TopCurrChem(2005)258:1–38 DOI10.1007/b137480 © Springer-VerlagBerlinHeidelberg2005 Publishedonline:8August2005 ChlorinsProgrammedforSelf-Assembly TeodorSilviuBalaban1 ((cid:1))·HitoshiTamiaki2 ·AlfredR.Holzwarth3 1InstituteforNanotechnology,ForschungszentrumKarlsruhe,Postfach3640, 76021Karlsruhe,Germany [email protected] 2DepartmentofBioscienceandBiotechnology,FacultyofScienceandEngineering, RitsumeikanUniversity,Kusatsu,525-8577Shiga,Japan [email protected] 3Max-Planck-InstitutfürBioanorganischeChemie(FormerMax-Planck-Institutfür Strahlenchemie),Stiftstr.34–36,Postfach101365, 45413Mülheima.d.Ruhr,Germany [email protected] 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1 ChlorinsastheMostAbundantNaturalPhotosyntheticChromophores . . 2 2 NaturalChlorin-ProteinComplexes . . . . . . . . . . . . . . . . . . . . . . 6 2.1 Self-Assembly:aProgramEncodedwithintheStructureoftheTectons . . 7 2.2 SupramolecularChemistryofChlorinsbyMetalLigation . . . . . . . . . . 8 2.3 SupramolecularChemistryofChlorinsbySelf-AssemblyofProtomers withinBacterialLight-HarvestingSystems . . . . . . . . . . . . . . . . . . 11 3 NaturalSelf-AssemblingChlorins:theChlorosomalBacteriochlorophylls. 13 4 SyntheticSelf-AssemblingPorphyrinsandChlorins . . . . . . . . . . . . . 18 4.1 SupramolecularChemistrybyHydrogenBondingandπ–π Interactions . . 18 4.2 Self-AssemblyofSyntheticChlorophyllsUsingHydrogenBonding, MetalCoordinationandπ–π InteractionsasLight-HarvestingAntenna ModelsofPhotosyntheticGreenBacteria . . . . . . . . . . . . . . . . . . . 20 4.3 SyntheticSelf-AssemblingChlorinsandPorphyrinsasMimics oftheChlorosomalBacteriochlorophylls . . . . . . . . . . . . . . . . . . . 28 5 SpectroscopicandFunctionalPropertiesofChlorinDyeSelf-Assemblies: ExcitonicCouplingandOpticalPropertiesofChlorosomalAggregates . . 32 6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Abstract Thesupramolecularchemistryofchlorinswhicharethemostabundantphoto- synthetic pigmentsisreviewed.Inchlorophyll-protein complexes,ligationofthecentral magnesiumatomcanoccurintwodiastereomericconfigurations.Light-harvestingcom- plexes of purple bacteria are formed by the self-assembly of short polypeptides which bindbacteriochlorophyllsintocircularstructures.Thelight-harvestingorganelleofgreen photosyntheticbacteria,theso-called“chlorosome”,isthemostefficientnaturalantenna systemandisformedbyself-assemblyofbacteriochlorophyllsc,dorewithoutthehelp of a protein scaffold. Semisynthetic and fully synthetic mimics of these self-assembling 2 T.S.Balabanetal. bacteriochlorophylls have been prepared and their self-assemblies havebeen studied in detail in view of artificial light-harvesting systems. From a single crystal X-ray diffrac- tionanalysis,onecouldputintoevidencehierarchicsupramolecularinteractionswithin such self-assembling systems. Interestingly, hydrogen bonding which allpresent models of bacteriochlorophyll self-assemblies contain as one of the important supramolecular interactionsisabsentinthefullysyntheticmimics. Keywords Self-assembly,chlorophyll,bacteriochlorophyll,antennacomplex,chlorosome, biomimeticmodels,porphyrinoids. 1 Introduction 1.1 ChlorinsastheMostAbundantNaturalPhotosyntheticChromophores Natureusestetrapyrrolessuchaschlorophyllsandbacteriochlorophyllsasthe mainchromophoresforlight-harvestinginphotosyntheticorganisms.While porphyrins have a fully conjugated 26 π electron system, in chlorins one of thepyrrolicdoublebondsisreducedandinbacteriochlorinstwosuchdouble bonds are reduced (Fig.1). In bacteriochlorins, the basic tetrapyrroleof the chlorophyllous ancestors, the single bonds are in opposite and not adjacent pyrrolerings.Corroleslackthe20-mesocarbonatomwhilephthalocyanines areveryrobustfullysyntheticpigmentswhichhavebenzo-annulatedpyrrole ringsandnitrogenbridgesinsteadofthefourmeso-methineunits. Chlorophylls(Chls)arechlorinswhichcarryanadditionalfive-membered ringhavingthusaphorbinskeletonandareusuallyencounteredincyanobac- Fig.1 Basiccyclictetrapyrrolesshownherewiththeusualnumberingsystem ChlorinsProgrammedforSelf-Assembly 3 teria, redalgae, green algae, and higher plants. Bacteriochlorophylls(BChls) occur in photosynthetic bacteria and also possess the annulated five- membered ring. While BChls typically derive from the bacteriochlorin structure (such as BChl a, Fig.2) some “bacteriochlorophylls” actually have a chlorin chemical and electronic structure (c.f. Fig.2). These chlorin- based “bacteriochlorophylls”—due to the fact that they are present in some photosynthetic bacteria—received their trivial name before their actual chemical structure was known. This chapter focuses on the properties of special (bacterio)-chlorins which have been endowed for supramolecular self-organization. Supramolecular chemistry or the “chemistry beyond the molecule”[1]iseffectedvianon-covalentinteractionssuchasmetal-ligation, hydrogen bonding, π-stacking and hydrophobic or dispersive interactions. All these can come into play with chlorins and often their combinations act cooperatively. In a supramolecular system the non-covalently bound assemblies have propertiesthat areoftendrasticallydifferent fromthoseoftheir monomeric constituents. Thus, ensemble characteristics are dominant and novel func- tions emerge. Since a chlorin molecule with its peripheral substituents is alittleover1nmindiameter, thetermfunctionalnanostructureisappropri- atefortheirsupramolecularassemblies. Chls and BChls are typically found as light-harvesting pigments in the membrane-bound antennasystems ofphotosyntheticorganisms[2]. Besides these cyclic tetrapyrroles,carotenoidsarealso encountered in mostantenna systems.Somespecialphotosyntheticorganismscontain,however,alsoextra- membraneous antenna systems which make use of different chromophores. These are the phycobilisomes of cyanobacteria and red algae which con- tain open chain tetrapyrroles as pigments, the so-called phycobilins which are covalently bound to proteins. The other notable exception are the so- called “chlorosomes” of the green bacteria, which are extra-membraneous antennasystemscontainingBChlsc,d,ore.Thephotosyntheticantennasys- temshavebeenoptimizedbyevolutionduringthepast2.6billionyears,after cyanobacteriaandeucaryotesevolvedfromthearchaebacteria[3].Cyanobac- teria were the first organisms capable of oxygenic photosynthesis and they evolved into the photosynthetic eukaryotes, a process which eventually led to the development of the higher plant kingdom. Light and oxygen can be extremely noxious to cells if the long-lived triplet excited states of chro- mophoresareallowedtogenerate singlet oxygen (O ∆1).Thisproblemwas 2 g solved during evolution by the incorporation of carotenoids which are able to efficiently quench both B(Chl) triplet states as well as singlet oxygen by thermal deactivation. The association of carotenoids with Chls is also ben- eficial for light-harvesting since carotenoids absorb well between 450 and 550nm, in the so-called Chl absorption gap (c.f. spectra in Fig.3). A third roleofcarotenoidsisprobablystructural:duetotheirextendedandrigidcon- formation they help in the assembly of chlorophyll-protein complexes (CP) 4 T.S.Balabanetal. ChlorinsProgrammedforSelf-Assembly 5 (cid:1) Fig.2 Naturalchromophoresinvolvedinlight-harvestingantennae.Phytolisthefattyalco- holexclusivelyesterifyingtheChlsinhigherplantsandotheroxygen-evolvingorganisms whilefarnesolisthemostabundantfortheBChls.AlsoencounteredinBChlsarestearol, cetol,phytol,geranyl-geraniol,andotherfattyalcohols.TheBChlsc,d,ande,oftheso- called“greenbacteria”whichareactuallyChlsaccordingtotheirelectronicstructure,occur usuallyashomologmixtureswithdifferentsidechainsinthe7,8,and12positions.The R8 substituentofBChlscanbeeithermethyl,ethyl,propylorisobutylwhiletheR12 sub- stituentcanbemethylorethyl.TheseBChlsalsocontainanadditionalstereocentreinthe C31positionandusuallyappearinmostgreenbacteriaasamixtureofepimers Fig.3 AbsorptionspectraofsomeofthechromophoresfromFigs.1and2.Upperpart:some naturalchromophores.Pheoastandsforpheophytina,thefreebaseofChlaafterreplace- mentofthemagnesiumionbytwoprotons;Lowerpart:somesyntheticchromophores:dark greentrace–nickeltetrasulfonatedphthalocyanine(PcS4)dissolvedinawaterDMSOmix- ture(notetheshouldersat640and600nmduetodimersandH-aggregates,respectively); magentatrace–meso-tetratolyl-porphyrin;cyantrace–zinctetratolyl-porphyrin.Notethe sharp420nm(Soret)bandsoftheporphyrinsandtheirverylowvisibleabsorptions(the Qbands)incomparisontoChlsandphthalocyanines. 6 T.S.Balabanetal. conferringrigidityandmechanicalstability.Carotenoidlessmutantsoftenas- semblemorelabileandphotochemicallyunstableantennasystems. Light-harvesting is the primary event in photosynthesis where special- ized chromophores, typically organized as pigment-protein complexes, ab- sorb parts of the solar radiation and become excited into their singlet ex- cited states. Excitation energy is then rapidly transferred among such chro- mophoresonatimescalefromhundredsoffemtosecondstotensofpicosec- onds in a partially directed random walk process and is eventually trapped withintheso-calledreactioncentres,wherephotoinduced chargeseparation occurs.Afterseveralsuccessiveelectrontransfersteps,theholeandtheelec- tron become separated on opposite sides of the photosynthetic membrane. Thiselectrochemicalpotentialisusedtopumpprotonsacrossthemembrane, whichultimatelydrivethesynthesisofATP.Inoxygenicphotosynthesiscon- comitantly the reductant NADPH is produced and these two compounds servetheorganismsasfuelandredoxequivalents,respectively,allowingthem toperformendergonicbiochemicaltransformations. Apart from cellulose, Chls are among the most abundant organic com- poundsinthebiosphereandarebeingcontinuouslysynthesized,degradedand recycled. Togetherwithcarotenoidsthephotosyntheticpigmentsaccountfor about10%ofthetotalbiomass.AsimplecalculationshowsthatifalltheChls andcarotenoidsproducedduringoneyearbytheSouthAmericanContinent were shipped by 100m-longoiltankers eachpresumed tocarry1000tonsof pigments,thenonewouldneedaconvoywhoselengthwouldspantheAtlantic oceanfromthesoutherntipofArgentinatoLondon(15000km).Thisdoesnot eventakeintoaccountthemarinealgaeandcyanobacteriawhich,accordingto remotesensingofchlorophyllfluorescence,canfurnishover10mgChla/m3 ofseawaterdowntoadepthofupto100m. 2 NaturalChlorin-ProteinComplexes Due to their molecular architecture, chlorins in general, but especially the naturallyoccurring(B)Chls c,d,andecaneasilyfunctionasbuilding blocks forsupramolecular interactions.Ahierarchyofseveralnon-covalentinterac- tionsareusedtoarrangethesebuildingblocksintodefinedarchitectures.The strongest such non-covalent bonding is metal ligation and metallo-chlorins, as well as other metallated tetrapyrroles possess a very rich coordination chemistry[4]. Hydrogenbondingisthenextstrongestsupramolecular interactioninthe hierarchy. In cases where multiple hydrogen bonds come into play in a co- operative manner in a supramolecular complex, very tight and directional binding can be effected. All (B)Chls carry in the fifth ring the 13-carbonyl group which can act as an acceptor group for hydrogen bonding and most ChlorinsProgrammedforSelf-Assembly 7 B(Chls), exceptforBChlc,d,ande,alsopossessamethoxycarbonylgroupin the 132 position whose carbonyl group is often involved in hydrogen bond- ing. In the ethyl chlorophyllidea dihydrate crystalstructure [5,6] one water molecule coordinatesthecentralmagnesium atomwhileasecondstructural water molecule is doubly hydrogen bonded between the first water and the 132-methoxycarbonylgroup.InBChlaorinChlbadditionalcarbonylgroups inthe31or71positions,respectively,canhelpinpositioningthesechlorinsin supramolecularcomplexesbyengaginginhydrogenbonding. Theextendedconjugatedmacrocyclesofchlorinsandporphyrinsarealso ideal for forming π–π interactions, another element in the hierarchy of the supramolecular interactions. The resulting strong dipole-dipole interactions are responsible forexcitonic couplingbetween groupsofchromophores(see below)whichforexampleplaysanimportantroleforengineeringthe“special pairs”of(B)Chlswhichareatthecoreofthephotosyntheticreactioncentres. Duetotheirspecialopticalandredoxpropertiesthesespecialpairsfunction aselectrondonors.Asimple but usefuldescriptionforπ–π interactions has beengivenbyHunterandSanders[7]. Finally, another important interaction which can account for a high sta- bilityofchlorin-containingsupramolecularcomplexeswithinphotosynthetic membranesisthehydrophobicinteraction.Longchainfattyalcoholsesterify the 17-propionic acid residue of all (B)Chls. These large residues are highly flexible and can thus adapt to occupy voids within hydrophobic pockets of proteinmatrices,ormayhelptosolvate(B)Chlsinnon-polarsolvents. An often ignored but highly important aspect for the self-organization of chlorins and related macrocyclic compounds is the cooperativity of the above-mentioned supramolecular interactions [8]. Positive cooperativity leads to thermodynamic stabilization of the supramolecular assemblies be- yondthemeresumoftheindividualnon-covalentbondingcontributions. 2.1 Self-Assembly:aProgramEncodedwithintheStructureoftheTectons When amoleculehasfunctionalgroupswhichallowittointeract withpart- ner molecules which may be of the same kind or different, and when the physicalconditionswhichinclude temperature, medium polarity,absence of inhibitors,etc.aresuchthatnon-covalentbondscanbeformed,self-assembly occurs. The algorithm which governs the process dictating the architecture andthusultimatelythefunctionofthefinalnanostructureisencodedwithin the molecular structure of the “bricks” or tectons [1]1. Viruses or the cor- rectpositioningofnucleotideswithinnucleiacidstrandsareexampleswhere Nature uses with perfection self-assembly mechanisms. Misplaced compo- nents usually have weaker binding constants such that under equilibrium 1tectonisderivedfromtheGreekτεκτωνmeaning“builder”. 8 T.S.Balabanetal. conditions they may be expelled from the ordered structures. Thus a repair mechanismoperateswhichensuresthatintheendacorrectlyperformedas- semblyprocessleadstothepre-programmednanostructure.Forthisefficient self-assembly to occur there must exist a very fine balance between the en- tropyand enthalpy terms which is dictated by the reaction conditions. With respect to the monomeric chromophores self-assembly is usually accompa- nied by an entropy loss. However, this lossis over-compensated typically by the desolvation of the tectons and the entropy gain of the solvent. Often the entropytermisthecontrollingfactorinthethermodynamicsofformationof themoststablenanostructure(s). 2.2 SupramolecularChemistryofChlorinsbyMetalLigation The central magnesium atom within chlorins provides an anchoring point viametalligation.WithinChl-proteincomplexes(CP),histidineresiduesare by far the most common ligands to the central Mg of B(Chls) but other amino acids like for example tyrosines, nitrogen atoms from glutamines or asparagines or even sulphur atoms from methionines may also take up that role. The second most frequent ligand is water. The respective Chls are typ- ically bound within the protein matrix by additional weak interactions, like forexample byhydrogenbonding tothemagnesium-bound water molecule, hydrogen bonding to the 13-carbonyl group at ring V, or for Chl b in the 7-position. Oneaspectwhichhasbeenneglectedsofarinthebiophysical/biochemical communityistheimportanceofthediastereotopicarrangementinwhichthe Chls are ligated within proteins [9–11]. Due to the presence of one or more chiralcarbonatomsin(B)Chlsandthenon-planarityofthemacrocycle,there existtwodiastereotopic configurationswhenthemagnesiumatomisligated, thus becoming five-coordinated: one configuration has the ligand above the tetrapyrrolicplaneandtheotherbelowthemacrocycle.Themetalcentrethus becomes anadditionalstereocentre andthetwodiastereoisomers must have different chemical and electronic properties, such as different absorption or emission wavelengths, radiative lifetimes, circular dichroism, NMR spectra, chromatographic retention times, etc. Figure 4 shows the formulae of these diastereoisomers.Providedthatthetimescaleoftheobservationmethodem- ployedisshorterthantheaverageligationlifetime,thediastereomersappear tobedifferent. InordertocomplywithcurrentIUPACnomenclaturerulesformetallated tetrapyrroles(ase.g.inhemes orVitaminB derivatives), theconfiguration 12 having the fifth metal ligand below the tetrapyrrolic macrocycle, which is numbered in a clockwise fashion is denoted as α, while the β configuration has the fifth ligand above the macrocycle. This nomenclature complies also with the one proposed by Sharpless for the direction of attack of an asym- ChlorinsProgrammedforSelf-Assembly 9 Fig.4 BChlawithchiralatomsindicatedbyasterisks.AtrighttheIUPACnumberingis given.Notethatintheαconfigurationtheligandtothemetalisontheoppositesideof themacrocycletothe17propionicacidresidue,whileintheβconfigurationtheyareon thesameside.Asamnemonicrule,theβ-configurationcanbederivedbyaleft-handrule where the thumb points to the ligand when the fingers are pointing in a clockwise (or nomenclature-wise)fashion metric epoxidation reagent to an olefin plane [12,13]. Furthermore, as with sugars or steroids, the α-substituent is below the molecular plane and the β-substituentisabovethisplane. When the metal ligand is a water molecule, or a group which binds only weakly(activationenergywithinkT),oriftheligandispresentinthesolution inrelativelylargeconcentrations,rapidinterconversionmayoccur.Whenim- idazolecoordinatesChl atheinterconversion isslowontheNMRtimescale and a splitting of signals is observed [14]. Within a protein matrix, the life- time of aparticular diastereomeric configurationcanbe considered infinite, sincea(B)Chlmoleculecannotbede-ligated,rotatedalongtheplane,andre- ligated by the same ligand from the other side of the macrocycle. Figure 5 shows two Chl a molecules with histidine ligands within the Photosystem I (PS I) core complex whose structure has been solved by X-ray analysis to 2.5˚Aresolution[15]. Sequencecomparisonshowedthatthediastereotopicnatureofthebinding sitesof(B)Chlswithinproteincomplexeshavebeenstrictlyconservedduring evolutioninthesofarknownantennacomplexes[9].Thusamongrelated,but even phyllogenetically quite distant organisms an α-bonded (B)Chl is never turnedintoaβ-ligatedoneorviceversa. Both from statistics [9] and from semi-empirical calculations [10,11] it follows that the α-coordination is by about 1kcal/mol more stable than the β-coordination.InPSIoutofthe96Chlamoleculesonly14areβ.Mostre- markably, almost all are part of the inner circle of the core antenna system, immediately surrounding the reaction centre. This might suggest that there existsanenergyfunnelfromtheouterantennaChlswhichareallα,totheβ ones.Figure6showsthislocationoftheβ-ChlsinthePSIcoreantenna. 10 T.S.Balabanetal. Fig.5 Left:Exampleofa“normal”α-chla;Right:aβ-Chlaasencounteredinthecoreof PSI[9,15] Fig.6 Locationoftheβchlorophylls(shownwiththickerlines)aroundtheelectrontrans- ferchainchlorophylls(shownwiththickcyanbonds)ofthereactioncentre.Alltheother chlorophyllsshownwiththinnerlinesareα-coordinated. Adaptedwithpermissionfrom Elsevier([9]) InPSItherearetwoβ,β-dimers(indicatedbyarrowsinFig.6)positioned symmetrically aroundthespecialpairP700absorbingaround700nm.Ithas beenspeculated thatthesedimersformtheso-called“redChls”whichactas energy-trappingsitesintheantenna,about1.8nmawayfromP700[16].