodologías de reacción de tipo click catalizada por Cu(I) (CuAAC) entre los grupo azida de los glicodendrones y los grupos alquino en la proteína, previamente derivatizada con este grupo funcional. Así, fue posible alcanzar dos grados de funcionalización, bajo (10-15 unidades de azúcar) y alto (30-35 unidades de azúcar). Las neoglicoproteínas sintetizadas han mostrado una buena actividad inhibitoria frente a lectinas lectinas humanas como Langerin y DC-SIGN. En particular, excelentes resultados fueron obtenidos con las neoglicoproteínas funcionalizadas con glicodendrones de αMan1,2Man. De hecho, estas estructuras han revelado una actividad, en terminos de inhibición de la actividad enzimática, en el rango bajo nanomolar. Esto demuestra que la proteína es una buena plataforma para exhibir, y de este manera, mejorar el reconocimiento de los glicanos frente a los receptor de azucares. • Diseño y desarrollo de una síntesis rápida que permita obtener el disacárido Mana1,2Man en gran escala y, con ello, permitir la preparación de estructuras de glicodendrones. Este objetivo se ha logrado mediante la adopción de una síntesis consecutiva de 5 etapas para obtener el aceptor de glicosilo, reduciéndose de manera significativa el coste total y el tiempo de preparación de este intermedio clave. Usando esta estrategia, fue posible obtener el disacárido deseado en escala de gramos y en menos de dos días y sobre todo con una mejoría del 30% sobre el rendimiento global, en comparación con la estrategia sintética tradicional paso a paso. Con este disacárido Manα1,2Man, se han preparado glicodendrones con biotina en la posición focal. Estos glicodendrones se van a utilizar, en el futuro, para estudios de interacción con lectinas empleando la resonancia de plasmón de superficie de imagen (SPRi).
Full text
CARBOHYDRATEMULTIVALENTSYSTEMSTO FUNCTIONALIZEPROTEINSANDSURFACESFORLECTIN INTERACTIONSTUDIES AntonioDiMaio Seville,2019
CentrodeInvestigaciones CientíficasIsladela Cartuja InstitutodeInvestigaciones Químicas CARBOHYDRATEMULTIVALENTSYSTEMSTO FUNCTIONALIZEPROTEINSANDSURFACESFORLECTIN INTERACTIONSTUDIES Doctoralthesispresentedby AntonioDiMaio,M.Sc. inordertoapplytotheDoctorofPhilosophyinChemistry Seville,2019
ThepresentworkhasbeencarriedoutinthreeyearsbyAntonioDiMaio,M.Sc., withintheinternationaltrainingnetworkIMMUNOSHAPEintheGlycosystem Laboratory,InstitutodeInvestigacionesQuímica,IIQ(CSIC‐UniversidaddeSevilla) attheCentrodeInvestigacionesCientíficasIsladelaCartuja,CICCartuja,Seville, Spain. ThisprojectreceivedfundingfromtheEuropeanUnion'sHorizon2020researchand innovationprogramundertheMarieSkłodowskaCuriegrantagreementNo642870.
Acknowledgments Ilmioprimoringraziamentoèneiconfrontidellepersoneamepiùcare,chemi hannopermessodinascere,crescereediventareciòchesono.Grazieaivostri sacrifici,ilvostroaiuto,ilvostroesempio,ivostriinsegnamenti,ilvostrosupporto, levostrelacrime,lavostraforza,lavostrasofferenza,lavostrapassione,lavostra educazione,lavostrapazienza,lavostraconoscenza,oggiiosonoquiascrivere questeparole.Grazieavoihoimparatocosasignifical’impegnoedildurolavoro, senzailqualenonsarebbestatopossibileraggiungereirisultatifinoradame ottenuti. Avoiilmioprimopensiero,perchéavoiquestatesidottoraleèdedicata. AmiamadreEldaemiaziaNirvana. Myfirstthanksisformydearestpeople,whoallowedmetobeborn,growand becomewhatIamnow.Thankstoyoursacrifices,yourhelp,yourexample,your teachings,yoursupport,yourtears,yourstrength,yoursufferings,yourpassion, youreducation,yourpatience,yourknowledge,todayIamwritingthesewords. ThankstoyouIlearnttoworkhard;without,itwouldnothavebeenpossibleto achievetheresultsobtainedsofar. Myfirstthoughtisforyou,becausethisdoctoralthesisisdedicatedtoyou. TomymotherEldaandmyauntNirvana. Insecondtime,IwouldliketothankmysupervisorDr.JavierRojofortheopportunity toworkinhisresearchgroupattheCiCCartujaandthehelpinthedevelopingofthis doctoralthesis.Continuing,theresearchersofthegroupJoseLuisdePaz,Pedro Nieto,JoseJuanReinaandAnaSousa. IwouldthankDr.NielsC.ReichardttokindlyhostmeinhislaboratoryinSan Sebastianandhisresearchgroup,Anna,Cristina(MissSpain),Olatz,theMariaS, Nerea,Alvaro,Raquel,Eli,Dorletta,Javier.Allofthemhelpmefromascientificand personalpointofviewintherealizationoftheworkdescribedinthechapter3of thisthesis. ThanktoDr.LudovicLandemarretokindlyhostmeinhiscompanyinOrléans,for thehelpduringmystayingandinthedevelopingoftheprojectdescribedinthe chapter4ofthisthesis. IwouldthankProf.FranckFieschi,forthehelpduringthesethreeyearsandforthe Lectinswhichallowedtherealizationofthebiologicalpartofthisdoctoralthesis.
IwouldthankProfAnaTeresaCarmonaAsenjoforthekindnessandhelpfulnessin thetutoringofthesedoctoralstudies. ThankstotheEuropeanfoundationthatallowmetoworkhereinSpainanddevelop thisresearchwork. AspecialthankstomadameMarieSkłodowskaCurie,forherworkandherbattles. Sheistheexampleofgreatwomanthatallthescientistsandresearchershaveto have. Donato,fratm’ena'vità.Avimm’fatttantàstradà‘nsieme,condividèndgioièe dulure,Gràzieassàjera'amiciziàtoja. Pedro,miamigo.UnadelassuertesquehetenidoenestomivivirenEspañahasido encontrarunapersonamaravillosacomotu.Graciasportodastusayudasysuporte enestosaños.Alprincipioencontréuncompañero,yahoratengounhermano... Blanka,thankyouveryverymuchmyCroatianfriend.Wewentthroughalot together,thankyouforyourpatienceandyourlessonsofbiochemistryand biotechnology,andforallthefantasticmomentsspenttogether. Inma,graciasati,otrapersonamaravillosaqueheencontradoenSevilla.Enitalia hayundicho:"Diosloshaceyluegolosune". Jorge,hemospasadojuntossolounapartedeestodoctoradoperohasidosuficiente paradesarrollarunaamistad.Aunque,congrandiferenciaencuantoapelo...nunca terindasniño!! Myriam,yolosequeestasmirandoestapaginaparabuscarelagradecimientopara ti..Graciasparadarmelaposibilidaddesertupsicólogopersonalenestosaños.Voy aechardemenosnuestraslargasconversaciones.. Noelia,misoídosyyotedamoslasgraciasportusfantásticoscánticosenel laboratorio,quealegrarontantoelambientecomomicorazón.Gracias,compañera dedoctorado. Noel,Kylo..graciasparalasrisashasidounverdaderoplacertrabajarcontigo. Ainhoa,graciasportusconsejosytusayudasquímicasypersonalesquemehan permisodeobtenerresultadosenmiprimerapartedeestodoctorado.Graciasmi profesoradeespañol.Comopuedesleerenestosagradecimientos,elproblemaera elestudiante…
MariaJoeee,graciasportucompañíaytusonrisayradiantesojosazulesquehizo losdíasenellaboratoriomásligeros. Leonarrrrrrdinho,parapoderhablarcontigoentodoslosdías,he“””aprendido”””el Español.Graciasportodos,miamigodeBrasil. Elena,lanuestraMiss,graciasportuamistad,belleza,palabrasyrisasenlos momentosdifícilesque(yocreosindartecuenta)mehanayudadomucho.Leecharé muchodemenos.. Rossana,ancheseseidiMilano,grazieperaverportatolatuaraggiantesimpatianel nostrolaboratorio. AloscompañerosdelCartuja,Antonio,Alberto,Carmen,Christian,Elena,Pablo, Pedro,Patricia,Tania,Veronicaylosotros(sorrysimeolvidoalguien)paralasrisas durantelascomidas,lascenasyloscaffè. AloscampeonesSevillistas,elgrandeRafayelCarlos,graciasparanuestras conversacionesylasrisasquemealegrabanmucholastardes.ForzaNapoliyForza Sevilla!Alosdelaseguridad(Antonio,ForzaBetis!),yaLuis,graciasporlaslargas conversaciones,quehemostenidodespuésdelas22enelencentrocompletamente vacio..Osecharémuchodemenoschicos!! ASuperMargaportodastusamableayudasduranteestosaños,almiticoFranque todossabeymehaayudadomuchissimo,aJoseManuelparalosvuelosa1euro, FelipeyBienvenidoparadivertirmeconsuspeleasentreellosyatodoslosotrosde lafamiliadelCartuja. Silvia,carissima…siamol’esempiochetraNordeSudcipuòessereunaamicizia… Valentin,moncherami,nousavonsétélepremiernoyaudecenetwork;mêmesila vienousdivise,jen'oublieraipasmonamifrançais. Joaooooo,quemomentosdivertidospassamosjuntos,especialmenteemCádiz; Obrigadopelasuaamizadeepeloseutrabalhoduro. Tanishta,myIndianfriend,Iwillmissyouverymuch…Nogood!!! AveryspecialthankstoalltheIMMUNOSHAPEnetwork,thebossesandinparticular theguys:Alonso,Arnaud,Ernesto,Laura,Márcia,Miguel,Ravi,Kun,Julie.Youare likeafamilyforme,thankyouforyourhelpintheseyearsandforthefunnytime spenttogetheraroundtheEuropewiththeImmunodollars!
ListofAbbreviations HATU 1‐[Bis(dimethylamino)methylene]‐ 1H‐1,2,3‐triazolo[4,5‐b]pyridinium3‐ oxidehexafluorophosphate HexHexane HIVHumanimmunodeficiencyvirus HSAHumanserumalbumin HSQCHeteronuclearsinglequantum coherencespectroscopy HSVHerpessimplexvirus IgGImmunoglobulineG ITOIndiumtinoxide JCouplingconstant mmultiplet MALDIMatrixassistedlaser desorptionionization ManMannose MeOHMethanol MFIMeanfluorescenceintensity MHCMajorhistocompatibilitycomplex MGLMacrophagegalactoselectin MRMannoserecptor MSMassspectrometry MWMicrowave m/zmasstochargeratio NaOMeSodiummethoxide NGLNeoglycolipid NGPNeoglycoproteinorneoglycopeptide NHSN‐Hydroxysuccinimide NISN‐Iodosuccinimide
ListofAbbreviations NLRNod‐likereceptor NMRNuclearmagneticresonance Ns2‐Nosyl ODPAOctadecylphosphonicacid OVAOvalbumin PAMAMPolyamidoamine PAMPsPathogenassociated molecularpatterns PBSPhosphatebufferedsaline ppmPartspermillion PPRPatternrecognitionreceptor ProProlin PyPyridine qquartet RIPRelativeinhibitorypotency RLRRIG‐I‐likereceptor rtRoomtemperature ssinglet SARStructureactivityrelationship SDS‐PageSodiumdodecylsulphate‐ polyacrylamidegelelectrophoresis SPAACStrain‐promotedalkyneazide cycloaddition SPRSurfaceplasmonresonance tTriplet T‐cellThymusderivedlymphocytes TBAITetrabutylammoniumiodide TBTATris(benzyltriazolylmethyl)amine t‐BuOOHtert‐Butylhydroperoxide
ListofAbbreviations TFATrifluoroaceticacid THFTetrahydrofuran TLRTolllikereceptors TMSOTfTrimethylsilyl trifluoromethanesulphonate TOFTime‐Of‐Flight UVUltraviolet
TableofContents 1.Introduction……………….……..…….……………………………………….……..…......1 1.1.Thehumanimmunesystem–OurAegisagainstpathogens……...…..1 1.2.DendriticCells…………….....………………………..…………….…………............2 1.3.Carbohydrateepitopesandproteinreceptors…………….….….............4 1.4.Lectins……………………...….……………………………..……….…...........….……..6 1.4.1.C‐typeLectins………….………..….................................................................9 1.4.2.Lectinclassification………….……..............................................................10 1.4.3.TheCRD………….….......….……………………………..….…………….…...................12 1.4.4.HumanC‐typelectinsinvolvedintheimmuneresponse…...................15 1.4.5.Langerin………….….......….……………………………..….…………….…...................12 1.4.6.HumanDC‐SIGN………….….......….…………………………….…….…...................16 1.5.C‐typeLectinsastherapeurictarget………….……………….…………........20 1.6.MultivalencyinCTLs………….……………………….….…………….……..........22 1.7.TheMultivalencyinCTLs………….………………………..…………..........…..24 1.8.DevelopmentoftoolstomodulateCarbohydrate‐Lectin interactions………….……………………….…………………………………………….28 1.9.DendrimersandDendrons……………….….……………………….……..........32 1.9.1Glycodendrons…………...........….….....….….....….….....….….....….….….........34 2.Theantecedentsandobjectives…….……….….…….………….………….…43 2.1.Theantecedents………………….………….………………….………….……………43 2.2.Objectives…………………...………….………………….………….…………………..52 3.On‐chipsynthesisofglycodendronstostudycarbohydrateproteininteractions…….….....…….….……….….…………….….…………….………55 3.1.Themicroarraytechnology….....…….….………...….…………..….………....55 3.1.1.Preparationofglycanarrays….…..………….…..…………….….…...........………56 3.1.2.Detectionmethods….………….….….……………….…..…………….….….…..........62
Esta parte de la tesis se publicará con un retraso de 12 meses desde su aprobación y defensa.
CHAPTER1 Introduction
Chapter1.Introduction 1 1. Introduction 1.1. Thehumanimmunesystem–OurAegisagainst pathogens TheAegis1thatprotectsthemankindagainstpathogensistheimmunesystem.The humanimmunesystemisacomplexstructureofcellsandtissueswhosemain functionsaretherecognitionandthesubsequenteliminationofforeignantigens, theformationofanimmunologicmemory,andthedevelopmentoftolerancetoselfantigens.Theimmuneresponsecouldbedividedintwotypes:theinnateandthe adaptiveimmuneresponse.2 Theinnateimmunesystemconsistsonthecellsthatfirstlyrecognizeand respondtopathogen‐associatedmolecularpatterns(PAMPs)anddamageassociatedmolecularpatterns(DAMPs)releasingcytokines,chemokinesand antimicrobialproteinswhichdriveinflammationandensuingimmune response.3 Theadaptiveimmunesystem,incontrastwiththeinnateone,requiresmore timetorespondtothethreatandismediatedbytwotypeoflymphocyte cellpopulations,thebone‐marrowderived(BlymphocytesorB‐cells)and thymusderivedlymphocytes(T‐cells).Itishighlyantigen‐specific4and developsanimmunologicalmemorythatprovidesprotectionagainst reinfectionofawiderangeofantigenspresentedonvirus,bacteria, parasitesandfungus.5 1TheAegisinLatin,ascitedintheIliad,isashieldoranarmorusedbyZeusandhisdaughter Athena. 2a)Palm,N.W.;Medzhitov,R.;Immunol.Rev.,2009,227,221–233;b)Medzhitov,R.; Janeway,C.A.Jr.;Curr.Opin.Immunol.,1997,9,4–9. 3Janeway,C.A.Jr;Medzhitov,R.;Annu.Rev.Immunol.,2002,20,197–216. 4a)Iwasaki,A.;Medzhitov,R.;Science,2010,327,291–295;b)Litman,G.W.etal.;Nat.Rev. Immunol.,2010,10,543–553. 5Walsh,K.P.;Mills,K.H.;TrendsImmunol.,2013,34,521‐30.
Chapter1.Introduction 2 Theseimmuneresponsesareinduced,coordinated,andregulated6byanimportant classofantigenpresentingcells(APCs),thedendriticcells(DCs).7Thesecellshave theabilitytobridgeinnateandadaptiveimmunityviapattern‐recognitionreceptors (PRRs),whichrecognizeandgeneratearesponsetothePAMPsorDAMPs.Hence, dependingonthepathogen,APCsregulateanddrivethefunctionoftheBandT cells,8andstarttheadaptiveimmuneresponse.9 1.2. Dendriticcells Thenameofthesecells“Dendritic”derivesfromtheGreekword“dendros”that meanstree,thisisduetothetypicalstellateshapesimilartothebranchesofatree.10 (Figure1.1) Figure1.1.A3DrepresentationofaDCinteractingwiththeHIV‐1(inred).11 Picturefromreference11. 6Steinman,R.M.;Annu.Rev.Immunol.,1991,9,271–296 7Niess,J.H.etal.,Science,2005,307,254–258. 8a)Marshall,N.A.etal.,CancerRes.,2012,72,581–591;b)Wilkinson,K.A.;Wilkinson,R.J.; Eur.J.Immunol.,2010,40,2139–2142;c)Banchereau,J.;Steinman,R.M.;Nature,1998, 392,245‐252. 9Carbohydrate‐basedvaccinesmethodsandprotocols,Ed.BerndLepenies,HumanaPress, 2015,Chapter1,Hütter,J.;Lepenies,1‐10. 10Steinman,R.M.;Cohn,Z.A.;J.Exp.Med.,1973,137,1142–1162. 11ImagecreatedbyDonaldBliss,NationalLibraryofMedicine,SriramSubramaniam,National CancerInstitute;Felts,R.L.etal.,ProcNatlAcadSciUSA,2010,107,13336‐13341.
Chapter1.Introduction 9 1.4.1. C‐typeLectins Theimmunesystem,throughtheuseofmultipleCTLs,isabletoreacttoabroad spectrumofpathogens.30ThisrelaysontheabilityofCTLstorecognizeandengage differentglycanpathogenligands,controlthemicrobicidalactivity,modulatethe genetranscriptionandaboveall,toregulatethefunctionofthecellsoftheimmune system.31 SomeCTLsareabletorecognizethemolecularsignaturesofpathogens,othersto detectcelldamage,recognizingmolecularelementsindicativeofabnormalityon theirenvironment,suchasoxidizedlipidsorheatshockproteins,signalsofapoptotic ornecrosiscellprocesses.Then,CTLsareabletostartabiomolecularpathwaythat leadtoengagetheendocyticandphagocyticcleanermachineryandstartthe immuneresponseagainstmicrobes,viruses,cancercellsorotherabnormalityinthe humanbody.30 Invertebrates,CTLshavetherelevant“onfirstline”roleof:interactandengagethe pathogencarbohydrateligands,modulatesignalsfromotherreceptorsand therefore,fine‐tuningtheadaptiveresponsetotheinfectionordamage.Indeed, CTLscanbesolubleormembraneboundandareexpressedbymyeloidcellsAPCs suchasDCs,granulocytesandmacrophages.32(Figure1.5)Theyareliterallythefirst molecularcontactamongthePAMPsandthecellsoftheimmunesystem.As explainedbefore,whentheDCs(orotherAPCs)senseapathogen,amolecular recognitiontakesplacethroughtheCTL(Figure1.5A).TheAPCinternalizesthe pathogenbyendocytosis,(Figure1.5B)processesthepathogenantigensand exposesthepathogen‐derivedpeptidesasMajorHistocompatibilityComplexes (MHCs)inordertoengagetheT‐cellandstarttheadaptiveresponse.33(Figure1.5C‐ D) 30Sancho,D.;ReiseSousa,C.;Annu.Rev.Immunol.,2012,30,491‐529. 31a)Klaver,E.J.;etal.,Int.J.Parasitol.,2013,43,191–200;b)Vautier,S,;MacCallum,D.M.; Brown,G.D.;Cytokine,2012,58,89–99;c)Lozach,P.Y.;Burleigh.L.;Staropoli,I.;Amara,A.; MethodsMol.Biol.,2007,379,51–68. 32GeijtenbeekT.B.;Gringhuis,S.I.;Nat.Rev.Immunol.,2009,9,465–479. 33Geijtenbeek,T.B.;Gringhuis,S.I.;Nat.Rev.Immunol.,2016,16,433–448.
Chapter1.Introduction 10 Figure1.5.SchematicpathogenrecognitionandroleofCTLsintheengagementof thePAMPs.Picturefromreference34a. Inaddition,CTLsact,inanindirectway,generatingsignalswithvariouseffector functionssuchastheproductionofpro‐inflammatorycytokines,whichleadthe activationofTcellsbutalsointeractingwithotherPRRlikeTLRs.34(Figure1.5E) 1.4.2. Lectinclassification In1988,KurtDrickamersuggestedtheideatoorganizeanimallectinsintoseveral categories.35Theterm“C‐typelectin”wasintroducedwiththeaimtodistinguishthe groupoftheCa2+dependentfromtheothertypesthatdonotrequireCa2+ionsfor 34a)Mayer,S.;Raulf,M.K.;Lepenies,B.;Histochem.Cell.Biol.,2017,147,223‐237;b) Kingeter,L.M.;Lin,X.;Cell.Mol.Immunol.,2012,9,105–112. 35Drickamer,K.;J.Biol.Chem.,1988,263,9557–9560.
Chapter1.Introduction 11 binding.ThelatterarenamedC‐typelectin‐likemolecules,missingtheabilitytobind thecarbohydrateligand.36 Themembrane‐boundCTLsareclassifiedintotwolargegroups:32 TypeICTLs.Thesearepartofthemannosereceptorfamily. TypeIICTLs.Thesearepartoftheasialoglycoproteins. TheIItypeofCTLsincludesseverallectinsassociatedwithDCsimmunoreceptors thatseemstopreferentiallyrecognizemannose,fucoseandglucans.This “promiscuity”allowstheDCstorecognizemosttypesofpathogens,including bacteria,fungi,virusesandparasites.37 ThelectinsofthegroupIIaretypeIItransmembraneCTLscontaininga transmembranedomain,ashortcytoplasmatictailandanextracellularstalkregion; andasingleCTLDcontainingtheCa2+ion,involvedintherecognition.Thesubgroups II,VandVIareexpressedbythemyeloidcells.38 ThesuperfamilyoftheCTLiscomposedbymorethan1000proteins,whichare furtherclassifiedin17subgroups(I‐XVII)baseonthedomainorganizationandthe phylogeny.38(Figure1.6) 36Drickamer,K.;Curr.Opin.Struct.Biol.,1999,9,585‐590. 37Drummond,R.A.;Brown,G.D.,PLoSPathog.,2013,9,e1003417 38Zelensky,A.N.;Gready,J.E.;FEBSJ.,2005,272,6179‐6217.
Chapter1.Introduction 12 Figure1.6.ClassificationofthesuperfamilyofCTLD‐containingproteins.Thesearesoluble aswellastransmembraneC‐typelectinreceptorsgroupedonthebasisoftheirstructural compositionandeffectorfunctions.Picturefromreference34a. 1.4.3. TheCRD TheCBPsofthelargefamilyofCTLsarecharacterizedbyverydifferentstructures; however,allofthemhaveacommondomaininvolvedintherecognizingofthe carbohydrateligand,calledC‐typelectin‐likedomain(CTLD)orcarbohydrate recognitiondomain(CRD).Thisisacompactglobularstructureresponsibleforthe selectivebindingoftheterminalunitsofthecarbohydrateepitopes.38 Fromastructuralpointofview,theCTLDischaracterizedbytwoproteinloops,38 wherethesecondmoreflexibleloopgenerallycontainstheproteindomaininvolved inthecarbohydratebinding.36FourCa2+bindingsitesarepresentintheCTLD structurebutonlythesite2ismainlyinvolvedinthecarbohydraterecognition.38 (Figure1.7)
Chapter1.Introduction 13 Figure1.7.AcartoonrepresentingatypicalCTLDstructure.Inblue,theloopregion;C1‐4 (inredcircles),representsthefoursitesinvolvedinthebinding.Picturefromreference38. TheCRDiscomposedbytwoaminoacidswithlongcarbonylsidechainsseparated byacisproline.ThiscarbonylsidechainscoordinateCa2+,forminghydrogenbonds withthecarbohydratesanddeterminingthebindingspecificity.Twoprincipalmotifs havebeenidentified: EPN.AminoacidsequenceGlu‐Pro‐Asn,specificformannose‐ligands QPD.AminoacidsequenceGln‐Pro‐Asp,specificforgalactose‐ligands However,theversatilityoftheCTLDisdemonstratedbythefactthatinseveralcases, itwasobservedthattheCa2+bindingsitescaninteractwithligandswhicharenot carbohydrates,likeproteins,lipidsorinorganicligands.38 1.4.4. HumanC‐typelectinsinvolvedintheimmuneresponse Mannose‐typelectinswhicharepartofthegroupIIandthereforehaveanEPN sequence,mediatemanybiologicaleventssuchascell‐cellorcell‐matrixadhesion, andglycoproteinturnover. TheseCTLshavearemarkablecontributiontotheinnateimmuneresponsethrough pathogenrecognitionandinternalizationofantigens.Furthermore,someCLTs
Chapter1.Introduction 14 possessintrinsicsignalingpropertiesthatenablethemtomodulatetheimmune reactions.39 Inparticular,DCsexpressallargevarietyofCTLswhichareoftendownregulated uponmaturation,butthatgivetheessentialabilitytoimmatureDCstobindand internalizeglycosylatedantigens.40ItisimportanttohighlightthatseveralCTLs expressedbyDCs(MR,DCAL‐2,CLEC‐2,DEC205,MGL,Dectin‐1,Mincle,Langerinand DC‐SIGN)havedemonstratedtodonotfunctionmerelyasantigenuptakereceptors buttheyhavetheabilitytofacilitatetheefficientloadingofantigensandtheTcell induction.41(Figure1.8) Figure1.8.APCsexpressCTLsinvolvedinuptakeandsignalingprocesses.Picturefrom reference39. 39vanVliet,S.J.;Garcia‐Vallejo,J.J.;vanKooyk,Y.;Immunol.CellBiol.,2008,86,580‐587. 40a)Wells,C.A.etal.,J.Immunol.,2008,180,7404‐7413;b)Weck,M.M.etal.,Blood,2008, 111,4264‐4272;c)Engering,A.etal.,J.Immunol.,2002,168,2118‐2126. 41a)Tacken,P.J.etal.,Blood,2005,106,1278‐1285;b)Bonifaz,L.C.etal.,J.Exp.Med.,2004, 199,815‐824.
Chapter1.Introduction 15 Inthisthesis,wedescribetwoofthemostimportantandstudiedHumanCTLs, LangerinandDC‐SIGN.(Figure1.8) IthasbeendemonstratedthatbothCTLsareinvolvedindifferentimportant pathwaysfromthepathogenrecognitiontothecell‐cellinteraction,involvingthe recognitionofspecificglycansexposedonpathogensorpathogenicstructures,such asmannosestructures,Lewis‐typeantigens,GalNAcorβ‐glucan.42 1.4.5. Langerin LangerinisatypeIItransmembraneCTLexpressedexclusivelybyLangerhanscells whichareDCspresentintheepitheliallayerandepidermis.Langerinislocalizedin BirbeckgranulesinthecytoplasmofLangerhanscells.43ThestructureofLangerinis characterizedbyanextracellulardomainthatconsistofaCRDandaneckregionthat stabilizestheformationoftrimersofCRDs.44(Figure1.9) Figure1.9.TwoviewsofthetrimericstructureofLangerin.Picturefromthereference44. TheCRDofLangerinhasaffinityforhighmannose,fucose,GlcNAcandβ‐glucansand sulfatedcarbohydrates.TheseaffinitiesmakeLangerinaproteinbinderof glycoproteinsandglycolipidspresentonseveralmicroorganismsandleadtothe 42a)deWitte,L.etal.,Nat.Med.,2007,13,367‐371;b)Tassaneetrithep,B.etal.,J.Exp. Med.,2003,197,823‐829. 43Valladeau,J.etal.,Immunity,2000,12,71–81. 44Chatwell,L.;Holla,A.;Kaufer,B.B.;Skerra,A.;Mol.Immunol.,2008,45,1981‐1994;Image obtainedbyPDB:3KQGin“BIOVIADiscoveryStudio4.5©”program.
Chapter1.Introduction 16 internalizationofthepathogenantigensintoBirbeckgranulesintroducingthemin processingpathways.Langerinligandsaremainlyexpressedindifferentpathogens. 43,45,46(Table1.1.) Ligand Ligandorigin Highmannose, fucose(Le y andLe B ) andGlcNAc ‐ Viruses:HIV‐1. ‐ Bacteria:M.leprae. ‐ Fungi:Candidaspp,Saccharomycesspp,Malasseziafurfur. ‐ TypeIprocollagen Table1.1.SelectiveligandsforLangerinandtheirorigin. IntheparticularcaseofHIV‐1,thebindingofthehighmannoseofgp120facilitates thedegradationofthevirus,incontrasttoDC‐SIGN,thatbindsthegp120facilitating theHIVtransmissiontoTcells.42aLangerinisabletodirectlycaptureHIV‐1through gp120bindingandsequentiallydegradingitintotheBirbeckgranulestopromoteT celleliminationofHIV‐1infection.ThissuggestthatLangerinplaysapleiotropicrole inHIVinfection.47,48 Besidesallthisinformation,manypathwaysinwhichLangerinisinvolved,likeitsrole intheregulationofDCactivation,remainsunknown.30 1.4.6. HumanDC‐SIGN HumanDendriticCell‐SpecificIntercellularadhesionmolecule‐3GrabbingNonintegrin(DC‐SIGN)ismainlyexpressedbymyeloidDCsbutalsoinalveolar,lymph nodes,sinusendothelialcells,Hofbauercellsintheplacentabydermal,interstitial andmonocyte‐derivedDCs,22andmacrophages.48AcloselyrelatedreceptoristheL‐ 45Liu,Y.;Liu,J.;Pang,X.;Liu,T.;Ning,Z.;Cheng,G.;Molecules,2015,20,2272–2295. 46a)deJong,M.A.etal.,Mol.Immunol.,2010,47,1216–25;b)deWitte,L.etal.,Nat.Med., 2007,13,367–371;c)Hunger,R.E.etal.,J.Clin.Investig.,2004,113,701–708. 47a)Schwartz,O.;Nat.Med.,2007,13,245–246;b)Turville,S.G.;Cameron,P.U.;Handley, A.;Lin,G.;Pöhlmann,S.;Doms,R.W.;Cunningham,A.L.;Nat.Immunol.,2002,3,975–983. 48Granelli‐Piperno,A.etal.,J.Immunol.,2005,175,4265–4273.
Chapter1.Introduction 17 SIGNorDC‐SIGN‐related(DC‐SIGNR)whichisfoundonendothelialcells,liver,lymph nodesandplacenta.49 TheysharethesamestructureformedbyasingleCRDglobularstructureconsisting oftwelveβ‐strands(inblue,Figure1.10A),twoαhelices(inred,Figure1.10A),and threedisulphidebridges.OntheCRD,thereisaloopthatprojectsbeyondtheprotein surfaceandformspartoftwoCa2+bindingsitesthatareresponsibleforthe coordinationofcarbohydratesattheCRD.(Figure1.10A)TheCRDischaracterized byahighlyconservedEPNmotifwhichdefinestheligandspecificityofthereceptor. BesidestheCRD,thereisaneckregion,responsibleofthetetramerizationofthe lectin,constitutedbysevencompleteandoneincompletetandemrepeats,anda transmembraneregionfollowedbyashortcytoplasmictail,whichisresponsiblefor signalingandinternalization.50,51,52(Figure1.10B) Figure1.10.A)CRDofDC‐SIGNwithadi‐mannose(yellow).ThebluespheresaretheCa2+ atoms.Picturefromreference51.B)DC‐SIGNschematicstructure,Picturemodifiedfrom reference50. However,DC‐SIGNisnotatotallyrigidmacromoleculeandshowsadegreeof flexibilitythatallowittoadapttoseveralligands.53Thislectinoligomerizesina 49Soilleux,E.J.;ClinicalSci.,2003,104,437‐46. 50Feinberg,H.;Castelli,R.;Drickamer,K.;Seeberger,P.H.;Weis,W.I.;J.Biol.Chem.,2007,282, 4202‐4209. 51ImageobtainedbyPDB:2IT6in“BIOVIADiscoveryStudio4.5©”program. 52Anderluh,M.;Jug,G.;Svajger,U.;Obermajer,N.;Cur.Med.Chem.,2012,19,992‐1007. 53Menon,S.etal.,Proc.Natl.Acad.Sci.USA,2009,106,11524‐11529. AB
Chapter1.Introduction 18 tetramericforminwhichtheCRDdomainsarespacedfromthemembranebyan extendedregioncontainingαhelices.48,54(Figure1.11) Figure1.11.TwoviewsofthecrystalstructureofafragmentofDC‐SIGNR,containingthe carbohydraterecognitiondomainandtworepeatsoftheneck.Imagefromreference54. TheoligomerizationstatusoftheDC‐SIGNandtheotherCTLsdependsonthe numberofhelicalrepeatsoftheneckregion.Indeed,atleast6repeatsareneeded fortetramerization.50 TheCRDrecognizesandbindsmainlyinternalmannosebranchedstructuresandthe terminalD‐mannoses,inparticulartheHighMannose(Man9GlcNAc2) oligosaccharide130‐and17‐foldmoretightlythanthesinglemannose.Inaddition, CRDrecognizesfucose‐bearingglycans,L‐fucoseandLewisantigensandGlcNAc.55 DC‐SIGNligandsareprincipallyexpressedinpathogens. 22,56 (Table1.2) 54Feinberg,H.;Guo,Y.;Mitchell,D.A.;Drickamer,K.;Weis,W.I.;J.Biol.Chem.,2005,280, 1327‐1335.ImageobtainedfromPDB:1XARbyNGLViewer (dio:10.1093/bioinformatics/bty419). 55a)Lai,W.K.etal.,Am.J.Pathol.,2006,169,200‐208;b)Geijtenbeek,T.B.etal.,Cell,2000, 100,575–585. 56a)Carroll,M.V.;Sim,R.B.;Bigi,F.;Jakel,A.;Antrobus,R.;Mitchell,D.A.;ProteinCell,2010, 1,859–870;b)Gringhuis,S,I,;denDunnen,J.;Litjens,M.;vanderVlist,M.;Geijtenbeek, T.B.H.;Nat.Immunol.,2009,10,1081–1088;c)Hodgesetal.,Nat.Immunol.,2007,8,569– 77;d)Zhang,P.etal.,J.Leukoc.Biol.,2006,79,731–738;e)Barreiro,L.B.etal.,Hum. Immunol.,2006,67,102–107;f)Kwon,D.S.;Gregorio,G.;Bitton,N.;Hendrickson,W.A.; Littman,D.R.;Immunity,2002,16,135–144;g)Alvarez,C.P.etal.,J.Virol.,2002,76,6841– 6844. B A
Chapter1.Introduction 25 Figure1.13.Monovalentinteractionmechanisms.Picturefromreference78. Foramultivalentinteraction,itcanbeconsidereddifferentmechanismsthat contributetothebindingevents,increasingthecomplexityoftheinteraction betweenamultivalentligandandamultivalentreceptor.Inaddition,onemajor problemistheformationofanequilibriuminvolvingallthesemechanismsandthe formationofadynamiccompetitionamongthem.77 Takingintoaccountthatanaturalmultivalentligandandareceptorinteractintheir naturalbiologicalenvironment,therealinteractioncanbetheresultofasumof mechanismswhichproducedifferentprocessesandbiologicalresponses.78 Firstofall,thenligandsfromamultivalentglycomoleculecansimultaneously interactwiththembindingsitesofthehomomultimericlectin,ina“chelate association”mechanism.Thismechanism,oneofthemoststudied,decreasetheoffrateandincreasethefunctionalaffinityinaremarkableway.79(Figure1.14A)A secondmechanismcalled"receptorclustering"happensinthesituationinwhicha monovalentlectinisanchoredtothecellmembrane.Inthiscase,thepresenceofa multivalentligandcausesthediffusionofotherreceptorsthroughthedynamic lipidicbilayer,leadingtoaclusteringamongthesinglemultivalentligandandthe receptors.ThiscanhappenduetotheextremeflexibilityofmanyCRDs.Inthiscase, 78a)Kiessling,L.L.;Gestwicki,J.E.;Strong,L.E.;Curr.Opin.Chem.Biol.,2000,4,696‐703;b) Mammen,M.;Choi,S.‐K.;Whitesides,G.M.;Angew.Chem.,Int.Ed.,1998,37,2754‐2794. 79Wittmann,V.;Pieters,R.J.;Chem.Soc.Rev.,2013,42,4492‐4503.
Chapter1.Introduction 26 theproximityoftheCTLsrecruitedcangeneratespecificsignaltransductionevents throughthemembrane.80(Figure1.14B) Athirdtypeisthe"subsitebinding"inwhichasecondarybindinginteractionis involvedinanotherregionoftheprotein,withadifferentaffinityandspecificity.This canalsoinvolveadifferentligandfromtheprimary,typicallypresentonhetero divalentligands.81(Figure1.14C) Inaddition,itisnecessarytotakeintoaccountthestatisticaleffectsofthere‐binding ofthemultivalentligands,favoredbyahighdensityofepitopesinproximitytothe bindingsite.Thiscouldhappenforareceptorofboth,ahomomultimericlectinora monomericlectin.80a,82(Figure1.14D) Figure1.14.Mechanismsofinteractionbetweenmultivalentligandsandmultivalent receptors.Picturemodifiedfromreference80a. Thebindingaffinityistheresultnotonlyofthemeresumofthenumberofsingle ligand‐receptorinteractions,butalsoanadditionalvalueduetoallthese mechanismsthatsynergicallyactbetweenthemultivalentligandandthemultivalent receptor. 80a)Kiessling,L.L.;Gestwicki,J.E.;Strong,L.E.;Angew.Chem.Int.Ed.,2006,45,2348‐2368; b)Bertozzi,C.R.;Kiessling,L.L.;Science,2001,291,2357‐2364;c)Gestwicki,J.E.;Kiessling, L.L.;Nature,2002,415,81‐84;d)Heldin,C.H.;Cell,1995,80,213‐223 81a)Guiard,J.;Fiege,B.;Kitov,P.I.;Peters,T.;Bundle,D.R.;Chem.Eur.J.,2011,17,7438‐ 7441;b)Rademacher,C.etal.,Chem.Eur.J.,2011,17,7442‐7453. 82Dam,T.K.;Gerken,T.A.;Brewer,C.F.;Biochemistry,2009,48,3822‐3827.
Chapter1.Introduction 27 Theconsequenceisthattheusuallyweakmonovalentcarbohydrate‐protein interactionsaretransformedintoverypotentattractiveforces,naturallyreinforced whenseveralcopiesoftheligandsarecloselypresentedonamultivalentscaffold. Thisphenomenonisknownasthe“Glycoclustereffect”.76f Thismultivalenteffectgivestothecellularreceptorstheabilitytoeasilyr discriminateamongstructurallysimilarrecognitionepitopeswhenmonovalent bindingeventspresentsareoflowaffinity.Finally,itismandatorytohighlighthow themultivalentsystemsallowahighfunctionalaffinitybutatthesametime,are characterizedbyauniquekinetics.Inmonovalentbindingevents,thedissociation constantisexclusivelydeterminedbytherateconstantfordissociation,without interferencebythepresenceofacompetingligand.Instead,multivalentbinding eventsexhibitagreaterreversibilityinthebinding,inthepresenceofcompeting ligands,duetothepresenceofamultiplenumberofcopies.83 Fromastructuralpointofview,itisfundamentaltoconsiderthatarchitectural changesinthespacingororientationofthecarbohydratesepitopescouldalterthe natureofaninteractionbutatthesametime,havenotanyinfluenceinthe recognitionelementsthemselves.Infact,ithasbeenshownthatthearchitectureof themultivalentscaffolddetermineswhetheramultivalentdisplayactsasan inhibitororaneffectorinacarbohydrate‐proteininteraction.77 Thisisextremelyrelevantinthedesignphaseofartificialmultivalentglycosystems, sincetheyallowcertain"degreesofliberty"intothestructuredispositionwithout, atthesametime,negativelyaffectthebindingphenomenontostudy. 83Rao,J.H.;Lahiri,J.;Isaacs,L.;Weis,R.M.;Whitesides,G.M.;Science,1998,280,708‐711.
Chapter1.Introduction 28 1.8. DevelopmentoftoolstomodulateCarbohydrate‐Lectin interactions Withtheaimtostudy,characterize,understand,andinterferthemechanismthat governingthemultivalentcarbohydrate‐proteininteractions,andtheprocesses wheretheyareinvolved,interestingandusefultoolshavebeengenerated.76a(Figure 1.15) Figure1.15.Mechanismofinhibitionbyamultivalentglycoconjugate.Picturemodified fromreference77. Thegoalistomimic,asgoodaspossible,thenaturaldisplayingofthe oligosaccharidespresentonthesurfaceofthehostcells.Thespatialproximityofthe singlesugarterminalepitopes,involvedinthebindingwiththemultivalentlectins, isessentialforthegenerationoftheappropriatestructuralrequirementsthatlead tothemultivalentaction,therefore,leadingtohighavidities. Theideaistocreateatoolabletocompetewithaparticularpathogenandthe priorityistoincorporateintothestructure,thecharacteristicsthatallowtohave highaffinityandagoodselectivitytospecificallyblockthelectinadhesiononthe targettissues.Therefore,thedesignhastobestronglyinfluencedbythenatural ligandofthetargetlectinintermsofvalency,topologyanddensityofcarbohydrates. Thedesignedsystemcouldbealsoabletoperturbthenormalassociationofalectin withitsnaturalligandandalsotomodulatethesignalingprocessesassociatedwith thelectin.75
Chapter1.Introduction 29 Theelectionofboth,amonovalentglycomimeticandamultivalentglycosylated structureapproaches,arevaliddependingontheindividualsituationandbothof themhaveadvantages,specificfeatures,andbenefits. Nonetheless,monovalentsyntheticglycoconjugateswouldnotreproduceexactly thevalencyandthetopologyofanaturalligandthat,asdescribedabove,isusually displayedinamultivalentform.However,thebindingpropertiesobtainedwiththe monovalentglycomimeticapproachare,inmostofthecases,ofremarkableimpact andthishasmadepossibletheirapplicationastherapeutics. Twowidelygeneralapproachesarecurrentlyadoptedtodesignamultivalent artificialligand:75 Lectin‐baseddesign.Afteradeepstudyofthemultivalentlectin,the multiglycosylatedstructurethatwouldbestfitthelectintopologyisbuilt. Ligand‐baseddesign.Thisapproachstartsfromthestudyofthenatural multivalentligandtodesignasimplifiedandoptimizedstructuretakinginto mindthenaturalvalency,topologyanddensityoftheligand.(Figure1.16) Figure1.16.Schematicpresentationofthenaturalcarbohydrateligandsmimickedby artificialcarbohydratemultivalentscaffolds.Picturefromreference75.
Chapter1.Introduction 30 Bothapproacheshavebeendemonstratedtobevalidforaddressinglectinstudies. Nevertheless,inourresearchgroup,theligand‐basedapproachistheonewidely exploredandapplied. Theligand‐basedapproachinvolvesthesimplificationofthecomplex oligosaccharidicstructure,anditspresentation,usingamultivalentcore appropriatelyselectedtomimicthenaturalscaffold.Atthesametime,thestructure hastoprovidethedesiredvalencyandthecorrectdisplayingoftheepitopestothe targetreceptor. Thefirstproblemistheselectionofthemultivalentcore.Alongthepast25years, chemistshavedesignedalargenumberofscaffoldsforthisaim.Ithasnotonlyto displaymultiplecopiesofthesugarepitopesbutaboveall,tocreateamultivalent presentationoftheseepitopesthatcanefficientlymimicthenaturalmodeofaffinity thatarisesfromseveralmultivalentmechanismsinvolvedbetweenthebinding proteinsandthecarbohydrateligands. Therangeofpossiblescaffoldstogeneratemultivalentglycoconjugatesisquitelarge andallofthesehavedemonstratedtobeexcellentcoreskeletonstodisplaythe sugarmoietyinagoodway.84Dependingonthescaffoldandtheconjugation chemistryused,theresultingglycoconjugatescanbedividedinpolydisperseand monodisperseglycoconjugates. Themultivalentglycomimeticstructuresderivedfromascaffoldthatpresentsthe carbohydratesinapolidispersiveform,areprobablytheclosestmimetictothe naturalligandofthelectin.Thisisduetothefactthatintheorganism,the glycosilationofproteinsandlipids,isusuallyapolidispersivemodification.Popular 84Bernardi,A.etal.,ChemSocRev.,2013,42,4709‐4727.
Chapter1.Introduction 31 examplesareneoglycoproteins,glycopolymers,85glyconanoparticles86or glyconanotubes.87(Figure1.17) Figure1.17.Somepolydispersemultivalentglycomimetics.Picturemodifiedfromreference 75. Onthecontrary,themultivalentglycomimeticstructuresderivedfromascaffold thatpresentstheminamonodisperseandwell‐definedform,mimicprobablyless faithfullythenaturalligands,butthisdoesnotmeanwithlesseffectiveness.The majorexamplesareglycofullerenes,88glycoclusters89andinparticular glycodendrimers(seesection1.9).(Figure1.18) Figure1.18.Monodispersemultivalentglycomimetics.Picturemodifiedfromreference75. Fromaquantitativepointofview,oneevidentadvantageofamultivalentexposition istheincreasingofthebindingaffinitypropertiesofmonovalentCTLsantagonists. 85Spain,S.G.;Gibson,M.I.;Cameron,N.R.;J.Polym.Sci.PartA:Polym.Chem.,2007,452059– 2072. 86a)delaFuente,J.M.;Penades,S.;Biochim.Biophys.Acta,2006,1760,636–651.b)dela Fuente,J.M.etal.,Angew.Chem.Int.Ed.,2001,41,1554–1557. 87Bianco,A.;Kostarelos,K.;Partidos,C.D.;Prato,M.;Chem.Commun.,2005,571–577. 88Bosi,S.;DaRos,T.;Spalluto,G.;Prato,M.;Eur.J.Med.Chem.,2003,38913–923. 89Patel,A.;Lindhorst,T.B.;Carbohydr.Res.,2006,341,1657–1668.
Chapter1.Introduction 32 Usually,monovalentglycomimeticsarecharacterizedbyapoorormodestaffinity forthelectin,whichcanbeconsiderablyincreasedwhentheyareconjugatedtoa scaffoldforapolyvalentpresentation. 1.9. DendrimersandDendrons DendrimerderivesfromthejoinoftwoGreekwords,"dendros"(treeorbranch)and "meros"(part).Thisnamewasgivenin1985,whenTomalia'sgrouppublishedthe synthesisofapoly(amidoamine)compound,thatiscurrentlyfamouswithits acronymPAMAM.90 However,thehistoryofthe"partsofatree"isabitolder,andstartsin1941when Flory91proposedtheidea,supportedbystatisticalcalculations,topreparetrivalent branchedmoleculesusingapolymericapproach.Butatthattime,thesyntheticand analyticaltechnologieswerenotsufficienttoexperimentallysupporthisintriguing idea.Thirty‐sevenyearspassedtoseethefirstpaperthatdescribedthesynthesisof lowmolecularweightdendriticpolyaminesbyVögtle,in1978.92Duringthefollowing 40years,thanksalsotothepioneeringworkofTomalia93andNewcome,94dendritic structureshavebeenwell‐establishedasanewclassofbranchedmonodisperse polymerswithagreatvalueformaterialandmedicinaltechnologies.95a Indeed,dendrimersanddendronswerequiteearlyrecognizedasveryuseful moleculestobeexcellentscaffoldforamultivalentpresentationandalsoforawide varietyofdifferentapplications.Atthemoment,thereisaremarkablyhighnumber ofpatentapplicationsaswellaspublicationsonscientificjournals,whichhighlight 90Tomalia,D.A.etal.,Polym.J.,1985,17,117–132. 91Flory,P.J.;J.Am.Chem.Soc.,1941,63,3091–3096. 92Buhleier,E.;Wehner,W.;Vögtle,F.;Synthesis,1978,155–158. 93Dendrimer‐BasedDrugDeliverySystems:FromTheorytoPractice,Eds.Cheng,Y.;Tomalia, D.A.,Wiley,2012,ISBN:978‐0‐470‐46005‐4. 94a) DendrimersandDendrons:Concepts,Syntheses,Applications,Eds.Newcome,G.R.; Moorefield,C.N.;Vögtle,F.,Wiley,2001,ISBN:3527299971/9783527299973:b)Newcome, G.R.;Zhong‐qi,Y.;Baker,G.R.;Gupta,V.K.;J.Org.Chem.,1985,50,2003–2004.
Chapter1.Introduction 33 theirapplicationsindifferentfieldssuchaschemistry,medicine,biology,physicsand materialengineering.95 Fromastructuralpointofview,thedendronsarehighlybranchedmonodisperseand polyfunctionalmacromolecules,constitutedbythreedistinctelements: Coreorfocalpoint Branchingpointorgeneration Endpointsortermini Thecoreisgenerallyasmallmoleculewithaprecisenumberoffunctionalgroups whichdefinesthenumberofbranchesofthedendrimer.Thesinglebranchiscalled “dendron”.Inthecaseofdendronsthereisnotacore,buta“focalpoint”whichcan consistofafunctionalgroupthathastheaimtoallowtheanchorageofthedendron toanothermolecule(suchasafluorescentprobe)ortoascaffold(aprotein,a surfaceetc.).Thebranchingpointsarecomposedofrepeatingmonomericunitsthat definethegeneration(GX).Thesizeofthedendrimer/dendronincreaseswiththe numberofgenerationandthegrowthprocessismainlyregulatebythechemistry used.Theendpointsofthedendrimersarethefunctionalgroupsthatmarkedly characterizethepropertiesoftheentirestructureandtheycanbemanipulatedfor thedesiredapplication.96Inthecaseofglycodendrons,theterminiarethe carbohydratestructures.(Figure1.19) 95a)Müller,C.;Despras,G.;Lindhorst,T.K.;Chem.Soc.Rev.,2016,45,3275‐3302;b) Carbohydratechemistry,ChemicalandBiologicalApproachesvol41,EdsRauter,A.P.; Lindhorst,T.K.;Albertina,C.;Queneau,Y.;TheRoyalSocietyofChemistry,2015,Chapter1, Sattin,S.;Bernardi,A.pg1‐25andL’Haridon,L.;Mallet,J.M.pg257‐269.c)Inoue,K.;Prog. Polym.Sci.,2000,25,453–571;b)Vögtle,F.;Gestermann,S.;Hesse,R.;Schwierz,H.; Windisch,B.;Prog.Polym.Sci.,2000,25,987–1041;d)Astruc,D.;Boisselier,E.;Ornelas,C.; Chem.Rev.,2010,110,1857–1959. 96FrontiersofNanoscience,Vol4;Chapter5,Sanchez‐Navarro,M.;Rojo,J.;2012,143‐156.
Chapter1.Introduction 34 Figure1.19.Structuralelementsofdendrimeranddendron. 1.9.1.Glycodendrons Thenano‐orpicomolarbindingaffinitieswhichusuallycharacterizethetherapeutic compoundsareinmostofthecasesdifficulttoreachusingglycomimeticsina monovalentform.Hence,asuccessfulstrategyhastoconsiderasyntheticapproach thatcouldbeconceivedinthisway: A) Thedesignofamonovalentglycomimeticbasedonanaturallectinbinding oligosaccharide. B) Multivalentpresentationofthemonosaccharide/oligosaccharideor glycomimeticonamultivalentscaffold.(Figure1.20) Moreover,thetwosequentialstepscanbeindependentlyappliedfromeachother. Infact,itispossibletofocustheresearchenergiesondevelopingasmallmonovalent glycomimeticwithastrongaffinity,ordevelopingamultivalentmacromoleculewith anexcellentpresentationofthemono/oligosaccharides.(Figure1.20)
CHAPTER2 Theantecedents andobjectives
Chapter2.Theantecedentsandobjectives 43 2. Theantecedentsandobjectives 2.1 Theantecedents Inthelast15years,ourresearchgrouphasdevelopednovelmultivalent glycosystems,mainlydendrimer‐based.Inparticular,Theyhavedevelopedthe generationofinnovativetoolstomodulateandbetterunderstandtheactionofCTLs likeDC‐SIGN. Inthiscontext,severaltypeofdendrimersanddendronshavebeenusedasscaffold tohaveamultivalentpresentationoftheselectedglycoepitopes.Indeed,thereare severalexampleswheretheglycosystems,createdinourgroup,havehad applicationsonthestudyofcarbohydrate‐lectininteractions,orhavebeenusedas vectorstointernalizeintoDCs,moleculesoftherapeuticinterestsuchaspeptides withanantiallergenicactivityandpotentialvaccineaction. Atthebeginningof2000,ourresearchgroupdesignedandprepareduseful glycodendrimersbasedonglycodendriticstructureswith16and32copiesof mannoseresiduesconjugatedtothecommercialavailablehyperbrancheddendritic polymerBoltornH20andBoltornH30,respectively.TheBoltorn‐typecompoundsare constructedusing2,2‐Bis(hydroxymethyl)propionicacid(Bis‐MPA)asbranchingunit andpentaerythritolasacore.Thecorrespondingglycodendriticpolymersof2ndand 3rdgenerationwereperfectlysolubleinphysiologicalconditions.103The glycodendrimerof3rdgeneration,having32mannoseresidues,(Figure2.1)wasable toblockDC‐SIGNmediatedEbolavirusinfectionresultinginanIC50=337nM. 103Lasala,F.;Arce,E.;Otero,J.R.;Rojo,J.;Delgado,R.;Antimicrob.AgentsChemother., 2003,47,3970‐3972.
Chapter2.Theantecedentsandobjectives 44 Figure2.1.Boltornhyperbranchedstructuresof2nd(BH20)and3rd(BH30)generationand thecorrespondingglycodendrimershaving30mannoseunits. Afterwards,theseglycodendrimerswerealsotestedbysurfaceplasmonresonance (SPR)incompetitionexperimentsinordertomeasuretheabilityofthese compoundstoinhibittheinteractionofHIV‐1gp120withDC‐SIGN,resultinginasubmicromolarrangeactivityforthecompoundwith32copies.104 Followingtheseinterestingresults,itwasestablishedacollaborationwithBernardi’s researchgroup,inordertogenerateamultivalentpresentationofDC‐SIGN antagonistsglycostructures.Thesemannosemimeticshadalreadydemonstrated,in amonovalentform,agoodinhibitoryactivityonEbolavirusentryintoJurkatcells expressingDC‐SIGN.64a,105 Inparticular,thepseudodisaccharidicandpseudotrisaccharidiccompounds,(Figure 2.2)wereinstalledonatetravalentplatformbasedinBis‐MPA,withaninhibition 104Tabarani,G.;Reina,J.J.;Ebel,C.;Vives,C.;Lortat‐Jacob,H.;Rojo,J.;Fieschi,F.;FEBSLett., 2006,580,2402‐2408. 105a)Sutkeviciute,I.etal.,ACSChem.Biol.,2014,9,1377−1385;b)Luczkowiak,J.etal., Bioconjug.Chem.,2011,22,1354–1365.
Chapter2.Theantecedentsandobjectives 45 actioninthelowmicromolarrange.105a‐bThetetravalentglycodendronsshowedto inhibitedtheDC‐SIGNgp120interaction,andinparticularthepseudo‐trisaccharide glycodendronblockedtheHIV‐1transfectiontoCD4+Tlymphocyteswitha percentage>94%.64a(Figure2.2) Figure2.2.Tetramericpresentationonbis‐MPAdendronsofglycomimeticscompounds. Theseexamplesshowtheactuallyefficacyofamultivalentpresentationofa glycomimeticcompound,inordertoobtainahighaffinityforaproteinreceptorlike DC‐SIGN. Takingadvantageofthepotentialofthecopper(I)‐catalyzedalkyne‐azide cycloaddition(CuAAC),alsocalled"click”reaction,106theresearchgrouphas developedseveralmonodisperseglycodendronandglycodendriticsystemswith differentvalences:fromthreetoeighteen,fullycharacterizedbyNMRandmass spectrometry.Thesedendrimersarecomposedbymannoseunitsattheendpoints andapentaerythritolcore.107(Figure2.3) 106a)Tiwari,V.K.;Mishra,B.B.;Mishra,K.B.;Mishra,N.;Singh,A.S.;Chen,X.;Chem.Soc. Rev.,2016,116,3086‐3240;b)Tabacaru,A.;Furdui,B.;Ghinea,I.O.;Cârâc,G.;Dinicâ,R.M.; Inorg.Chim.Acta,2016,455,329‐349;c)Singh,M.S.;Chowdhury,S.;Koley,S.;Tetrahedron, 2016,72,5257‐5283;d)Liang,L.;Astruc,D.;Coord.Chem.Rev.,2011,255,2933–2945. 107Varga,N.etal.,Biomaterials,2014,35,4175‐4184.
Chapter2.Theantecedentsandobjectives 46 O O O O O NN N O N N N O N NN O N N N O O O O O NN N O N N N O N NN O N 3 O O O O O NN N ONNN ONNN O O O O N N N O N N N O N N N O O O O O N N N O N NN O NN N ON O NNO OOO O O O NN N O N N N O N N N O N N N 3 III III IV O HO HO OH O OH Figure2.3.Mannoseglycodendrimersoffirstgenerationpreviouslysynthetizedinour laboratories. However,itwaspossibletoincreasethedendrimergeneration,takingadvantageby thepossibilitytohaveanazidogroupatthefocalpositionoftheglycodendronV, andbyasecondclickreactionondifferentcores(VI‐IX).(Scheme2.1)
Chapter2.Theantecedentsandobjectives 47 O O O O O O OO O O OO O O CuAAC O O O O O O N N N O O N N N O O N NN O N 3 O O OO O O N NN O O N N N OO NN N OO N NN n n = 3, 4, 6 O O OOO O O Cl VI VII VIII IX V Scheme2.1.Clickreactionamongtheglycodendronandseveralcores. Withthisstrategyadopted,severalglycodendronsofsecondgeneratuionwith differentcarbohydrateepitopeswereprepared,suchasthoseX‐XIIdescribedin Figure2.4.107
Chapter2.Theantecedentsandobjectives 48 O O O O O O O O N N N O N NN ONN N O N NN O O O O ONN NO N N N O N N N O N N N O O O OO N N N O N N N O N NN O N N N X O O O O O N N N O N N N O N NN O O O O ON N N NN N 7N N O O O O O N N N O NN N O N N N O OO O O O NN N O N NNO N N N O N NN O O OO O N NN O N N N O N N N O XI O O O O N N N XII O O O O N N N O N N N O N N N O N N N O O O O N N N O N N N O N N N O N N N O O O O N N N O N N N O N N N O O O O O NNN O O O O NN N O NNN ONNN O NN N O O O O N N N O NN N O N NN O NN N O O O O NNN O N NN O NN N O HO HO OH O OH Figure2.4.Mannoseglycodendronsofsecondgenerationpreviouslypreparedin ourlaboratory. Thepossibilityandtheversatilitytohaveanazidoorachloridegroupatthefocal positionhaveallowedtheconjugationoftrivalent(V)andnonavalentglycodendrons (derivedfromscaffoldVII)onotherscaffoldssuchas: Graphene.DemonstratingaselectiveinteractionwiththeplantlectinConA, testedbyAFM,FluorescenceandUV‐Visstudies.108(Figure2.5A) 108Ragoussi,M.E.;Casado,S.;Ribeiro‐Viana,R.;delaTorre,G.;Rojo,J.;Torres,T.;Chem.Sci., 2013,4,4035‐4041.
Chapter2.Theantecedentsandobjectives 49 Fullerene.Demonstratingforthefirsttimethatafullerenedecoratedby monovalentortrivalentglycodendronsofmannoseisabletoinhibit efficientlytheEbolainfectionprocess.109Figure2.5B Goldnanoclusters.Demonstratingthatagoldnanoparticlewithtrivalent mannoseglycodendronsonthesurface,showanincreasingintheuptaking onhuman‐monocyte‐derivedDCs,incomparisonwithnon‐carbohydrate goldnanoclusters.110Figure2.5C Withafluorophoremarker.DemonstratingtheuptakeprocessintoDCsvia areceptor‐dependentmechanismofglycodendronsofmannose,fucose andmaltotriose.111(Figure2.5D)Provingtheactuallyefficacyof glycodendronsofmannose,fucose,orN‐acetyl‐galactosamineinthestudy ofthemicroalgaeinteractionmechanisms.112Figure2.5E 109Luczkowiak,J.etal.,Biomacromolecules,2013,14,431‐437. 110LeGuével,X.etal.,ACSAppl.Mater.Interfaces,2015,7,20945−20956. 111Ribeiro‐Viana,R;Garcia‐Vallejo,J.J.;Collado,D.;Perez‐Inestrosa,E.;Bloem,K.;vanKooyk, Y.;Rojo,J.;Biomacromolecules,2012,13,3209−3219. 112deAlmeidaGonçalves,C.;CunhaFigueiredo,R.;Giani,A.;Collado,D.;Pérez‐Inestrosa,E.; Rojo,J.;CunhaFigueredo,C.;J.Appl.Psychol.,2018,1‐11.
Chapter2.Theantecedentsandobjectives 50 Figure2.5.Severalconstructsgeneratedbytheconjugationofglycodendrons.
Esta parte de la tesis se publicará con un retraso de 12 meses desde su aprobación y defensa.
CHAPTER5 Glycodendronstructuresbasedon α1,2mannobiosidedisaccharide .
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 221 5. Glycodendronstructuresbasedonα1,2 mannobiosidedisaccharide. 5.1 Mannosedisaccharide,animportantepitopeinglycoscience Agreatefforthasbeeninvestedintheglycosciencefieldconcerningthedesignand thesynthesisofefficienttoolsabletomimicthenaturalstructurespresentinliving organisms.Inparticular,thedevelopmentofsyntheticstrategiesenablingtoobtain thedesiredglycansingoodquantityand,aboveall,assimilaraspossibletothe naturalsystems. OneoftheprincipaltargetsofthesestudiesaretheN‐glycanstructures,duetotheir abilitytoaffectinatremendousway,manypropertiesoftheglycoproteinsinwhich theyareattached,likesolubility, conformation,activity,antigenicity,andespecially, therecognitionbyglycan‐bindingproteins(GBPs). Ineukaryotes,alltheN‐glycanscontainacommoncoresequence,Man3GlcNAc2Asn, andtheyareclassifiedintothreetypes316(Figure5.1): OligomannoseorHigh‐Mannose.Thecoreisextendedonlybymannose residues. Hybrid.ThecoreisextendedbymannosethroughtheManα1‐6armofthe coreandoneortwoGlcNAcextendtheManα1‐3arm. Complex.TheantennaeinitiatedbyGlcNAc,extendthecore. 316EssentialsinGlycobiology,EdVarki,A.;Cummings,R.D.;Esko,J.D.;Freeze,H.H.; Stanley,P.;Bertozzi,C.R.;Hart,G.W.;Etzler,M.E.;ColdSpringHarborLaboratoryPress, Plainview,2009.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 222 Figure5.1.TypesofN‐glycans. AmongthesetypesofN‐glycans,agreatattentionhasbeenfocusedonthe oligomannoseglycans;duetothefactthatmanyimportantclassesoflectins recognizeligandscontainingmannose,orhighermannosestructures.317Thehigh mannoseoligosaccharidesareusuallycharacterizedbytwotosixα‐mannose residuesboundtothecore.Thus,thelargesthigh‐mannose,discoveredforthefirst timeinthe1977,318containsninemannoseandtwoN‐acetylglucosamine.(Figure 5.1)ItturnouttobeonethemaincarbohydrateligandrecognizedbyDC‐SIGNand Langerin.Forthisreason,thestructureofhighmannosehasbeenoneofthe principalsourcesofinspirationforthedesignofhighaffinityligandsforthese importantlectins.319 Hence,inthepasttwodecadesoneofthetargetsforthedevelopingofGBPligands hasbeenthetotalsynthesisofthe(Man)9(GlcNAc)2.225c,320However,thehigh complexityofthisclassofglycanstructureshasrequiredtheuseofalternative 317Kwon,D.S.,etal.,Immunity,2002,16,135–144. 318Ito,S.;Yamashita,K.;Spiro,R.G.;Kobata,A.;J.Biochem.,1977,81,1621‐1631. 319Guo,Y.etal.,Nat.Struct.Mol.Biol.,2004,11,591‐598. 320a)Lee,H.K.,etal.,Angew.Chem.Int.Ed.,2004,43,1000–1003;b)Geng,X.;Dudkin,V.Y.; Mandal,M.;Danishefsky,S.J.;Angew.Chem.Int.Ed.,2004,43,2562‐2565;c)Grice,P.;Ley, S.V.;Pietruszka,J.;Priepke,H.W.M.;Angew.Chem.Int.Ed.,1996,35,197–200.;d)Merritt, J.R.;Naisang,E.;Fraser‐Reid,B.;J.Org.Chem.,1994,59,4443‐4449.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 223 syntheticstructuresinordertooffsetthepossibilitytohavethisligandinlarge amountandaddressbiologicalstudies.321 Infact,syntheticglycanmimeticshasbeenwidelyadoptedinthestudyofthe relevantbiologicalinteractionsthatinvolvehigh‐mannoseligandsanditsnatural receptor,withtheaimtobetterunderstandtheroleandthestructure‐activity relationshipsofthecarbohydratesinbiologicalprocesses.322Onestrategywasthe useofthesingleantennasalternativelytothefullMan9molecule.Thisdisaccharidic ortrisaccharidicepitopescouldbeactuallyinstalledonmultivalentscaffoldinorder tomimicthenaturalmultivalentpresentationoftheseglycans.(Figure5.2) Figure5.2.High‐mannoseanditsprincipalantennas. Theuseofthisapproachissupportedbyseveralstudiesperformedinthepasttwo decades,whichhavedemonstratedhowtheuseofthedisaccharideMan2orthe trisaccharideMan3,canbeefficientlyadoptedinsteadofthewholestructure.50First ofall,anstructuralstudyonthecrystalofacomplexincludingthehighmannose (Man)9(GlcNAc)2andaFabfragmentofthegp120antibody2G12shownthatthe85% 321CarbohydrateChemistry:StateoftheArtandChallengesforDrugDevelopment,EdCipolla, L.,ImperialCollegePress,2016;Chapter15,Sattin,S.;Fieschi,F.,Bernardi,A.;379‐394. 322a)Reina,J.;Bernardi,A.;MiniRev.Med.Chem.,2012,12,1434‐1442.;b)Reina,J.; Bernardi,A.;Clerici,M.;Rojo,J.;Fut.Med.Chem.,2010,2,1141–1159.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 224 oftheproteincontactwereconstitutedbythedisaccharideManα1,2Man.323In addition,Seeberger’sgroupdemonstrated,byamicroarraystudyusingseveral oligomannoseligands(Man9included),thatahighdensityofManα1‐2Manwasable tobindDC‐SIGNaseffectiveastheentirehighmannosestructure.324Therefore,the useoftheManα1,2Manfragmentcanbeconsideredasaninterestingstartingpoint atthemomenttodesignnewmimeticsystemswiththeaimtostudyinteraction processeswithproteinreceptors.Thisispreciselythecaseofmimickinghighmannoseinteractions,involvingmannosecompoundsasepitopes. Thepreparationofmultivalentsystemsusingdifferentscaffoldssuchasproteinsor surfacestoperformthedesiredapplicationwiththisdisaccharidicepitoperequired anefficientsynthesisoftheManα1,2Manepitope.Inthisrespect,several approacheshavebeendescribedintheliteraturetoprepareα1,2mannobioside derivatives.325Thesynthesisofthesecompoundsiscomplicatedbythefactthatthe hydroxylgroupattheC‐2positionofthemannosemoietyisnotdirectlyaccessible byaregioselectiveglycosylation.326Thus,thesynthesisrequiredtheuseofmany reactionstepsofselectiveprotectionsanddeprotectionswithseveralandtedious purificationstepsoftheintermediateproducts. Therefore,theseclassicalproceduresrequirealargetimeconsumptionintermof synthesisandpurification,highcostintermofreagentsandsolvents,andin 323Calarese,D.A.,etal.,Science,2003,300,2065‐2071. 324Adams,E.W.;Ratner,D.M.;Bokesch,H.R.;MacMahon,J.B.;O’Keefe,B.R.;Seeberger, P.H.;Chem.Biol.,2004,11,875‐881. 325a)Patel,M.K.;Vijayakrishnan,B.;Koeppe,J.R.;Chalker,J.M.;Doores,K.J.;Davis,B.G.; Chem.Commun.,2010,9119–9121;b)Jaipuri,F.A.;Pohl,N.L.;Org.Biomol.Chem.,2008,6, 2686–2691;c)Mathew,F.;Mach,M.;Hazen,K.C.;Fraser‐Reid,B.;Synlett,2003,9,1319‐ 1322;d)Ogawa,T.;Nukada,T.;Carbohydr.Res.,1985,136,135–152. 326a)Pratt,M.R.;Bertozzi,C.R.;J.Am.Chem.Soc.,2003,125,6149‐6159;b)Matsuo,I.; Wada,M.;Manabe,S.;Yamaguchi,Y.;Otake,K.;Kato,K.;Ito,Y.;J.Am.Chem.Soc.,2003,125, 3402‐3403;c)Ogawa,T.;Sasajima,K.;Carbohydr.Res.,1981,93,67‐81;d)Ogawa,T.; Nakabayashi,S.;Agric.Biol.Chem.,1981,45,2329‐2335;d)Arnarp,J.;Haraldsson,M.; Lönngren,J.J.Chem.Soc.,PerkinTrans.1,1985,535‐539.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 225 particular,theseveralpurificationstepsaffectinatremendousmannertheglobal yieldofthefinalproduct. Infact,aone‐potprocedurecouldovercometheseproblemssuchaspurificationand thenumerousstepsandcouldmaximizethefinalyield,diminishingthetime requiredforthesynthesis.ThisisthecaseoftheworkdonebythegroupsofWang327 andDavis328morerecently.Inthelatercase,therewasapolymerizationstrategy veryefficient;however,itisimportanttohighlightthattheseprotocolsresultinvery goodoverallyieldonlyinsamecases.Indeed,onlygoodyieldswereachievedinthis polymerizationprocedurewhenatthereducingterminustherewasanalcoholwith aparticularlengthandsubstituents.Thesechemicalconstraintsareanevident disadvantagewhichmakesdifficulttheapplicationofthisstrategytothesynthesis ofManα1,2Manwithadesiredgroupattheanomericposition. Withthisinmind,ourresearchgroupplannedthedevelopmentofanewstrategy thatallowedtoobtainα1,2mannobiosidederivativesingram‐scale,notonlywitha highyieldbutalsoinarapid,straightforwardandversatilemanner.Inourapproach, a2‐azidoethylgroupwasintroducedattheα‐anomericpositiontoperformlaterthe couplingofthisdisaccharidewithalkyneorcyclooctynegroupsinaclickreaction procedure,inordertoaffordthepreparationofmultivalentstructures. Inparticular,inthischapteritwillbedescribedthesynthesisofα1,2mannobioside compoundsandwithoneofthesedisaccharides,thesynthesisofglycondendrons structureshavingabiotinatthefocalposition. 5.2. SynthesisofManα1,2Mandisaccharides Inthissyntheticstrategy,twoglycosylacceptorhavebeenselected,onehavinga methoxygroupattheanomericposition,andtheotherwithanazidoethylspacer forafurtherconjugation.Thesetwodifferentgroupscanbeusedtodemonstrate thattheefficacyofthestrategyisnotinfluencedbythegroupatthereducingend. 327Wang,C.C.;Zuluetab,M.M.L.;Hung,S.C.;Chimia,2011,65,54‐58. 328Schuster,H.J.;Vijayakrishnan,B.;Davis,B.G.;Carbohydr.Res.,2015,403,135‐141.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 226 Inaddition,tofurtherverifytheversatilityofthestrategy,twoglycosyldonors,peracetylated(117)andper‐benzoylated,(118)wereselected.(Scheme5.1) O HO HO OH OR O HO HO OH OH O 23 R= 2-azidoethyl 114 R= Me O R'O R'O OR' OR' O O BzO BzO OBz OR OH NH CCl 3 O HO HO OH OH OH 1 2 3 45 6 115 R= 2-azidoethyl 116 R= Me O HO HO OH OR OH 20 R= 2-azidoethyl O R'O R'O OR' OR' OR' 117 R'= Acetyl 118 R'= Benzoyl 119 R'= Acetyl 120 R'= Benzoyl Glycosyl donor Glycosyl acceptor Scheme5.1.RetrosyntheticschemeforthesynthesisofManα1,2Mandisaccharides. Firstofall,ithasbeennecessarytoselecttheorthogonalprotectivegroupstrategy onthehydroxylgroupsoftheglycosyldonorandtheglycosylacceptor.(Scheme5.1) Theacetateprotectinggroupwasselectedattheposition2oftheglycosylacceptor andbenzoatesgroupswereselectedaspermanentprotectinggroupsintheother hydroxylgroups.Thesetwoorthogonalgroupsarecleavableunderdifferent conditions,191allowingthestabilityofthebenzoatesinallthestepsofthesynthesis untilthefinaltotaldeprotection.Alltheseprotectivegroupsarecompatiblewiththe presenceoftheazidogrouponthechainattheanomericposition. Inthisapproach,thelimitingfactor,intermsoftimeandyield,isthesynthesisofthe glycosylacceptors(115‐116).Thisisduetothefactthattheproductionofamannose orthogonallyprotectedwithanacetateinpositionC2andbenzoategroupsinthe otherpositionsmayberelativelycomplicated.Nevertheless,thesynthesisofthe glycosyldonorsarmedwithatrichloroacetimidategroupattheanomericposition (117‐118)isrelativesimpleandstraightforward.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 233 group,beingverysensibletothehydrolysisdegradation.By1H‐NMR,theappearance ofthetypicalsingletat8.50‐9.00ppmcorrespondingtotheNHgroupofthe trichloroacetimidateconfirmedwithoutdoubtstheformationofthedesired products. O R'O R'O OR' OR' OH O R'O R'O OR' OR' O NH CCl 3 0°C -> r.t. ; 3h 67% Cl 3 CCN, DBU NH 2 NH 2 AcOH, DMF , 24h, 121 R'= OAc 74% 122 R'= OBz 70% 117 R'= OAc 60% 118 R'= OBz 67% 119 r.t. 120 60°C O R'O R'O OR' OR' OR' 119 R'= OAc Commercial 120 R'= OBz Quant. Scheme5.5.Synthesisoftheglycosyldonors117and118. Themannosederivativessoarmedwerereadytoreactwiththeglycosylacceptors 115and116.Forthisaim,acetylcompound117wasuseasadonor,inorderto obtainthedisaccharides123and124. O AcO AcO OAc OAc O NH CCl 3 117 O BzO BzO OBz O OH 115 O BzO BzO OBz O OH 116 O BzO BzO OBz O O O AcO AcO OAc OAc O BzO BzO OBz O O O AcO AcO OAc OAc 123 124 N 3 CH 3 N 3 CH 3 Scheme5.6.Schemeofthepreparationofprotecteddisaccharides123and124.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 234 Toset‐uptheoptimizedconditionsforthisreaction,firsttrialswerecarriedoutwith 2‐azidoethylmannosederivative115asglycosylacceptor,usingaspromoterthe trimethylsilyltriflate(TMSOTf).331Avaryingrangeoftemperatureswere examinated,indichloromethaneunderargonatmosphereandusingmolecular sieves4Å.(Table5.1) Glycosy l donor Glycosy l acceptorTMSOTf(eq)T (°C)Yield(%) 1 1171150.2 ‐4125 2‐2047 3059 4r.t.13 Table5.1.Reactionconditionsfortheglycosylationtoobtainthecompound123. By1HNMR,itwaspossibletodeterminatethecorrectglycosylationoftheproduct throughthedetectionoftwopeaksat5.31ppmcorrespondingtotheanomeric protonoftheglycosyldonormoietyandat5.24ppmcorrespondingtothe correspondingglycosylacceptor.(Figure5.6) 331a)Francesconi,O;Nativi,C.;Gabrielli,G.;Gentili,M.;Palchetti,M.;Bonora,B.;Roelens,s.; Chem.Eur.J.,2013,339,11742‐11752;b)Depras,G.;Robert,R.;Sendid,B.;Machez,E.; Poulain,D.;Mallet,J.M.;Bioorg.Med.Chem.,2012,20,1817‐1823;c)Heng,L.;Ning,J.;Kong, F.;J.Carbohydr.Chem.,2001,20,285‐296.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 235 Figure5.6.1H‐NMRspectrumofthedisaccharide123. Fromthesetrials,itwasclearthattheuseofroomtemperature(entry4Table5.1), couldcausetherapiddegradationoftheglycosyldonor.Indeed,intheTLC,atthis temperature,appearedalargespotcorrespondingtothehydrolysisofthedonor. Theuseofaverylowtemperature(‐41°Centry1Table5.1)resultedinayieldof25% andusingahighertemperature(‐20°Centry2Table5.1)theyieldincreasedupto 47%.Thebestresultswereobtainedat0°C(entry3Table5.1),usingalso0.2 equivalentsofTMSOTf,incomparisonwiththeglycosylacceptor,thatwasindefect. Inthiscase,theyieldoftheglycosylationreactionwas59%. Withtheaimtominimizetheformationoftheorthoestercompound,atypicalsubproductofthisreaction,(Figure5.7)asmallamountofTMSOTfwasused.This subproductcomesoutfromanintramolecularreactionoftheglycosyldonor,in whichthereisaparticipationoftheacetylgroupinpositionC2.(Scheme5.7)
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 236 O BzO BzO OBz O O N 3 O OAc OAc OAc O O O AcO AcO OAcOAc O NH CCl 3 TMSOTf, 4Å MS CH 2 Cl 2 dry O BzO BzO OBz O OH 117 N 3 115 Scheme5.7.Possiblesub‐productoftheglycosylation. Thesereactionconditionswereappliedalsotothesynthesisofthedisaccharide havingthemethoxygroupattheanomericposition124,reachingayieldofthe46%. (Scheme5.8)Oncethepreparationofthedisaccharidederivedfromper‐acetylated donor117wasoptimized,itwassetupthepreparationofthedisaccharideusingthe per‐benzoylated118asglycosyldonor.Inthiscase,theorthoesterformationisnot possibleoratleastitisreducedtoaminimum,duetothefactthatthebenzoylgroup hasamajorsterichindranceincomparisonwiththeacetylgroup. Takingadvantagefromtheexperienceobtainedwiththedisaccharide123,thesame conditionswereusedtopreparethedisaccharides125and126.Inthiscase,as expected,therewasastrongincreasingoftheglycosylationyieldwhichconfirmthe hypothesesdescribedabove.Infact,changingtheprotectivegroupoftheglycosyl donor,fromacetatestobenzoates,theyieldincreasedfrom46‐59%to79%forthe disaccharide125andto75%forthedisaccharide126.(Scheme5.8)
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 237 O BzO BzO OBz OR OH 115 R= 2-azidoethyl 116 R= Me O AcO AcO OAc OAc O NH CCl 3 117 O BzO BzO OBz OR O O AcO AcO OAc OAc 123 R= 2-azidoethyl 124 R= Me TMSOTf, 4Å MS CH 2 Cl 2 dry 0°C; 45 min 59% 46% TMSOTf, 4Å MS CH 2 Cl 2 dry 0°C; 45 min O BzO BzO OBz OR O O BzO BzO OBz OBz 125 R= 2-azidoethyl 126 R= Me 79% 75% O HO HO OH OR O O HO HO OH OH 23 R= 2-azidoethyl 114 R= Me MeOH r.t., 45 min Quant. Quant. MeOH r.t., 45 min MeONa MeONa O BzO BzO OBz OBz O NH CCl 3 118 Scheme5.8.Synthesisanddeprotectionofdisaccharidescompounds23and114. Deprotectionofdisaccharideshavingthe2azidoethylgroup,andthemethoxy group,wereachievedusingtheclassicalZemplenconditions,193sodiummethoxide inmethanol.TheneutralpHwasrestoredusinganAmberliteIRA120‐H+resinand afterfiltration,thedeprotectionaffordedthefinaldisaccharidesinquantitative yield.(Scheme5.8)Thisprocedureallowedarapidsynthesistoproducethe disaccharide23ingramscale. 5.3. Synthesisofglycodendronswithbiotinatthefocal position OncetheManα1‐2Mandisaccharidehasbeenprepared,weaffordedthe preparationofmultivalentdendriticcompoundsusingthiscarbohydrateasaligand. Forthis,wehavesynthesizedafamilyofglycodendronsofvalencenineandtriand thecorrespondingmonovalentcompound,allhavingabiotinatthefocalpositionof thedendronscaffolds.(Figure5.7)
Chapter 5. Glycodendron structures based on α1,2 mannobioside disaccharide. 238 Figure 5.7. Glycodendrons with a biotin group at the focal position 127-129. The synthetic strategy to address the preparation of these glycodendrons was based on the previous experience of our research group for the preparation of first and second generation mannosylated glycodendrons and glycodendrimers using the CuAAC coupling. 111, 113,292 The retrosynthetic strategy for the preparation of these glycodendrons is depicted in Scheme 5.9
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 239 OOO O H N OOO ONH NN N O HO HO OH O O O HO HO OH OH O O O O O N N N O N N N O N NN O O O NH O HO HO HO O O HO HO HO OH O OH OH HO OO OH OH HO OH OOH OH OH O OOH OH OH HO N 3 O HO HO OH O O O S HN NH H HO O S HN NH H HO O O O O O O O NH O S HN NH H HO O O O O O N N N O N N N O N NN O N 3 O HO HO HO O O HO HO HO OH O OH OH HO OO OH OH HO OH OOH OH OH O OOH OH OH HO O S HN NH H HO O HO HO OH OH O O O O O N N N O N N N O N NN O O HO HO HO O O HO HO HO OH O OH OH HO OO OH OH HO OH OOH OH OH O O OH OH OH HO OO O O O N N N N N N N NN O O O HN O O S HN NH H HO O O O OO N N N O NN N O N N N O HO HO HO OO HO HO HO OH O OH OH OH O O OH OH OH HO O OH OH OH O O HO HO OH HO O O O O O N NN O N N N O NN N O OH OH OH O O OHOH OH HO O HO HO HO O O HO HO HO OH O HO HO HO OO OH OH HO OH Scheme5.9.Retrosyntheticrouteforthepreparationofglycodendrons127‐129. 5.3.1. Synthesisofbuildingblocks Thepreparationofthebuildingblockstoachievethesynthesisoftheglycodendrons neededtheManα(1,2)Manwithanazidogroupattheanomericposition(23)and thealkynylatedscaffoldsnecessarytoperformtheclickreactions.Respecttothe alkynebuildingblocks,itwasselectedatrialkynepentaerythritolfunctionalizedwith ashortdiethyleneglycollinkerwithaterminalchloride76,atrialkyne pentaerythritolfunctionalizedwithalongtetraethyleneglycolwithaterminalbiotin 130,andalineartetraethyleneglycol,functionalizedwithabiotinandapropargyl groupattheends131.(Figure5.8)Thesynthesisofthesecompoundswere OH HO OH H O OOHO OCl Cl OCl HO Br HO O S HN NH H HO N3 O HO HO OH O O O HO HO OH OH
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 240 performedusingastrategy,appropriatelymodified,developedinourgroup113,332 fromaprecedentworkofRoyetal.294 OOO O H N O O O O O O O NH O S HN NH H HO O S HN NH H HO O O O O O Cl 130 131 76 Figure5.8.Alkynebuildingblocks76,130and131. Thesynthesisofthescaffold76wasdescribedinthepreviouslyChapter4,section 4.4.3.Thepreparationofcompounds130and131derivedfromthebiotinylationof thecompounds77and78aredepitectedinScheme5.10. 130 O O O O O O O H2N 77 O O O O O O O HN O S HN NH H H O Biotin, HATU, DIPEA DMF r.t.; 22h 88% 131 O O O O H2N 78 HN O S HN NH H H O O O O O Biotin, HATU, DIPEA DMF r.t.; 22h 87% Scheme5.10.Syntheticrouteforthepreparationofthebuildingblocks130and131. 332Sanchez‐Navarro,M.etal.,Chem.Eur.J.,2011,17,766‐769.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 241 Thecouplingoftheaminogroupatthefocalpositionwithbiotin,wasperformed usinghexafluorophosphateazabenzotriazoletetramethyluronium(HATU),333to promotetheamideformation.Afterthepurificationbysilicagelcolumn chromatography,thebuildingblock130wasobtainedwitha87%,yield.(Scheme 5.10)Theformationoftheimportantbuildingblockwasconfirmedboth,byNMR analysisandmassspectrometry(Figure5.9). Figure5.9.ESI‐MSoftheaminocompound130. Inordertoobtainthemonoalkynebuildingblock131,thesameprocedurewas applied,obtainingthemonoalkynecompoundwithagoodyield.(Scheme3.10) 5.3.2. Synthesisoftheglycodendronswithbiotin Withthecarbohydratesandthealkynebuildingblocksonhands,theconjugationof bothentitieswasperformedusingclickchemistryreactiontopreparethe correspondingManα1,2Manglycodendronstructures. The1,3dipolarcycloaddiction,catalyzedbycopper,betweenthetrialkyne130and theManα1,2Mandisaccharide23wasperformedusingtheexperimentalconditions explainedinthesection4.4.4;exceptfortheuseofmicrowaveheatinginorderto speedupthereactionreducingthetimenormallyrequiredforthistypeof reaction.334Thismicrowavewarmingwasrecentlyappliedbyourresearchgroup witharemarkablesuccessinthesynthesisofmannosylatedglycodendronswith differentvalencyanddendriticgeneration.292 333Zhou,Y.,etal.,Analyst,2017,142,345–350. 334Bock,V.D.;Hiemstra,H.;vanMaarseveen,J.H.;Eur.J.Org.Chem.,2006,51–68.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 242 Thesameconditionswereapplied,usingtheprotectedformofthedisaccharide23, thebenzoylateddisaccharide125,334withtheideatoremovetheprotectionafter thepurificationphase.Tocarryonthereaction,CuSO4·5H2O(0.3equivalents)were used,thismeans,0.1equivalentperalkynegroup.TBTA(0.6equivalents)and sodiumascorbate(0.6equivalentstoreducetheCu(II)toCu(I)),wereadded.The reactionwasheatedundermicrowaveirradiationat60°Cfor30mininDMSO.After thistime,theTLCrevealedthecompleteabsenceofanyreactionproduct.(Scheme 5.11) CuSO 4 5H 2 O TBTA, NaAsc, MW 60°C; 30min DMSO N3 O BzO BzO OBz O O O BzO BzO BzO OBz O O O O O N N N O N N N O N NN O O O HN O BzO BzO OBz O O BzO BzO OBzOBz O BzO BzO OBz OO OBz BzO OBz OBz OOBz OBz BzO O OOBz OBz BzO BzO O S HN NH O H H O O O O HN O O O O S HN NH O H H 130 125 132 N Bn NN N 3 TBTA X Scheme5.11Afirstapproachtothepreparationoftheprotectedtrivalentglycodendron 132. Asecondtrialwasperformeddoublingthenumberofequivalentsofthereagents andalso,adoublecycleofmicrowaveheatingaftertheadditionofmorecatalyst. Noneofthesevariationsinthesynthesisallowedtoobservechangesonthenegative finalresult.Studyingtheclickreactionswithmicrowavereportedinourresearch groupintherecentpast,oneofthedifferencewastheuseofcarbohydrates protectedwithacetylgroupinsteadofthebenzoyls.Probably,thearomaticringof thebenzoylprotectinggrouphasthecapacitytochelatethecopper,decreasingthe availableamountofcatalystinthereaction. Forthisreason,itwasdecidedtousethedeprotecteddisaccharide23,inorderto avoidthepossiblechelationoftheCu.Thesamereactionconditionsusedbefore
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 249 Glycodendron105constitutesthebranchesofthenonavalentstructurethatare displayedonacentralpentaerhytritolcore,havingthreealkynesgroupsandalong linkerwiththebiotinatthefocalposition(compound130).Usingasecondclick reaction,itwaspossibletoincreasethegenerationbycoupling105onthisscaffold. (Scheme5.15) Theconjugationconditionsweresimilartothepreviousoneswiththeexceptionthat inthiscase,thelargenumberoffreehydroxylgroupspresentinthetrivalent105 requiredanexcessoftheCu(I)catalyst,addedinsmallportionswithanintervalof 1h.TheESI‐MSofthecrudeallowedtoverifythecompleteformationofthe nonavalentproduct.Afterremovingtheexcessofcopperbyaddingascavenger resin,thecrudewaspurifiedbysizeexclusionchromatography. Thecharacterizationofthecompound129hasbeendonebytheNMRanalysisdue toitsstructuralcharacteristics.Indeed,thenonavalentglycodendronshavetwokind oftriazolerings,aninternalonecorrespondingtothecore,andanexternalone correspondingtothethreebranches.Inthe1HNMRspectrum,itwaspossibleto observe,around8ppm,twosingletscorrespondingtotheprotonsoftheinternal triazolerings(indicatedinblue,Figure5.12)andoftheexternalones(indicatedin red,Figure5.12)ofthestructurewitharatioof3:9,respectively.Inthisway,itwas possibletoverifythecompletefunctionalizationofthecorebyintegratingthese signalsandalsobytheirmultiplicity,becausetheincompleteproductswouldgive differentsignals(lossofsymmetry).Moreover,inthe13CNMRspectrumwerevisible twopeakscorrespondingtothecarbonatomsofthetriazolering,verycloseoneto eachother.InFigure5.12,itispossibletoobservetwopeaks.Around125ppm,there arethepeakscorrespondingtotheCHcarbonsofthetriazole,andaround144ppm, thosecorrespondingtothequaternarycarbons.Inblue,areindicatedthepeakof theinternaltriazoleringsandinred,thecorrespondingtotheexternalones.The HSQCspectrumwasusedtoconfirmthiscorrelation.
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 250 Figure5.12.1Hand13CNMRspectraofthenonavalentglycodendron129. UsingESI‐MS,itwaspossibletoconfirmtheformationoftheproduct,identifyingthe peakshavingtripleandquadruplecharge.Figure5.13
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 251 Figure5.13.ESI‐MSspectrumofthenonavalentglycodendron129. 5.4. Discussion Adoptingaconsecutivesynthesisoftheglycosylacceptor,thetotalcostandthetime ofpreparationofthefinaldeprotecteddisaccharidehasbeenreducedinasignificant manner.Thisapproachallowedtheincreaseoftheyieldandthedecreaseofthe timethecostwasalsoreducedduetotheminimizingofthepurificationprocedures toonlyonecolumnchromatographyafterfiveconsecutivesteps. Inthisway,theManα1,2Manhasbeenpreparedinagram‐scaleandinlessthan2 days,inaveryeasyandreproducibleway.Theversatilityofthisapproachwas demonstratedusingasaglycongroupthemethylandthe2‐azidoethyl.The availabilityofthisdisaccharideinlargequantityhasallowedtheeasysetupofthe synthesisofothercompoundssuchastheglycodendrons.335 Manα1,2Manglycodendronsofvalencenineandtriandthemonovalentcompound weredesignedandsynthetizedwithabiotinatthefocalposition.Infact,taking advantagefromthestrongnon‐covalentbondofbiotin‐streptavidin,oneofthe applicationofthesecompounds,inthefuture,willbethestudyoftheimportance ofthemultivalencyonseveralplantlectinsusingthesurfaceplasmonresonance imaging,conjugatingthesebiotynilatedglycodendronsonastreptavidinsurface. Moreover,theseglycodendronshavebeenusedtostartacollaborationwithDr. FrankMomburgandhisPhDstudentMárciaGonçalves,attheGermanCancer ResearchCenter(DFKZ),Germany.Thegoalwillbetomeasurethebindingofthe 335Reina,J.J.;DiMaio,A.;Ramos‐Soriano,J.;Figueiredo,R.‐C.;Rojo,J.;Org.Biomol.Chem., 2016,14,2873‐2882. [M+3Na] 3+ [M+4Na] 3+
Chapter5.Glycodendronstructuresbasedonα1,2mannobiosidedisaccharide. 252 glycodendronsonnativehumanbloodleukocytes(peripheralbloodmononuclear cells,“buffycoat”preparations)byflowcytometry.Cellsurfacebindingofthe glycanswillbemeasuredbybiotin‐streptavidininteraction,thetypesofcellswithin thelymphocytepreparationwillthenbefurtherdefinedbytheirspecific(CD) markers,sowecandifferentiatebindingtoT‐(CD4/CD8),B‐,NKcellsandmonocytes. Theseexperimentsshouldgiveusafirstestimationaboutthecapacityofthese glycodendronstobindtocertaintypesofbloodlymphocytesandtheirpossible impactonthecellularactivationstage.
CHAPTER6 Conclusions
Esta parte de la tesis se publicará con un retraso de 12 meses desde su aprobación y defensa.
CHAPTER7 ExperimentalSection
Chapter7.Experimentalsection 261 7. Experimentalsection 7.1 .Reagents,solventsandanalyticaltechniques ChemicalswerepurchasedfromSigma‐Aldrich,Merck,DextraorCarboSynthand usedwithoutfurtherpurification,unlessotherwiseindicated.Solventswere purchasedfromFisherscientific,ScharLabandCarloErba.Anhydroussolventswere purchasedfromSigma‐Aldrich®withacontentofwater≤0.005%.H2Owaspurified withaMilli‐QpurificationsystemfromMillipore(18.3Ω).MicroplatesHB896well werepurchasedbyBiomat,Italy. Allreactionsthatneededdryconditionswerecarriedoutusingstandardtechniques underaninertatmosphereofoxygen‐freeArgonorNitrogen,unlessotherwise stated.TLCwereperformedusingpre‐coatedaluminumchromate‐platesSilicaGel 60F254Merckof0.25mmthick.CompoundswerevisualizedbyusingUVlight(254 nm)andrevealedbyimmersionintosolutionsofmolybdenumblue(20gof ammoniummolybdate(VI)tetrahydrate,0.4gofsulphateofhydratedceriumand 10%sulfuricacidin400mLofH2O)andheatingat100ºC,or0.1%ninhydrinin ethanol,orabasicsolution(10%w/wK2CO3inwater)ofKMnO4. PurificationsbyFlashcolumnchromatographywereperformedusingSilicagel60 (particlesize0,063‐0,200nmor0,015‐0,040mm),fromMerck,elutingbygravityor subjectingittolightpressure.PurificationsbySize‐ExclusionChromatographyhas beencarriedoutincolumnsfilledwithSephadexLH‐20orG‐25(GEHealthcareLife Science),elutingbygravity. NMRspectra,1Hand13C,wererecordedat298KonaBrukerDRX300,DRX400and DRX500spectrometers.Allchemicalshiftswerereportedinppm(δ)usingthe residualprotonsolventpeaksasinternalstandards.Theseabbreviationswereused toindicatethemultiplicities:s=singlet,d=doublet,dd=doubletofdoublets,t= triplet,td=tripletofdoublets,m=multplet.
Chapter7.Experimentalsection 358 –3.81(m,4H,H6andH5),2.37–1.79(m,12H,CH3).13CNMR(75MHz,CDCl3):δ 170.8,170.1,170.0,169.9(CO),160.0(C7),94.8(C1),90.8(C8),71.53(C5),69.1(C3), 68.2(C2),65.7(C4),62.3(C6),21.1,21.0,20.9(CH3). 2,3,4,6‐Tetra‐O‐benzoyl‐α‐D‐mannopyranose(122)330a O BzO BzO OBzOBz OH Toasolutionofα‐D‐mannopyranosepentabenzoate120(5.00g,7.14mmol)in anhydrousDMF(50mL)undernitrogenatmosphere,hydrazineacetate(0.78g,8.57 mmol)wasaddedinportions.After24hofstirringat60°C,themixturewascooled atroomtemperatureandthecrudewaspurifiedbycolumnchromatographyon silicagel(EtOAc:Hex1:2)affording122asafluffywhitesolid(2.9g,70%).1HNMR (400MHz,CDCl3):δ=8.13–7.84(m,8H,HBz),7.60–7.25(m,12H,HBz),6.18(t,J= 10.1Hz,1H,H2),6.01(dd,J=10.1,3.3Hz,1H,H3),5.75(dd,J=3.3,1.9Hz,1H,H4), 5.54(dJ=1.9Hz,1H,H1),4.77(dd,J=12.2,2.7Hz,1H,H6),4.68(m,1H,H5),4.46 (dd,J=12.2,3.7Hz,1H,H6’).13CNMR(100.6MHz,CDCl3):δ=166.5,165.7,165.6 (CO),133.5,133.3,133.2,130.0,129.4,129.2,129.2,128.7,128.6,128.4(Carom), 92.5(C1),71.0(C5),70.0(C3),69.0(C2),67.0(C4),62.9(C6). 2,3,4,6‐Tetra‐O‐benzoyl‐α‐D‐mannopyranosyltrichloroacetimidate(118)330a O BzO BzO OBz OBz O NH CCl 3 1 2 3 45 6 7 8 Undernitrogenatmosphere,trichloroacetonitrile(2.42g,16.8mmol)wasdropped slowlytoasolutionof122(1.4g,4.0mmol)andDBU(0.78g,5.04mmol)at0°C.The reactionmixturewaswarmedslowlyatroomtemperatureandstirredfor3h.Then, itwasconcentratedundervaccumandthecrudeproductwaspurifiedbyflash columnchromatographyonsilicagel(EtOAc/Hex,1:2)toafford118(2.71g,67%)as
Chapter7.Experimentalsection 359 afluffycolourlesssolid.1HNMR(400MHz,CDCl3):δ=8.87(s,1H,NH),8.12‐7.83(m, 8H,HBz),7.65‐7.25(m,12H,HBz),6.58(d,J=1.8Hz,1H,H1),6.18(m,J=10.1Hz, 1H,H2),6.01(dd,J=10.1,3.3Hz,1H,H3),5.75(dd,J=3.3,1.9Hz,1H,H4),4.77(dd, 1H,J=12.2,2.7Hz,1H,H6),4.68(m,1H,H5),4.46(dd,J=12.2,3.7Hz,1H,H6’).13C NMR(100.6MHz,CDCl3):δ=166.0,165.5,165.4,165.1(CO),159.9(C7),133.6, 133.3,129.0,128.8,128.7,128.4(Carom),94.7(C1),90.6(C8),71.5(C5),69.8(C3), 68.9(C2),66.1(C4),62.4(C6). 2‐AzidoethylO‐(2,3,4,6‐tetra‐O‐acetyl‐α‐D‐mannopyranosyl)‐(1‐2)‐3,4,6‐tri‐O‐ benzoyl‐α‐D‐mannopyranoside(123) O BzO BzO OBz O O N 3 O AcO AcO OAc OAc 1 2 3 45 6 1 2 3 45 6 7 8 A B Amixtureoftheacceptor115(120mg,0.214mmol)andthedonorper‐acetylated mannosetrichloroacetimidate117(158mg,0.321mmol)wasco‐evaporatedfrom toluenethreetimes.Powderedandactivated4Åmolecularsieveswereadded,and themixturewaskeptundervacuumforfewhoursandthendissolvedinCH2Cl2(8 mL).Themixturewascooledto0°Cfor15min,followedbytheadditionofTMSOTf (8.5μL,0.040mmol),andstirredfor30minat0°C.Thereactionwasquenchedby theadditionofEt3N,filteredoverapadofCeliteanddriedundervacuum.Thecrude waspurifiedbyflashcolumnchromatographyonsilicagel(DCM:MeOH,100:1)to obtain123asawhitesolid(112mg,59%).1HNMR(400MHz,CDCl3):δ8.06(d,J= 7.1Hz,2H,HBz),7.98(d,J=7.1Hz,2H,HBz),7.95(d,J=7.0Hz,2H,HBz),7.68–6.99 (m,9H,HBz),5.94(t,J=9.9Hz,1H,H4ManA),5.84(dd,J=9.9,3.2Hz,1H,H4ManA), 5.49–5.43(m,2H,H2ManB+H3ManB),5.26(t,J=9.5Hz,1H,H4ManB),5.15(d,J= 1.9Hz,1H,H1ManA),4.98(d,J=1.5Hz,1H,H1ManB),4.63(dd,J=12.2,3.0Hz,1H, H6ManA),4.52(dd,J=12.1,5.4Hz,1H,H6ManA),4.41(ddd,J=10.1,5.4,2.9Hz, 1H,H5ManA),4.34(dd,J=3.2,1.9Hz,1H,H2ManA),4.25(dd,J=11.9,5.4Hz,1H,
Chapter7.Experimentalsection 360 H6ManB),4.17(ddd,J=12.1,7.4,3.3Hz,1H,H5ManB),4.11(dd,J=11.9,2.5Hz,1H, H6ManB),4.01(dt,J=10.3,4.7Hz,1H,H7),3.82–3.72(m,1H,H7),3.54(t,J=5.0Hz, 2H,H8),2.10(s,3H,COOCH3),2.06(s,3H,COOCH3),2.04(s,3H,COOCH3),2.01(s, 3H,COOCH3).13CNMR(101MHz,CDCl3):δ170.51,169.80,169.48,169.42(COAc), 166.22,165.50,165.19(COBz),133.43,133.32,133.02,129.92,129.86,129.70 (CHBz),128.97(CBz),128.82(CBz),128.55,128.47,128.36(CHBz),99.46(C1ManB), 98.65(C1ManA),76.50(C2ManA),70.62(C3ManA),69.30(C2ManB),69.2 (C5ManB),69.1(C5ManA),68.8(C3ManB),67.2(C7),67.1(C4ManA),66.3 (C4ManB),63.6(C6ManA),62.6(C6ManB),50.4(C8),20.70,20.67(CH3).ESI‐MS calcd.C43H45N3O18:891.3[M]+;found:914.3[M+Na]+;ESI‐HRMSm/zcalcd.for C43H45N3O18Na:914.2596;found;914.2590. MethylO‐(2,3,4,6‐tetra‐O‐acetyl‐α‐D‐mannopyranosyl)‐(1‐2)‐3,4,6‐tri‐O‐benzoyl‐ α‐D‐mannopyranoside(124) O BzO BzO OBz OCH 3 O O AcO AcO OAc OAc Amixtureoftheacceptor116(150mg,0.296mmol)andthedonorper‐acetylated mannosetrichloroacetimidate117(219mg,0.445mmol)wasco‐evaporatedfrom toluenethreetimes.Powderedandactivated4Åmolecularsieveswereadded,and themixturewaskeptundervacuumforfewhoursandthendissolvedinDCM(10 mL).Themixturewascooledto−0°Cfor15min,followedbytheaddionofTMSOTf (13μL,0.059mmol),andstirredfor30minat0°C.Thereactionwasquenchedby theadditionofEt3N,filteredoverapadofCeliteanddriedundervacuum.Thecrude waspurifiedbyflashcolumnchromatographyonsilicagel(DCM:MeOH,100:1)to obtain124asanwhitesolid(114mg,46%).1HNMR(400MHz,CDCl3):δ8.15–8.07 (m,4H,HBz),8.04(d,J=7.0Hz,2H,HBz),8.02(d,J=7.0Hz,2H,HBz),8.00–7.95(m, 4H,HBz),7.90(d,J=6.9Hz,2H,HBz),7.65–7.30(m,21H,HBz),6.12–6.05(m,2H, H3ManB+H4ManB),6.02(t,J=9.9Hz,1H,H4ManA),5.94(dd,J=2.3,1.8Hz,1H,
Chapter7.Experimentalsection 361 H2ManB),5.91(dd,J=9.9,3.2Hz,1H,H3ManA),5.29(d,J=1.8Hz,1H,H1ManB), 5.09(d,J=1.8Hz,1H,H1ManA),4.73‐4.64(m,3H,H5ManB+H6ManB+H6ManA), 4.60(dd,J=12.2,5.5Hz,1H,H6ManA),4.50(dd,J=12.0,5.3Hz,1H,H6ManB),4.41 (dd,J=2.6,1.8Hz,H2ManA),4.41‐4.35(m,1H,H5ManA),3.42(s,3H,OMe).13CNMR (101MHz,CDCl3):δ170.52,169.84,169.48,169.40(COAc),166.26,165.55,165.20 (COBz),133.39,133.27,132.98,129.91,129.84,129.70(CHBz),128.87,128.54, 128.45(CBz),99.47(C1ManB),98.65(C1ManA),72.50(C2ManA),70.84(C3ManA), 69.29(C2ManB),69.22(C5ManB),69.1(C5ManA),68.82(C3ManB),68.68(C7), 67.30(C4ManA),66.27(C4ManB),63.69(C6ManA),62.65(C6ManB),50.31(C8), 29.71,20.72,20.69(CH3).ESI‐MSm/zcalcd.forC42H44O18:836.2[M]+;found:859.3 [M+Na]+;ESI‐HRMSm/zcalcd.forC42H44O18Na:859.2425;found:859.2413. 2‐AzidoethylO‐(2,3,4,6‐tetra‐O‐benzoyl‐α‐Dmannopyranosyl)‐(1‐2)‐3,4,6‐tri‐O‐ benzoyl‐α‐D‐mannopyranoside(125) O BzO BzO OBz O O N 3 O BzO BzO OBz OBz Amixtureoftheacceptor115(250mg,0.440mmol)andthedonor118(494mg, 0.670mmol)wasco‐evaporatedfromtoluenethreetimes.Powderedandactivated 4Åmolecularsieveswereadded,andthemixturewaskeptundervacuumforfew hoursandthendissolvedinDCM(12mL).Themixturewascooledto0°Cfor15min, followedbytheadditionofTMSOTf(20μL,0.088mmol),andstirredfor30minat0 °C.ThereactionwasquenchedbytheadditionofEt3N,filteredoverapadofCelite anddriedundervacuum.Thecrudewaspurifiedbyflashcolumnchromatography onsilicagel(DCM:MeOH,100:1)toobtain125asawhitesolid(396mg,79%).1H NMR(400MHz,CDCl3):δ8.13(d,J=7.3Hz,2H,HBz),8.09(d,J=7.3Hz,2H,HBz), 8.06(d,J=7.3Hz,2H,HBz):δ8.03–7.95(m,6H,HBz),7.91(d,J=7.6Hz,2H,HBz),
Chapter7.Experimentalsection 362 7.65–7.29(m,21H,HBz),6.15–6.02(m,3H,H4ManA+H3ManB+H4ManB),5.97– 5.90(m,2H,H2ManB+H3ManA),5.31(d,J=1.8Hz,1H,H1ManB),5.24(d,J=1.8 Hz,1H,H1ManA),4.74–4.60(m,3H,H5ManB+H6ManB+H6ManA),4.53(dd,J= 12.0,4.9Hz,1H,H6ManA),4.49(m,2H,H2ManA+H6ManB),3.91(ddd,J=10.5,6.7, 3.8Hz1H,H7),3.59(ddd,J=10.2,6.0,3.5Hz1H,H7),3.52–3.37(m,2H,H8).13C NMR(101MHz,CDCl3):δ166.3,166.1,165.6,165.3,165.1,164.9(COBz),133.5, 133.4,133.3,133.3,133.1,133.1,130.0,130.0,129.9,129.8,129.8,129.7(CHBz), 129.2,129.0,128.8,128.6(CBz),128.5,128.5,128.4,128.4,128.3(CHBz),99.7 (C1ManA),98.7(C1ManB),76.9(C2ManA),70.6,70.1,69.8,69.7,69.2,67.5,67.0, 66.9,63.7(C6ManA),63.1(C6ManB),50.3(C8).ESI‐MSm/zcalcd.forC63H53N3O18: 1139.3[M]+;found:1162.5[M+Na]+;ESI‐HRMSm/zcalcd.forC63H53N3O18Na: 1162.3222;found:1162.3212. MethylO‐(2,3,4,6‐tetra‐O‐benzoyl‐α‐Dmannopyranosyl)‐(1‐2)‐3,4,6‐tri‐O‐benzoyl‐ α‐D‐mannopyranoside(126) O BzO BzO OBz OCH 3 O O BzO BzO OBz OBz Amixtureoftheacceptor116(60mg,0.119mmol)andthedonor118(105mg, 0.142mmol)wasco‐evaporatedfromtoluenethreetimes.Powderedandactivated 4Åmolecularsieveswereadded,andthemixturewaskeptundervacuumforfew hoursandthendissolvedinDCM(5mL).Themixturewascooledto0ºCfor15min, followedbytheadditionofTMSOTf(4,5μL,mmol,0.024mmol),andstirredfor30 minat0ºC.ThereactionwasquenchedbytheadditionofEt3N,filteredoverapad ofceliteanddriedundervacuum.Thecrudewaspurifiedbyflashcolumn chromatographyonsilicagel(DCM:MeOH,100:1)toobtain126asanwhitesolid(97 mg,75%).1HNMR(400MHz,CDCl3):δ8.15‐8.07(m,4H,HBz),8.04(d,J=7.0Hz, 2H,HBz),8.02(d,J=7.0Hz,2H,HBz):δ8.00‐7.95(m,4H,HBz),7.90(d,J=6.9Hz,
Chapter7.Experimentalsection 363 2H,HBz),7.65‐7.30(m,21H,HBz),6.12‐6.05(m,2H,H3ManB+H4ManB),6.02(t,J =9.9Hz,1H,H4ManA),5.94(dd,J=2.3,1.8Hz,1H,H2ManB),5.91(dd,J=9.9,3.2 Hz,1H,H3ManA),5.29(d,J=1.8Hz,1H,H1ManB),5.09(d,J=1.8Hz,1H,H1ManA), 4.73‐4.64(m,3H,H5ManB+H6ManB+H6ManA),4.60(dd,J=12.2,5.5Hz,1H, H6ManA),4.50(dd,J=12.0,5.3Hz,1H,H6ManB),4.41(dd,J=2.6,1.8Hz,H2ManA), 4.41‐4.35(m,1H,H5ManA),3.42(s,3H,OMe).13CNMR(101MHz,CDCl3):δ166.4, 166.1,165.6,165.3,165.0,165.0(COBz),133.5,133.4,133.3,133.,133.1,133.0, 130.0,130.0,129.9,129.8,129.8,129.7(CHBz),129.2,129.1,128.9,128.8(CBz), 128.5,128.5,128.4,128.4,128.3(CHBz),99.6(C1ManA),99.6(C1ManB),76.9 (C2ManA),70.8,70.1,69.8,69.7,68.8,67.6,67.0(C2,3,4,5ManA‐B),63.8(C6ManA), 63.1(C6ManB),55.2(CH3).ESI‐MSm/zcalcd.forC62H52O18:1084.3[M]+;found: 1107.3[M+Na]+;ESI‐HRMSm/zcalcd.forC62H52O18Na:1107.3051;found: 1107.3039. 2‐Azidoethylα‐D‐mannopyranosyl‐(1‐2)‐α‐Dmannopyranoside(23) O HO HO OH O O N3 O HO HO OH OH Fromacetylateddonor123.Toasolutionof123(100mg,0,112mmol)indry methanol,undernitrogenatroomtemperature,wasaddeda1Msolutionofsodium methoxideinMeOH(2equiv.)andthereactionwasstirredfor1hour.Then,the reactionmixturewasneutralizedwithAmberliteIRA120‐H+resin.Theresinwas filteredoffandthefiltratewasconcentratedunderreducedpressure.Thecrudewas dilutedinH2O(10mL),washedwithDCM(10mL)andtheaqueousphasewas lyophilizedtoobtain23asawhitesolid(45mg,quant.).
Chapter7.Experimentalsection 364 Frombenzoylateddonor125.Toasolutionof125(390mg,0.342mmol)indry methanol,undernitrogenatroomtemperature,wasaddeda1Msolutionofsodium methoxideinMeOH(2equiv.)andthereactionwasstirredfor1hour.Then,the reactionmixturewasneutralizedwithAmberliteIRA120‐H+resin.Theresinwas filteredoffandthefiltratewasconcentratedunderreducedpressure.Thecrudewas dilutedinH2O(10mL),washedwithCH2Cl2(10mL)andtheaqueousphasewas lyophilizedtoobtain23asawhitesolid(140mg,quant.). 1HNMR(400MHz,D2O):δ5.08(d,J=1.8Hz,1H,H1ManB),4.95(d,J=1.9Hz,1H, H1ManA),3.99(dd,J=3.4,1.8Hz,1H,H2ManB),3.92(dd,J=3.3,1.8Hz,1H, H2ManA),3.89–3.74(m,4H,H3ManA+H3ManB+H6ManA+H4ManA+H7),3.73– 3.50(m,7H,H4ManB+H5ManA+H5ManB+H6ManA+2H6ManB+H7),3.49–3.35 (m,2H,H8);13CNMR(101MHz,D2O):δ102.3(C1ManB),98.2(C1ManA),78.6 (C2ManA),73.3(C5ManB),72.9(C5ManA),70.3(C3ManB),70.0(C3ManA),69.9 (C2ManB),66.9(C4ManB),66.9(C4ManA),66.4(C7),61.1(C6ManA),60.9(C6ManB), 50.2(C8);ESI‐MSm/zcalcd.forC14H25N3O11:411.1[M]+;found:434.2[M+Na]+;ESIHRMSm/zcalcd.forC14H25O11N3Na:434.1381;found:434.1372. Methylα‐D‐mannopyranosyl‐(1‐2)‐α‐Dmannopyranoside(114) O HO HO OH OCH 3 O O HO HO OH OH Fromacetylateddonor124.Toasolutionof124(100mg,0.119mmol)indry methanol,undernitrogenatroomtemperature,wasaddeda1Msolutionofsodium methoxideinMeOH(2equiv.)andthereactionwasstirredfor1hour.Then,the reactionmixturewasneutralizedwithAmberliteIRA120‐H+resin.Theresinwas filteredoffandthefiltratewasconcentratedunderreducedpressure.Thecrudewas
Chapter7.Experimentalsection 365 dilutedinH2O(10mL),washedwithDCM(10mL)andtheaqueousphasewas lyophilizedtoobtain114asawhitesolid(42mg,quant.). Frombenzoylateddonor126.Toasolutionof126(90mg,0.081mmol)indry methanol,undernitrogenatroomtemperature,wasaddeda1Msolutionofsodium methoxideinMeOH(2equiv.)andthereactionwasstirredfor1hour.Then,the reactionmixturewasneutralizedwithAmberliteIRA120‐H+resin.Theresinwas filteredoffandthefiltratewasconcentratedunderreducedpressure.Thecrudewas dilutedinH2O(10mL),washedwithDCM(10mL)andtheaqueousphasewas lyophilizedtoobtain114asawhitesolid(29mg,quant.). 1HNMR(400MHz,D2O):δ4.95(d,J=1.8Hz,1H,H1ManB),4.92(d,J=2.0Hz,1H, H1ManB),3.99(dd,J=3.3,1.8Hz,1H,H2ManB),3.88(dd,J=3.3,1.8Hz,1H, H2ManA),3,85–375(m,4H,H3ManA+H3ManB+H6ManA+H4ManA),3.74–3.50 (m,6H,H4ManB+H5ManA+H5ManB+H6ManA+2H6ManB),3,33(s,3H,OCH3); 13CNMR(101MHz,D2O):δ102.3(C1ManB),99.3(C1ManA),78.5(C2ManA),73.2 (C5ManB),72.5(C5ManA),70.3(C3ManB),70.3(C3ManA),69.9(C2ManB),66.9 (C4ManB),66.8(C4ManA),61.1(C6ManA),60.9(C6ManB),54.8(CH3);ESI‐MSm/z calcd.forC13H24O11:356.1[M]+;found:379.2[M+Na]+;ESI‐HRMSm/zcalcd.for C13H24O11Na:379.1211;found;379.1204. 7.15. PreparationofGlycodendronswithbiotinatthefocal position Compound130 NHO O O O OOO O S HN NH H HO 1234 56 7 8 910 11 12 14 13 15 16 17 18 19 20 ToasolutionofD‐Biotin(0.103g,0.423mmol)inDMF(2mL),HATU(0.193g,0.51 mmol)andDIPEA(0.131g,1.026mmol)wereadded.After10minutes,thescaffold
Chapter7.Experimentalsection 366 77(0.180g,0.423mmol)inDMF(1mL)wasadded,andthereactionmixturewas stirredatroomtemperaturefor22h.Aftercheckingthecompletediseppearenceof thestartingproduct,thecrudeofreactionwasdriedundervacuumandthe purificationwasaffordedbycolumnchromatographyonsilicagel(DCM:MeOH99:1) togive130likeayellowoil.(0.169g,87%)1HNMR(300MHz,CDCl3):δ6.87(s,1H, H16),6.45(s,1H,H18),4.51(dd,J=7.9,4.6Hz,1H,H19),4.32(dd,J=7.8,4.6Hz, 1H,1H,H15),4.11(d,J=2.4Hz,6H,H3),3.75–3.36(m,24H,H4,H6,H7,H8),3.23 –3.08(m,1H,H14),2.90(dd,J=12.9,4.8Hz,1H,H18),2.75(d,J=12.8Hz,1H,H18’), 2.43(t,J=2.4Hz,3H,H1),2.23(t,J=7.4Hz,2H,H10),1.83–1.56(m,4H,H11,H13), 1.22(d,J=11.8Hz,2H,H12).13CNMR(75MHz,CDCl3):δ175.2(C9),164.1(C17), 80.1(C2),75.0(C1),70.2,70.1,70.0,69.9(C7),69.7(C6),69.0(C4),61.7(C15),60.2 (C19),59.1(C3),55.5(C14),45.0(C5),43.4(C20),39.1(C8),35.8(C10),28.2(C12), 27.9(C13),24.3(C11).ESI‐MSm/zcalcd.forC32H49N3O9S:651.8[M]+;found:674.4 [M+Na]+. Compound131 O O O O HN O S HN NH H HO 1 23 4 5 67 8 9 10 11 12 13 14 15 16 17 ToasolutionofD‐Biotin(0.458g,1.88mmol)inDMF(8mL),HATU(0.858g,2.26 mmol)andDIPEA(0.485g,3.76mmol)wereadded.After10minutesthescaffold78 (0.434g,1.88mmol)inDMF(2mL)wasaddedandthereactionmixturewasstirred atroomtemperaturefor22h.Aftercheckingofthecompletedisappearanceofthe startingproduct,thecrudeofreactionwasdriedundervacuumandthepurification wasaffordedbycolumnchromatographyonsilicagel(DCM:MeOH99:1)togive131 asayellowoil.(0.757g,88%)1HNMR(400MHz,CDCl3):δ6.97(m,1H,H15),6.83
Chapter7.Experimentalsection 367 (s,1H,H13),6.04(s,1H,NH),4.48(dd,J=7.9,4.7Hz,1H,H16),4.28(dd,J=8.0,4.6 Hz,1H,H12),4.17(d,J=2.4Hz,2H,H3),3.79–3.48(m,14H,CPEG),3.40(m,2H,H5), 3.11(td,J=7.2,4.3Hz,1H,H11),2.87(dd,J=12.8,4.8Hz,1H,H17),2.72(d,J=12.7 Hz,1H,H17’),2.46(t,J=2.4Hz,1H,H1),2.20(t,J=7.5Hz,2H,H7),1.69(m,4H,H8 andH10),1.41(m,2H,H9).13CNMR(101MHz,CDCl3):δ173.4(C6),164.3(C14),79.6 (C2),74.7(C1),70.5,70.4,70.3,70.0,69.9,69.0(C4),61.7(C12),60.2(C16),58.4 (C3),55.7(C11),40.5(C17),39.1(C5),35.9(C7),28.3(C9),28.1(C10),25.6(C8).ESIMSm/zcalcd.forC21H35N3O6S:457.5[M]+;found:480.4[M+H]+. DimannoseTrivalentdendron127 O O O O O N N N O N N N O N NN O O O HN O HO HO HO O O HO HO HO OH O OH OH HO OO OH OH HO OH OOH OH OH O O OH OH OH HO O S HN NH O H H A B 1 2 3 4 5 6 1 2 4 3 5 6 7 89 10 11 12 13 14 15 16 17 18 18 20 21 22 24 27 23 25 26 Toasolutionofdisaccharide23(0.026g,0.066mmol)andscaffold130(0.010g, 0.0098mmol)inamixtureofDMSO/H2O(0.5mL/0.25mL)CuSO4·5H2O(0.003g, 0.009mmol),sodiumascorbate(0.005g,0.027mmol)andTBTA(0.009g,0.018 mmol),allthreedissolvedinDMSO(1.0mL),wereadded.Thereactionwastreated undermicrowaveirradiationat60°Cfor30min.Thenoneportionmoreof CuSO4·5H2O(0.3eq)wasaddedandthereactionwasirradiatedagainwithMWfor
Chapter8.References 375 8. References [1]TheAegisinlatin,ascitedintheIliad,isashieldoranarmorusedbyZeusandhis daughterAthena. [2]a)Palm,N.W.;Medzhitov,R.;Immunol.Rev.,2009,227,221–233;b)Medzhitov, R.;Janeway,C.A.Jr.;Curr.Opin.Immunol.,1997,9,4–9. [3]Janeway,C.A.Jr;Medzhitov,R.;Annu.Rev.Immunol.,2002,20,197–216. [4]a)Iwasaki,A.;Medzhitov,R.;Science,2010,327,291–295;b)Litman,G.W.etal., Nat.Rev.Immunol.,2010,10,543–553. [5]Walsh,K.P.;Mills,K.H.;TrendsImmunol.,2013,34,521‐30. [6]Steinman,R.M.;Annu.Rev.Immunol.,1991,9,271–296 [7]Niess,J.H.etal.,Science,2005,307,254–258. [8]a)Marshall,N.A.etal.,CancerRes.,2012,72,581–591;b)Wilkinson,K.A.; Wilkinson,R.J.;Eur.J.Immunol.,2010,40,2139–2142;c)Banchereau,J.;Steinman, R.M.;Nature,1998,392,245‐252. [9]Carbohydrate‐basedvaccinesmethodsandprotocols,Ed.BerndLepenies, HumanaPress,2015,Chapter1,Hütter,J.;Lepenies,1‐10. [10]Steinman,R.M.;Cohn,Z.A.;J.Exp.Med.,1973,137,1142–1162. [11]ImagecreatedbyDonaldBliss,NationalLibraryofMedicine,Sriram Subramaniam,NationalCancerInstitute;Felts,R.L.etal.,ProcNatlAcadSciUSA, 2010,107,13336‐13341. [12]Banchereau,J.etal.,Annu.Rev.Immunol.,2000,18,767–811. [13]Mellman,I.;Steinman,R.M.;Cell,2001,106,255–258.
Chapter8.References 376 [14]a)Randolph,G.J.;Angeli,V.;Swartz,M.A.;Nat.Rev.Immunol.,2005,5,617– 628;b)Cyster,J.G.;J.Exp.Med.,1999,189,447–450. [15]Banchereau,J.;Palucka,A.K.;Nat.Rev.Immunol.,2005,5,296‐306. [16]Lennon‐Dumenil,A.M.;Bakker,A.H.;Wolf‐Bryant,P.;Ploegh,H.L.;Lagaudriere‐ Gesbert,C.;Curr.Opin.Immunol.,2002,14,15–21. [17]FundamentalImmunology(7thedition),Eds.Paul,W.E.,Steinman,R.M.in Lippincott‐Raven,Philadelphia,2012,ISBN9781451117837. [18]a)Kadowaki,N.etal.,J.Exp.Med.,2001,193,1221–1226;b)Fernandez,N.C. etal.,Nat.Med.,1999,5,405–411. [19]Laffont,S.;Powrie,F.;Nature,2009,462,732‐733. [20]Querec,T.etal.,J.Exp.Med.,2006,203,413–424. [21]Albert,M.L.;Sauter,B.;Bhardwaj,N.;Nature,1998,392,86–89. [22]Geijtenbeek,T.B.H.etal.,Cell,2000,100,587–597. [23]Reuter,G.;Gabius,H‐J.;Cell.Mol.LifeSci.,1999,55,368–422. [24]Gabius,H.J.;Andre,S.;Kaltner,H.;Siebert,H‐C.;Biochim.Biophys.Acta,2002, 1572,165–177. [25]Gabius,H.J.;Siebert,H.C.;Andre,S.;Jimenez‐Barbero,J.;Rudiger,H.; ChemBioChem,2004,5,740–764. [26]a)Broz,P.;Monack,D.M.;Nat.Rev.Immunol.,2013,13,551–565;b)Figdor, C.G.;vanKooyk,Y.;Adema,G.J.;Nat.Rev.Immunol.,2002,2,77–84. [27]CellularandMolecularImmunology(9thedition),Eds.Abbas.A.;Lichtman,A.H.; Pillai,S.;Elsevier,2017,ISBN:9780323479783. [28]Ernst,B.;Magnani,J.L.;Nat.Rev.DrugDiscover.,2009,8,661‐677. [29]Sharon,N.;Lis,H.;Glycobiology,2004,14,53R‐62R. [30]Sancho,D.;ReiseSousa,C.;Annu.Rev.Immunol.,2012,30,491‐529.
Chapter8.References 377 [31]a)Klaver,E.J.;etal.,Int.J.Parasitol.,2013,43,191–200;b)Vautier,S,; MacCallum,D.M.;Brown,G.D.;Cytokine,2012,58,89–99;c)Lozach,P.Y.;Burleigh. L.;Staropoli,I.;Amara,A.;MethodsMol.Biol.,2007,379,51–68. [32]GeijtenbeekT.B.;Gringhuis,S.I.;Nat.Rev.Immunol.,2009,9,465–479. [33]Geijtenbeek,T.B.;Gringhuis,S.I.;Nat.Rev.Immunol.,2016,16,433–448. [34]a)Mayer,S.;Raulf,M.K.;Lepenies,B.;Histochem.Cell.Biol.,2017,147,223‐237; b)Kingeter,L.M.;Lin,X.;Cell.Mol.Immunol.,2012,9,105–112. [35]Drickamer,K.;J.Biol.Chem.,1988,263,9557–9560. [36]Drickamer,K.;Curr.Opin.Struct.Biol.,1999,9,585‐590. [37]Drummond,R.A.;Brown,G.D.,PLoSPathog.,2013,9,e1003417 [38]Zelensky,A.N.;Gready,J.E.;FEBSJ.,2005,272,6179‐6217. [39]vanVliet,S.J.;Garcia‐Vallejo,J.J.;vanKooyk,Y.;Immunol.CellBiol.,2008,86, 580‐587. [40]a)Wells,C.A.etal.,J.Immunol.,2008,180,7404‐7413;b)Weck,M.M.etal., Blood,2008,111,4264‐4272;c)Engering,A.etal.,J.Immunol.,2002,168,2118‐ 2126. [41]a)Tacken,P.J.etal.,Blood,2005,106,1278‐1285;b)Bonifaz,L.C.etal.,J.Exp. Med.,2004,199,815‐824. [42]a)deWitte,L.etal.,Nat.Med.,2007,13,367‐371;b)Tassaneetrithep,B.etal., J.Exp.Med.,2003,197,823‐829. [43]Valladeau,J.etal.,Immunity,2000,12,71–81. [44]Chatwell,L.;Holla,A.;Kaufer,B.B.;Skerra,A.;Mol.Immunol.,2008,45,1981‐ 1994;ImageobtainedbyPDB:3KQGin“BIOVIADiscoveryStudio4.5©”program. [45]Liu,Y.;Liu,J.;Pang,X.;Liu,T.;Ning,Z.;Cheng,G.;Molecules,2015,20,2272– 2295.
Chapter8.References 378 [46]a)deJong,M.A.etal.,Mol.Immunol.,2010,47,1216–25;b)deWitte,L.etal.; Nat.Med.,2007,13,367–371;c)Hunger,R.E.etal.,J.Clin.Investig.,2004,113,701– 708. [47]a)Schwartz,O.;Nat.Med.,2007,13,245–246;b)Turville,S.G.;Cameron,P.U.; Handley,A.;Lin,G.;Pöhlmann,S.;Doms,R.W.;Cunningham,A.L.;Nat.Immunol., 2002,3,975–983. [48]Granelli‐Piperno,A.etal.,J.Immunol.,2005,175,4265–4273. [49]Soilleux,E.J.;ClinicalSci.,2003,104,437‐46. [50]Feinberg,H.;Castelli,R.;Drickamer,K.;Seeberger,P.H.;Weis,W.I.;J.Biol.Chem., 2007,282,4202‐4209. [51]ImageobtainedbyPDB:2IT6in“BIOVIADiscoveryStudio4.5©”program. [52]Anderluh,M.;Jug,G.;Svajger,U.;Obermajer,N.;Cur.Med.Chem.,2012,19, 992‐1007. [53]Menon,S.etal.,Proc.Natl.Acad.Sci.USA,2009,106,11524‐11529. [54]Feinberg,H.;Guo,Y.;Mitchell,D.A.;Drickamer,K.;Weis,W.I.;J.Biol.Chem., 2005,280,1327‐1335.ImageobtainedfromPDB:1XARbyNGLViewer (dio:10.1093/bioinformatics/bty419). [55]a)Lai,W.K.etal.,Am.J.Pathol.,2006,169,200‐208;b)Geijtenbeek,T.B.etal., Cell,2000,100,575–585. [56]a)Carroll,M.V.;Sim,R.B.;Bigi,F.;Jakel,A.;Antrobus,R.;Mitchell,D.A.;Protein Cell,2010,1,859–870;b)Gringhuis,S,I,;denDunnen,J.;Litjens,M.;vanderVlist, M.;Geijtenbeek,T.B.H.;Nat.Immunol.,2009,10,1081–1088;c)Hodgesetal.,Nat. Immunol.,2007,8,569–77;d)Zhang,P.etal.,J.Leukoc.Biol.,2006,79,731–738;e) Barreiro,L.B.etal.,Hum.Immunol.,2006,67,102–107;f)Kwon,D.S.;Gregorio,G.; Bitton,N.;Hendrickson,W.A.;Littman,D.R.;Immunity,2002,16,135–144;g) Alvarez,C.P.etal.,J.Virol.,2002,76,6841–6844. [57]Geijtenbeek,T.B.H.etal.,J.Exp.Med.,2003,197,7–17. [58]Geijtenbeek,T.B.H.etal.,Nat.Immunol.,2000,1,353–357.
Chapter8.References 379 [59]Cambi,A.C.;Figdor,C.G.;Curr.Opin.Cell.Biol.,2003,15,539‐546. [60]Doores,K.J.etal.,Proc.Natl.Acad.Sci.USA,2010,107,17107‐17112. [61]McDonald,D.;Wu,L.;Bohks,S.M.;KewalRamani,V.N.;Unutmaz,D.;Hope,T.J. Science,2003,300,1295–1297. [62]vanKooyk,Y.;Engering,A.;Lekkerkerker,A.N.;Ludwig,I.S.;Geijtenbeek,T.B.H.; Curr.Opin.Immunol.,2004,16,488–493. [63]Khoo,U.S.;Chan,K.Y.;Chan,V.S.;Lin,C.L.;J.Mol.Med.,2008,86,861‐874. [64]a)Sattin,S.etal.,ACSChem.Biol.,2010,5,301‐312;b)Requena,M.etal., Immunology,2008,123,508‐518. [65]Alen,M.M.F.;Kaptein,S.J.F.;DeBurghgraeve,T.;Balzarini,J.;Neyts,J.;Schols, D.;Virology,2009,387,67‐75. [66]a)Dai,B.;Yang,L.;Yang,H.;Hu,B.;Baltimore,D.;Wang,P.;Proc.Natl.Acad.Sci. USA,2009,106,20382‐20387;b)Yang,L.etal.,Nat.Biotechnol.,2008,26,326‐334. [67]Johannssen,T;Lepenies,B.;TrendsBiotechnol.,2017,35,334‐346. [68]Teillet,F.;Dublet,B.;Andrieu,J.P.;Gaboriaud,C.;Arland,G.J.;Thielens,N.M.;J. Immunol.,2005,174,2870‐2877. [69]Rini,J.M.;Biophys.Biomol.Struct.,1995,24,551‐577. [70]Iborra,S.;Sancho,D.;Immunobiology,2014,220,175‐184. [71]a)Dam,T.K.;Roy,R.;Page,D.;Brewer,C.F.;Biochemistry,2002,41,1359–1363; b)Mann,D.A.;Kanai,M.;Maly,D.J.;Kiessling,L.L.;J.Am.Chem.Soc.,1998,120, 10575‐10582. [72]Mitchell,D.A.;Fadden,A.J.;Drickamer,K.;J.Biol.Chem.,2001,276,28939‐ 28945. [73]Appelmelk,B.J.;vanDie,I.;vanVliet,S.J.;Vandenbroucke‐Grauls,C.M.; Geijtenbeek,T.B.;vanKooyk,Y.;J.Immunol.,2003,170,1635‐1639. [74]Rojo,J.;Delgado,R.;J.Antimicrob.Chemother.,2004,54,579‐581.
Chapter8.References 380 [75]Cecioni,S.;Imberty,A.;Vidal,S.;Chem.Rev.,2015,115,525‐561. [76]a)Chabre,Y.M.;Roy,R.;Chem.Soc.Rev.,2013,42,4657‐4708;b)Pieters,R.J.; Org.Biomol.Chem.,2009,7,2013‐2025;c)Deniaud,D.;Julienne,K.;Gouin,S.G.; Org.Biomol.Chem.,2011,9,966‐979;d)Jayaraman,N.;Chem.Soc.Rev.,2009,38, 3463‐3483;e)Lahmann,M.;Top.Curr.Chem.,2009,288,17‐65;f)Lundquist,J.J.; Toone,E.J.;Chem.Rev.;2002,102,555‐578. [77]Gestwicki,J.E.;Cairo,C.W.;Strong,L.E.;Oetjen,K.A.;Kiessling,L.L.;J.Am.Chem. Soc.,2002,124,14922‐14933. [78]a)Kiessling,L.L.;Gestwicki,J.E.;Strong,L.E.;Curr.Opin.Chem.Biol.,2000,4, 696‐703;b)Mammen,M.;Choi,S.‐K.;Whitesides,G.M.;Angew.Chem.,Int.Ed., 1998,37,2754‐2794. [79]Wittmann,V.;Pieters,R.J.;Chem.Soc.Rev.,2013,42,4492‐4503. [80]a)Kiessling,L.L.;Gestwicki,J.E.;Strong,L.E.;Angew.Chem.Int.Ed.,2006,45, 2348‐2368;b)Bertozzi,C.R.;Kiessling,L.L.;Science,2001,291,2357‐2364;c) Gestwicki,J.E.;Kiessling,L.L.;Nature,2002,415,81‐84;d)Heldin,C.H.;Cell,1995, 80,213‐223 [81]a)Guiard,J.;Fiege,B.;Kitov,P.I.;Peters,T.;Bundle,D.R.;Chem.Eur.J.,2011, 17,7438‐7441;b)Rademacher,C.etal.,Chem.Eur.J.,2011,17,7442‐7453. [82]Dam,T.K.;Gerken,T.A.;Brewer,C.F.;Biochemistry,2009,48,3822‐3827. [83]Rao,J.H.;Lahiri,J.;Isaacs,L.;Weis,R.M.;Whitesides,G.M.;Science,1998,280, 708‐711. [84]Bernardi,A.etal.,ChemSocRev.,2013,42,4709‐4727. [85]Spain,S.G.;Gibson,M.I.;Cameron,N.R.;J.Polym.Sci.PartA:Polym.Chem., 2007,452059–2072. [86]a)delaFuente,J.M.;Penades,S.;Biochim.Biophys.Acta,2006,1760,636–651. b)delaFuente,J.M.etal.,Angew.Chem.Int.Ed.,2001,41,1554–1557. [87]Bianco,A.;Kostarelos,K.;Partidos,C.D.;Prato,M.;Chem.Commun.,2005,571– 577.
Chapter8.References 381 [88]Bosi,S.;DaRos,T.;Spalluto,G.;Prato,M.;Eur.J.Med.Chem.,2003,38913– 923. [89]Patel,A.;Lindhorst,T.B.;Carbohydr.Res.,2006,341,1657–1668. [90]Tomalia,D.A.etal.,Polym.J.,1985,17,117–132. [91]Flory,P.J.;J.Am.Chem.Soc.,1941,63,3091–3096. [92]Buhleier,E.;Wehner,W.;Vögtle,F.;Synthesis,1978,155–158. [93]Dendrimer‐BasedDrugDeliverySystems:FromTheorytoPractice,Eds.Cheng, Y.;Tomalia,D.A.,Wiley,2012,ISBN:978‐0‐470‐46005‐4. [94]a)DendrimersandDendrons:Concepts,Syntheses,Applications,Eds.Newcome, G.R.;Moorefield,C.N.;Vögtle,F.,Wiley,2001,ISBN:3527299971/9783527299973: b)Newcome,G.R.;Zhong‐qi,Y.;Baker,G.R.;Gupta,V.K.;J.Org.Chem.,1985,50, 2003–2004. [95]a)Müller,C.;Despras,G.;Lindhorst,T.K.;Chem.Soc.Rev.,2016,45,3275‐3302; b)Carbohydratechemistry,ChemicalandBiologicalApproachesvol41,EdsRauter, A.P.;Lindhorst,T.K.;Albertina,C.;Queneau,Y.;TheRoyalSocietyofChemistry,2015, Chapter1,Sattin,S.;Bernardi,A.pg1‐25andL’Haridon,L.;Mallet,J.M.pg257‐269. c)Inoue,K.;Prog.Polym.Sci.,2000,25,453–571;b)Vögtle,F.;Gestermann,S.; Hesse,R.;Schwierz,H.;Windisch,B.;Prog.Polym.Sci.,2000,25,987–1041;d)Astruc, D.;Boisselier,E.;Ornelas,C.;Chem.Rev.,2010,110,1857–1959. [96]FrontiersofNanoscience,Vol4;Chapter5,Sanchez‐Navarro,M.;Rojo,J.;2012, 143‐156. [97]Roy,R.;Shiao.T.C.;Chem.Soc.Rev.,2015,44,3924. [98]a)Appelhans,D.etal.,Chem.Soc.Rev.,2015,44,3968;b)Reina,J.J.etal.,Braz. J.Pharm.Sci.,2015,49,109‐124;c)Malkoch,M.etal.,Macromolecules,2005,38, 3663–3678;d)Wu,P.;Chem.Commun.,2005,5775–5777. [99]Roy,R.;Zanini,D.;Meunier,S.J.;Romanowska,A.;Chem.Comm.,1993,24, 1869‐1872.
Chapter8.References 382 [100]Wang,S.K.;Liang,P.H.;Astronomo,R.D.;Hsu,T.L.;Hsieh,S.L.;Burton,D.R.; Wong,C.H.;Proc.Natl.Acad.Sci.USA,2008,105,3690‐3695. [101]Branderhorst,H.M.;Ruijtenbeek,R.;Liskamp,R.M.J.;Pieters,R.J.; ChemBioChem,2008,9,1836–1844. [102]Dehuyser,L.;Schaeffer,E.;Chaloin,O.;Mueller,C.G.;Baati,R.;Wagner,A.; Bioconjug.Chem.,2012,23,1731–1739. [103]Lasala,F.;Arce,E.;Otero,J.R.;Rojo,J.;Delgado,R.;Antimicrob.Agents Chemother.,2003,47,3970‐3972. [104]Tabarani,G.;Reina,J.J.;Ebel,C.;Vives,C.;Lortat‐Jacob,H.;Rojo,J.;Fieschi,F.; FEBSLett.,2006,580,2402‐2408. [105]a)Sutkeviciute,I.etal.,ACSChem.Biol.,2014,9,1377−1385;b)Luczkowiak,J. etal.,Bioconjug.Chem.,2011,22,1354–1365. [106]a)Tiwari,V.K.;Mishra,B.B.;Mishra,K.B.;Mishra,N.;Singh,A.S.;Chen,X.; Chem.Soc.Rev.,2016,116,3086‐3240;b)Tabacaru,A.;Furdui,B.;Ghinea,I.O.; Cârâc,G.;Dinicâ,R.M.;Inorg.Chim.Acta,2016,455,329‐349;c)Singh,M.S.; Chowdhury,S.;Koley,S.;Tetrahedron,2016,72,5257‐5283;d)Liang,L.;Astruc,D.; Coord.Chem.Rev.,2011,255,2933–2945. [107]Varga,N.etal.,Biomaterials,2014,35,4175‐4184. [108]Ragoussi,M.E.;Casado,S.;Ribeiro‐Viana,R.;delaTorre,G.;Rojo,J.;Torres,T.; Chem.Sci.,2013,4,4035‐4041. [109]Luczkowiak,J.etal.,Biomacromolecules,2013,14,431‐437. [110]LeGuével,X.etal.,ACSAppl.Mater.Interfaces,2015,7,20945−20956. [111]Ribeiro‐Viana,R;Garcia‐Vallejo,J.J.;Collado,D.;Perez‐Inestrosa,E.;Bloem,K.; vanKooyk,Y.;Rojo,J.;Biomacromolecules,2012,13,3209−3219. [112]deAlmeidaGonçalves,C.;CunhaFigueiredo,R.;Giani,A.;Collado,D.;Pérez‐ Inestrosa,E.;Rojo,J.;CunhaFigueredo,C.;J.Appl.Psychol.,2018,1‐11. [113]Ribeiro‐Viana,R.;Sánchez‐Navarro,M.;Luczkowiak,J.;Koeppe,J.R.;Delgado, R.;Rojo,R.;Davis,B.G.;Nat.Commun.,2012,3,1303‐1310.
Chapter8.References 383 [115]Hyun,J.Y.,Pai,J.&Shin,I.;Acc.Chem.Res.,2017,50,1069–1078. [116]Park,S.;Lee,M.R.;Shin,I.;Chem.Commun.,2008,4389–4399. [117]a)Liang,P.H.,Wang,S.K.,Wong,C.H.;J.Am.Chem.Soc.,2007,129,11177–84; b)PareraPera,N.etal.,ChemBioChem,2010,11,1896–1904. [118]a)Blixt,O.;Allin,K.;Bohorov,O.;Liu,X.F.;Andersson‐Sand,H.;Hoffmann,J.; Razi,N.;Glycoconj.J.,2008,25,59−68;b)Pedersen,J.W.etal.,J.Biol.Chem.,2011, 286,32684–32696;c)Serna,S.,Yan,S.,Martin‐Lomas,M.;Wilson,I.B.H.,Reichardt, N.‐C.;J.Am.Chem.Soc.,2011,133,16495–16502;c)Ban,L.etal.,Nat.Chem.Biol., 2012,8,769−773. [119]a)Rillahan,C.D.;Schwartz,E.;McBride,R.;Fokin,V.V.;Paulson,J.C.;Angew. Chem.Int.Ed.,2012,51,11014‐11018;b)Rillahan,C.D.;Schwartz,E.;Rademacher, C.;McBride,R.;Rangarajan,J.;Fokin,V.V.;Paulson,J.C.;ACSChem.Biol.,2013,8, 1417‐1422;c)Hyun,J.Y.etal.,ChemBioChem,2017,18,1077‐1082. [120]a)Tang,P.W.;Gooi,H.C.;Hardy,M.;Lee,Y.C.;Feizi,T.;Biochem.Biophys.Res. Commun.,1985,132,474–480;b)Liang,R.etal.,Science,1996,274,1520–1522;c) Roy,R.;Curr.Opin.Struct.Biol.,1996,6,692–702. [121]Park,S.,Gildersleeve,J.C.,Blixt,O.;Shin,I.;Chem.Soc.Rev.,2013,42,1441– 1466. [122]Blixt,O.etal.,Proc.Natl.Acad.Sci.USA.;2004,101,17033–17038. [123]Drickamer,K.;Taylor,M.E.GenomeBiol.,2002,3,1034.1–1034.4 [124]Katrik,J.;Juraj,S.;Gemeiner,P.;Kozar,T.;Tkac,J.;Med.Res.Rev.,2010,30, 394–418. [125]Feizi,T.;Chai,W.;Nat.Rev.Mol.CellBiol.,2004,5,582–588. [126]Feizi,T.;Fazio,F.;Chai,W.;Wong,C.H.;Curr.Opin.Struct.Biol.,2003,13,637‐ 645. [127]Ratner,D.M.etal.,ChemBioChem,2004,5,379–382. [128]Geissner,A.;Seeberger,P.H.;Annu.Rev.Anal.Chem.,2016,9,223–247.