Synthesis and Self-Assembly of Novel ABC Miktoarm Star Terpolymers

Synthesis and Self-Assembly of Novel ABC Miktoarm Star Terpolymers DISSERTATION zur Erlangung des akademisches Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) im Fach Chemie an der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften der Universität Bayreuth vorgelegt von Andreas Hanisch Geboren in Kulmbach Bayreuth, 2013 Die vorliegende Arbeit wurde in der Zeit von Juli 2008 bis Januar 2013 in Bayreuth am Lehrstuhl Makromolekulare Chemie II unter Betreuung von Herrn Prof. Dr. Axel H. E. Müller angefertigt. Vollständiger Abdruck der von der Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.). Dissertation eingereicht am: 25.01.2013 Zulassung durch die Promotionskomission: 13.02.2013 Wissenschaftliches Kolloquium: 26.04.2013 Amtierender Dekan: Prof. Dr. Beate Lohnert Prüfungsausschuss: Prof. Dr. Axel H. E. Müller (Erstgutachter) Prof. Dr. Stephan Förster (Zweitgutachter) Prof. Dr. Matthias Karg (Vorsitz) Prof. Dr. Karlheinz Seifert “ Life is like riding a bicycle. To keep your balance you must keep moving. ” Albert Einstein Meiner Familie Greta und Oskar Table of Contents I Table of Contents Summary 1 Zusammenfassung 3 Glossary 7 1 – Introduction 9 1.1 Solution Based Self-Assembly in Polymer Systems 9 1.1.1 General Aspects of Self-Assembly 9 1.1.2 Multicompartment Structures from Ternary Systems 10 1.1.2.1 Linear Triblock Terpolymers and Directed Self-Assembly 12 1.1.2.2 Miktoarm Star Terpolymers 16 1.2 Miktoarm Star Polymers 19 1.2.1 Synthesis 19 1.2.1.1 ABC Miktoarm Star Terpolymers as Model Systems 19 1.2.1.2 Other Miktoarm Star Polymer Systems 26 1.2.2 Self-Assembly in Bulk 27 1.2.3 Application of ABC Miktoarm Star Terpolymers for Functional Materials 29 1.3 Synthetic Strategies in Polymer Science 31 1.3.1 DPE Chemistry in Anionic Polymerization 31 1.3.2 Click Chemistry 32 1.4 Aim of this Thesis 34 1.5 References 35 2 – Overview of the thesis 41 2.1 Modular Synthesis of Miktoarm Star Terpolymers 42 2.2 Counterion-Mediated Hierarchical Self-Assembly of an ABC Miktoarm Star Terpolymer Containing a Poly(N-methyl-2-vinylpyridinium iodide) Segment 45 2.3 Application of the Triiodide-Directed Self-Assembly to other ABC and ABA’ Miktoarm Star Polymers with a Poly(N-methyl-2-vinylpyridinium iodide) Segment 47 2.4 Individual Contributions to Joint Publications 51 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers via a New Alkyne-Substituted Diphenylethylene Derivative 53 3.1 Introduction 54 3.2 Experimental Section 57 3.3 Results and Discussion 60 3.3.1 Synthesis of 1-[(4-(tert-Butyldimethylsilyl)ethynyl)-phenyl]-1-phenylethylene (click-DPE) 60 3.3.2 Synthesis of Alkyne Mid-Functionalized Diblock Copolymers 61 3.3.3 Hydrolysis of the Alkyne Mid-Functionalized Diblock Copolymers 64 3.3.4 Synthesis of ω-Azido Homopolymers 66 Zusammenfassung 4 gregaten umlagern. Aufgrund ihrer Erscheinung in elektronenmikrospopischen Aufnahmen wurde für diese der Begriff „woodlouse“ („Kellerassel“) eingeführt. Die Aggregate sind nicht hohl und bestehen aus PB/PtBMA Lamellen, die über eine teilweise gequollene P2VPq Phase miteinander verbunden und dadurch stabilisiert werden. Die PB/PtBMA Lamellen besitzen dabei eine teilweise entmischte innere Struktur. Dieses Konzept wurde erfolgreich auf zwei weitere Miktoarmstern-Systeme angewandt. Für eine Serie von ABC-Miktoarmstern-Terpolymeren aus PS, PB und P2VPq (µ-BVqS) wurde eine Abhängigkeit der Mizellform vom hydrophilen Anteil beobachtet; analog zu Diblock-Copolymeren. Einerseits wurden für lange P2VPq-Blöcke Aggregate aus gestapelten Lamellen/Scheiben erhalten, die aus Kugelmizellen als Grundbausteinen entstanden sind; andererseits wurde für kurze P2VPq Blöcke die Ausbildung multilamellarer Vesikel durch Fusion unilamellarer Vesikel beobachtet. Anders als durch die Polymerarchitektur erwartet, konnte für die Vesikelwand keine Kompartmentalisierung beobachtet werden, sondern es wird eine lamellare Zusammensetzung mit einem PB Kern angenommen, die durch eine dünne PS-Schicht vor der P2VPq-Korona geschützt wird. Für einen der µ-BVqS-Miktoarmsterne wurde erfolgreich eine Nanohybridstruktur mit Goldnanopartikeln gebildet. Für ein zweites Polymersystem wurde das elastische PB-Segment durch einen weiteren glasartigen PS-Block mit unterschiedlicher Länge ersetzt (µ-SVqS‘). Obwohl mit Iodid als Gegenion Vesikel erhalten wurden, bildeten sich bei der Triiodid-induzierten Überstrukturbildung anisotrope Aggregate aus deformierten Vesikeln. Die Abwesenheit von multilamellaren Vesikeln wird dabei auf die reduzierte Dynamik der kernbildenden, glasartigen PS-Blöcke zurückgeführt. Ähnlich zu den „woodlouse“ Partikeln von µ-BVqT, wurden somit für µ-SVqS‘ längliche Aggregate mit einer lamellaren Struktur erhalten, jedoch über Vesikel als Grundbausteine. Somit ist dieses über das Triiodid-Ion beeinflussbare Konzept der gesteuerten Selbstaggregation nicht nur auf Miktoarmstern Systeme mit einem elastischen Block beschränkt, da die Umordnungsprozesse während des Dialyse stattfinden, wo das anfangs vorhandene organische Lösungsmittel eine ausreichende Mobilität der den Mizellkern bildenden Blöcke zur Folge hat. Zusammenfassung 5 Neben einer neuen Synthesestrategie für Miktoarmstern-Terpolymere, basierend auf einem vielseitigen Alkin-substituierten DPE Derivat, konnte somit die durch das TriiodidIon induzierte Überstrukturbildung als neuartiges Konzept für gesteuerte Selbstanordnungsprozesse in wässrigen Lösungen aufgezeigt werden. 6 Glossary 7 Glossary 2VP 2-vinylpyridine AAO anodic aluminium oxide ATRP atom transfer radical polymerization CoQ10 coenzyme Q10 CRP controlled radical polymerization cryo-TEM cryogenic transmission electron microscopy CuAAC copper-assisted azide-alkyne cycloaddition DNA deoxyribonucleic acid DPE 1,1-diphenylethylene DVB divinylbenzene f w volume fraction of water IPEC interpolyelectrolyte complex MCM multicompartment micelles NRC nitroxide radical coupling SET-LRP single electron transfer living radical polymerization PAA poly(acrylic acid) PB polybutadiene PBLL poly(ε-tert-butyloxycarbonyl-L-lysine) PCEMA poly(2-cinnamoyloxyethyl methacrylate) PDMAEA poly(N,N-dimethylaminoethyl acrylate) PDMAEMA poly(N,N-dimethylaminoethyl methacrylate) PEO poly(ethylene oxide) PEE polyethylethylene PI polyisoprene PISC poly((sulfamate-carboxylate)isoprene) PMA poly(methyl acrylate) PMAA poly(methacrylic acid) PMCL poly(γ-methyl-ε-caprolactone) PnBu poly(n-butyl acrylate) PNiPAAm poly(N-isopropylacrylamide) PS polystyrene PSGA poly(sucinnated glyceryl monomethacrylate) PtBA poly(tert-butyl acrylate) PtBMA poly(tert-butyl methacrylate) PVBFP poly(pentaflurorphenyl 4-vinylbenzyl ether) P2VP poly(2-vinylpyridine) P2VPq poly(N-methyl-2-vinylpyridinium) ROP ring-opening polymerization TEM transmission electron microscopy THF tetrahydrofuran TPPBr triphenylphosphonium bromide ΔG agg. Gibbs free energy of micelle formation ΔH agg. enthalpy of micelle formation ΔS agg. entropy of micelle formation 8 1 – Introduction 9 1 – Introduction 1.1 Solution Based Self-Assembly in Polymer Systems 1.1.1 General Aspects of Self-Assembly Self-assembly describes the well-defined organization of one species or a set of identical molecular building units into highly ordered aggregates by non covalent interactions. Here, the complexity and diversity achieved in nature displays an impressive prototype for such structures at diverse length scales. 1-3 Examples of biological structures of different dimensions, built up with the concept of self-assembly include e.g. DNA, 4 viruses 5 or spider silk. 6 The engineering of natural building units resembles a remarkable pathway for the programmed construction of nanoscale objects in a bottom-up approach. 7 An outstanding example is DNA-origami, which is based on the combination of a long scaffold strand of virus-DNA with small, designed staple strands of adequate base-sequence to obtain directed folding into diverse two-dimensional 8 or three-dimensional structures. 9 Despite the synthetic advances in polymer chemistry, achieving such a distinct complexity with polymer systems has not been reached. Nevertheless, it represents a desirable paradigm for the construction of complex functional structures of different dimension in analogy to systems in nature (Figure 1-1A). 10 In the case of block copolymers as most simplified polymeric system two chemically different blocks are covalently linked together. If the polymer now is exposed to a solvent selective for only one block, the individual molecules have to self-organize to shield the solvophobic block. The gain in free energy (ΔH agg. < 0) for minimizing the unfavorable interaction between the solvent and the insoluble block overcomes the loss of entropy (ΔS agg. > 0), leading to an overall negative Gibbs free energy ΔG agg. . ΔG agg. = ΔH agg. - TΔS agg. < 0 Depending on the fraction of the stabilizing block the polymers assemble into spherical micelles, cylindrical micelles or vesicles (Figure 1-1-B). 11-14 Despite the structural simplicity of these aggregates the tailored synthesis of block copolymers enables constructing functional material. These micellar structures can serve as nanocarriers 15,16 or 1 - Introduction 10 nanoreactors, 17-19 or as scaffold for e.g. hybrid materials. 20-22 Vesicles (or also called polymersomes) resemble the synthetic analogues of liposomes and are therefore of interest for biological applications. 23-25 Even though this thesis deals with the selfassembly in solution it should be mentioned that the same principles also apply for the bulk state, provided that the thermodynamic incompatibility of the two blocks is sufficiently high. The covalent linkage prevents phase separation on the macroscale and therefore forces the polymer to align in nanoscopic structures, i.e. spherical, cylindrical, gyroid or (perforated) lamellar morphologies. 26,27 Figure 1-1. (A) Length scales of molecular self-assembled structures in synthetic and biological systems with increasing complexity. 10 (B) Schematic representation of the structures of AB diblock copolymers in a solvent selective for the B block. Depending on the volume fraction of the soluble block, f B , the polymer self assembles into spherical micelles, cylindrical micelles or vesicles. 11 1.1.2 Multicompartment Structures from Ternary Systems Extending the functionality of block copolymer systems with a third chemically differing block can be accomplished in two manners. The linear attachment leads to triblock terpolymers, whereas by nonlinear conjunction of the third block miktoarm star terpolymers (from the greek word ικτός for mixed) are achieved. 28 The synthesis of the latter will be discussed in chapter 1.2. In contrast to diblock copolymers different blockselective solvents are possible for these ternary systems, which induce either a compartmentalization of the core or the corona. 29 In the following a short overview of possible structures at the example of spherical micelles is given. If linear terpolymers are exposed to a solvent selective for the end-blocks micelles with an either mixed, patchy or Janus-type corona are obtained (Figure 1-2A). 30 However, accessing the region of exclusively Janus-type micelles from ABC triblock terpolymers in endblock-selective solvents 1 – Introduction 11 is challenging and mostly leads to mixtures with patchy micelles. 30,31 Additionally, in solvents selective for the middle and one end-block core-shell-corona structures will be obtained (Figure 1-2B). 32 In contrast to their linear analogues the influence of block sequence is eliminated for miktoarm star terpolymers as a consequence of the polymer architecture. Hence core-shell-corona structures are not possible and for the coronacompartmentalized structures Janus-type micelles are rather unfavored due to the sterical frustration of the two soluble blocks. Figure 1-2. Overview of corona- (A, B) and core-compartmentalized structures (C) obtained from ABC miktoarm star terpolymers and linear ABC terpolymers. The structures depicted are micelles with a mixed, patchy or Janus-type corona (A, from left to right), core-shell-corona micelles (B) for solvents selective for the end-blocks of linear terpolymers and multicompartment or “onion”-like micelles (C, from left to right). (D) shows a schematic illustration of the chain packing for ABC miktoarm star and linear terpolymers to obtain multicompartment micelles. On the other hand, solvents selective for only one block lead to a compartmentalization of the micellar core (Figure 1-2C). Inspired by biological systems, in 1999 Ringsdorf introduced the concept of multicompartment micelles (MCM’s) for synthetic polymer systems. 33 Separated compartments within the core would allow for storage of two different payloads within one micelle, while simultaneously maintaining access to the surrounding medium. When triblock terpolymers self-assemble in solvents selective for one end block, the interfacial energies between each of the individual core-forming blocks and the corona-forming block determine whether micelles with simple concentric compartments (“onion”-like structures, right-hand structure in Figure 1-2C) 34 or micelles 1 - Introduction 12 with non-concentric nano-structured cores (multicompartment micelles, left-hand structure in Figure 1-2C) are obtained. 35 However, attributed to their frustrated architecture, miktoarm star terpolymers are inherently forced to self-assemble in structures with compartmentalized cores upon dissolution in a solvent selective for one block, disregarding different pairs of interfacial energies (Figure 1-2D). The first example for visualization of the individual core-segments of multicompartment micelles and worms was given by Hillmyer, Lodge and co-workers in 2004 for a miktoarm star terpolymer with highly incompatible segments, poly(ethylethylene)-arm-poly(ethylene oxide)-armpoly(perfluoropropylene oxide), µ-EOF. 36,37 In cryo-TEM the perfluorinated segments were in-situ detectable owing to their increased electron density. In contrast to this miktoarm system, for linear terpolymers, besides special chemical composition, interpolyelectrolyte complexation, the use of additives, solvent mixtures or adequate assembly pathways have to be applied to guide the sequentially connected blocks towards multi-compartmentalized structures. In the following two sections I will first give an overview of the directed self-assembly of linear triblock terpolymers into multicompartment structures (1.1.2.1) and then highlight the well-defined morphologies obtained by different miktoarm star terpolymer systems (1.1.2.2). 1.1.2.1 Linear Triblock Terpolymers and Directed Self-Assembly In the field of linear triblock terpolymers Laschewsky et al. presented the first successful in-situ visualization of the multicompartment character in 2005 for a poly(4-methyl-4-(4vinylbenzyl)morpholin-4-ium chloride)-block-polystyrene-block-poly(pentafluorophenyl 4-vinylbenzyl ether) (PVBM-b-PS-b-PVBFP) system by cryo-TEM. 38 After dialysis to water the terpolymer assembled into micelles with a core consisting of spherical domains (~3 nm in diameter) of the pentafluorophenyl group within a hydrocarbon matrix formed by both the polystyrene block and the aromatic moiety of the fluorinated block (Figure 1-3A). Therefore segregation within the PVBFP block took place. They extended this polymer design to other terpolymers based on acrylate-type monomers to obtain spherical compartmentalized structures. 39-41 Besides systems with a solvophilic endblock, also systems with a solvophilic midblock were shown to self-assemble into multicompartment structures. 40-42 Even though simulations of triblock systems in mid- 1 – Introduction 13 block selective solvents predict multicore micelles, where the two chemically different cores are separated by the solvophilic block, 43 the presence of multicompartment micelles here might be attributed to the short lengths of the solvophobic blocks and their chemical nature. Interestingly, instead of using triblock terpolymers, most recently, Langlois and co-workers followed another approach utilizing a statistical terpolymer based on biocompatible poly(3-hydroxyalkanoates). 44 Again, the segregation is induced by the hydrophilic, lipophilic and fluorophilic character of the attached sidechains. Nanoprecipitation in water forced to polymer to form micelles (18 and 79 nm in diameter for two different systems) with distinct fluorinated subdomains in the core. Another possibility to induce phase segregation within the core is based on segments carrying charged functions. As a consequence of the charge neutrality of poly((sulfamate-carboxylate)isoprene) at low pH, poly((sulfamate-carboxylate)isoprene)- block-polystyrene-block-poly(ethylene oxide) (PISC-b-PS-b-PEO) was reported to yield micelles with a “raspberry”-like PISC core with spherical PS domains under acidic conditions. 45 The remaining isoprene units within the PISC domain and the formation of hydrogen-bonds with PEO are supposed to further reduce its water solubility and additionally the transition into micelles with a mixed PISC/PEO corona was demonstrated at increased pH. Schacher et al. showed for zwitterionic polybutadiene-block-poly(N-methyl2-vinylpyridinium)-block-poly(methacrylic acid) (PB-b-P2VPq-b-PMAA) that in aqueous media patchy intermicellar IPEC domains of P2VPq/PMAA are formed on the PB core. 46 Surprinsingly, the non-quaternized and non-hydrolyzed precursor polymer also formed multicompartment micelles with a “sphere on sphere” morphology in acetone as selective solvent for the poly(tert-butyl methacrylate) block. 47 Due to the strong incompatibility between PB and P2VP, the system is assumed to aim surface minimization of the PB/P2VP interface. Apart from the spherical multicompartment micelles discussed so far, Fang et al. demonstrated the hierarchical self-assembly of pre-formed corona-compartmentalized micelles into one-dimensional core-compartmentalized structures upon reducing the solvent quality. 48 Depending on the corona structure (patchy or Janus-type) of the micelles from poly(4-tert-butoxystyrene)-block-polybutadiene-block-poly(tert-butyl methacrylate) with a perfluoro-modified midblock insoluble in dioxane, the dialysis into etha- 1 - Introduction 20 or end-functionalization of homopolymers (1), with the stoichometry of adequate linking reactions with pre-formed polymeric building blocks or initiator molecules (2). Even though in literature different classifications have been used, the synthesis of ABC miktoarm star terpolymers can be divided into four approaches of fundamentally differing chemistries. For the first three types anionic polymerization is utilized for the introduction of special functionalities in defined positions and/or selective reactivity of living anionic chain ends (A-C), whereas the fourth is based on multifunctional core molecules (D). (A) Chlorosilane Method Similar to the synthesis of regular stars by reaction of living anionic polymer chains with chlorosilanes, 85,86 these compounds can be used as linking agents for the construction of ABC miktoarm star terpolymers. Therefore, living anionic chain ends of different reactivity toward chlorine-silicon bonds have to be used to allow for step-wise substitution. Iatrou et al. synthesized a miktoarm star with polyisoprene, polystyrene and polybutadiene segments with trichloromethylsilane as trifunctional linking agent. 87 However, the synthesis can only be conducted in the specific sequence PS > PI > PB, due to the reactivity of the living anion toward the chlorosilane functionality. The less reactive and most sterically hindered polymer anion has to be added first, whereas the less sterically hindered and most reactive polymer anion has to be added at the end to guarantee full conversion. Even with this sequence (Scheme 1-2) the last step is timeconsuming with reaction times of up to 4 weeks and for each step the stoichometry is of outermost importance. Similarly, an ABCD miktoarm star quaterpolymer was synthesized by expanding the system with poly(4-methyl styrene) and using tetrachlorosilane. 88 The application of this approach to other less reactive polymer anions like poly(methyl methacrylate) or poly(2-vinylpyridine) requires a postor pre-modification of the chlorosilane compound, respectively. 89,90 1 – Introduction 21 Scheme 1-2. Synthesis of an ABC miktoarm star terpolymer by the successive reaction of living polymer anions with trichlorosilane as linking agent. (B) Macromonomer Method Apart from the special chemistry of chlorosilanes, diphenylethylene (DPE) and its double-diphenylethylene derivatives represent a powerful class of compounds, as their inability to homopolymerize permits selective functionalization of diblock copolymers. 91 Preformed macromonomers with a terminal DPE functionality can be applied for the sequential living anionic polymerization of diblock copolymers. By sequential addition of the macromonomer after polymerization of the first block, ABC miktoarm star terpolymers are accessible under control of the stoichometry (Scheme 1-3A). For this purpose Quirk et al. used 1,4-bis(1-phenyl-ethenyl)benzene (Scheme 1-3B) to synthesize A 2 B and ABC miktoarm star polymers. 92 When the DPE derivative was used in two-fold excess for the linking reaction with polystyryl-lithium primarily monoaddition took place to generate the polystyrene macromonomer. Similarly, Hückstädt et al. demonstrated 1- (4-bromomethylphenyl)-1-phenylethylene (Scheme 1-3B) to be a suitable termination agent for living anions of polybutadiene 93 and polystyrene 94 . These served as macromonomers for the synthesis of a polybutadiene-arm-polystyrene-arm-poly(methyl methacrylate) miktoarm star terpolymer and series of polystyrene-arm-polybuatdienearm-poly(2-vinylpyridine) miktoarm star terpolymers, respectively. However, also in the case of the bromo-functionalized DPE, formation of dimeric macromonomers can occur under inadequate reaction conditions as a result of Wurtz-analogous side reactions. Besides the use of DPE derivatives as termination agents for the macromonomer synthesis, Quirk and co-workers proved 1-(4-hydroxypropylphenyl)-1-phenylethylene to be a suitable initator for the synthesis of poly(ethylene oxide) macromonomers after deprotonation with triphenylmethylpotassium (Scheme 1-3B). 95 In this way a polystyrene-arm-poly(ethylene oxide)-arm-poly(tert-butyl methacrylate) miktoarm star terpolymer was successfully synthesized. In a similar strategy a polystyrene-armpoly(dimethylsiloxane)-arm-poly(tert-butyl methacrylate) miktoarm star terpolymer was 1 - Introduction 22 accessible by ring opening polymerization of hexamethylcyclotrisiloxane with lithiated para-(dimethylhydroxy)silyl-α-phenylstyrene as initiatior. 96 For the synthesis involving macromonomers the stoichometry of the endcapping reaction has to be considered to allow for simple purification of possible side products. Furthermore, the choice of monomer sequence is limited to the different reactivities of living anionic polymer chains. Scheme 1-3. (A) Schematic representation of the synthesis of ABC miktoarm star terpolymers via sequential anionic polymerization utilizing macromonomers. (B) DPE-derivatives used for the synthesis of macromonomers: 1,4-bis(1-phenyl-ethenyl)benzene, 92 1-(4-bromomethylphenyl)-1-phenylethylene, 93,94 1- (4-hydroxypropylphenyl)-1-phenylethylene deprotonated with trimethylphenylpotassium 95 (from left to right) (C) Mid-Functionalized Diblock Copolymers Another possibility of constructing ABC miktoarm star terpolymers is the synthesis of mid-functional diblock copolymers. The third arm then is attached by adequate reactions with the functional group (Scheme 1-4). Again, DPE chemistry is advantageous within this context, as homopolymerization is excluded and therefore monofunctionalization is guaranteed under appropriate reaction conditions and polymerizations sequences. Exclusively hydroxyl-functionalized DPE’s in their protected form have been used so far. 97,98 Lambert et al. synthesized mid-functional polystyreneblock-poly(ethylene oxide) and polystyrene-block-poly(methyl methacrylate) by sequential anionic polymerization with 1-[4-(2-tert-butyldimethylsiloxy)ethyl]phenyl-1phenylethylene (Scheme 1-4A). After deprotection and deprotonation the hydroxylfunction served as initiator for the anionic ring opening polymerization of ɛcaprolactone 97 or L-lactide 98 in the case of polystyrene-block-poly(ethylene oxide) as diblock copolymer, or of ethylene oxide 98 in the case of polystyrene-block-poly(methyl 1 – Introduction 23 methacrylate). Using a similar DPE derivative, Hirao and co-workers synthesized hydroxy mid-functionalized polystyrene-block-poly(2-(perfluorooctyl)-ethyl methacrylate) diblock copolymers (Scheme 1-4B). 99 However, after transformation of the silyl-protected hydroxyl function into benzyl bromide, living anionic polymers of 2-vinylpyridine and methyl methacrylate were grafted to the diblock copolymers by anionic coupling reactions. Modification of the synthetic strategy and using a dual hydroxy-functionalized DPE further yielded A 3 B, ABC 2 and ABCD miktoarm star polymers. Another elegant way for introduction of a hydroxyl function at the border of two blocks is the use of 2methoxymethoxymethyloxirane for the endcapping of living polymer anions (Scheme 14C). 62,64,100 The hydroxy function inherently generated during the coupling reaction with the endcapper is used for the ring opening polymerization of ethylene oxide as second block. After deprotection the second hydroxy function was used to attach a carboxylterminated poly(perfluoropropylene oxide) via esterification 100 or “grafting-from” of γmethyl-ɛ-caprolactone 62 or N,N-dimethylaminoethyl acrylate 64 after post-modification of the alcohol to obtain amphiphilic ABC miktoarm star terpolymers. In a similar manner polystyrene-block-poly(ethylene oxide) diblock copolymers bearing an primary amino101 or allyl-function 102 at the block border were synthesized by Frey and co-workers from the corresponding functionalized glycidyl ethers. Due to their defined mid-functionality, these are of possible future interest for the construction of miktoarm star polymers. However, for such glycidyl compounds as endcapping agents, the polymerization method of the second block is restricted to anionic ring-opening polymerization. Also for the approaches utilizing DPE to generate the mid-functionality the choice of monomer sequence is dependent on the reactivity of the monomers. 91 1 - Introduction 24 Scheme 1-4. Overview of mid-functional diblock copolymers for the construction of ABC miktoarm star terpolymers. Utilizing DPE chemistry (A,B) 97-99 or a glycidyl ether (C) 62,64,100 hydroxyl mid-functionalized diblock copolymers were synthesized which serve as precursor for the attachment of the third block via “grafting-from” or “grafting-to” approaches. (D) Heterofunctional Core Molecules All the examples discussed up to now take advantage of anionic polymerizations steps to generate well-defined and functionalized polymers as building blocks for the miktoarm star terpolymer synthesis. However, in the past decade, an increasing number of reactions fulfilling the criteria of click chemistry were utilized in polymer chemistry for the construction of various polymer architectures in combination with controlled radical polymerization methods. 103 Herein, heterofunctional core molecules can serve as common junction point for the “grafting-from” and “grafting-to” of different polymer segments by a combination of such click reactions with standard polymerization methods to construct miktoarm star terpolymers. For example Zhang et al. synthesized a trifunctional core molecule bearing an alkyne-, hydroxyland bromine-function. 104 Due to the compatibility and tolerance of the reaction conditions simultaneous azide-alkyne click chemistry, ring-opening polymerization and ATRP was possible to synthesize polystyrene-arm-poly(ɛ-caprolactone)-arm-poly(N,N-dimetylaminoethyl methacrylate) or poly(ethylene oxide)-arm-poly(ɛ-caprolactone)-arm-poly(N,N-dimetylaminoethyl methacrylate) in a one-pot reaction (Scheme 1-5A). Based on the same principles, diverse other strategies are reported in literature where consecutive ATRP, ring-opening polymerization, click reactions like azide-alkyne or thiol-ene click chemistry, esterification and transformation reactions were combined to construct ABC miktoarm star terpolymers. 66-68,77,105-110 Furthermore, Tunca and co-workers presented an approach utilizing three orthogonal click reactions for the construction of a poly(ethylene oxide)- arm-poly(ɛ-caprolactone)-arm-poly(N-butyl oxanorboneneimide) miktoarm star 1 – Introduction 25 (Scheme 1-5B). 111 First the poly(ethylene oxide) segment was attached to the core molecule by a Diels-Alder click reaction, followed by the simultaneous ligation of the other two blocks by azide-alkyne cycloaddition and nitroxide radical coupling click reaction. Huan et. al reported a facile strategy for the construction of supramolecular ABC miktoarm star terpolymers using β-cyclodextrin as core molecule. 112 Recently, the group of Li demonstrated the Passerini three-component reaction to be a powerful method to simultaneously introduce an ATRP initator and an alkyne-function to aldehyde endfunctionalized poly(ethylene oxide). 113 Starting with this dual-functionalized PEO diverse ABC miktoarm star terpolymers were accessible either by consecutive ATRP and azidealkyne cycloaddition or simultaneous SET-LRP and click reaction (Scheme 1-5C). Additionally, in literature different other miktoarm star architectures like ABCD, 114 star115 and H-shaped ABCDE miktoarm stars 116 and the first ABC miktoarm star terpolymer with cyclic arms 117 are reported by modifications of these strategies with heterofunctional core molecules. Scheme 1-5. Synthetic strategies for the construction of ABC miktoarm star terpolymers with heterofunctional core molecules via simultaneous ATRP, ROP and CuAAC (A), 104 consecutive triple click reactions (B) 111 or three-component Passerini reaction (C). 113 1 - Introduction 26 1.2.1.2 Other Miktoarm Star Polymer Systems Hirao and co-workers designed special reaction sequences which enable the iterative synthesis of asymmetric star branched polymers by repeating the sequence. 76 These individual steps involve the linking reaction of a living polymer anion with a polymer containing a suitable functional group (1), which is followed by the regeneration of the functional group at the reaction site (2). Based on the silyl-protected hydroxylfunctionalized DPE shown in Scheme 1-4B such an iterative methodology is possible, starting with a living polymer anion endcapped with this DPE derivative. After transformation of the protected hydroxy group into a benzyl bromide function a second living anion endcapped with this DPE is reacted with the bromide, which resembles step 1. Regeneration of the bromide function at the core from the newly introduced silylprotected hydroxyl-function resembles step 2, which allows for repeating the cycle (Scheme 1-6). In this manner an ABCD miktoarm star polymer was synthesized. 118 However, as a prerequisite for the re-introduction of the functional group the living polymer anion used in the last step has to be nucleophilic enough to copolymerize with DPE. Therefore, for less nucleophilic living anions of polymers like poly(methyl methacrylate), a reintroduction of the functional group is not possible and they have to be attached in the last step. This methodology was also applied to bromide-functionalized DPE 119 or bromide-functionalized 1,3-butadiene, 120 which are used to re-introduce a nonhomopolymerizable DPE or butadiene end-functionality. Repeating these steps, stars containing up to seven different arms could be achieved. Another functionality capable of such linking reactions without undergoing homopolymerization is the α-phenyl acrylate, which can also copolymerize with less reactive monomers like tert-butyl methacrylate. Using a dual hydroxy-functionalized DPE derivative, which protecting groups can be selectively cleaved to esterify them with the α-phenylacrylic acid in separate steps, ABCDE miktoarm stars were synthesized with an iterative methodology in the group of Hirao. 121 For all these strategies additional fractionation is necessary to obtain the pure miktoarm star polymers as the linking reactions are conducted with excess of the living anionic polymer chain to guarantee 100% conversion. 1 – Introduction 27 Scheme 1-6. Iterative methodology for the synthesis of star branched polymers via reaction of living polymer anions with a benzyl bromide function. 118 Another widely used method is the divinylbenzene (DVB) method, based on the ability of preformed polystyrene arms to anionically copolymerize with divinylbenzene to form a core with active reaction sites. 122 Addition of a second monomer leads to polymer chains growing from this core. Therefore, with this “in-out” method, star systems of the type A n B n with symmetrical composition and different second blocks were easily accessible as shown by Tsitsilianis and co-workers. 123-126 By the addition of a third monomer furthermore A n (B-block-C) n miktoarm star systems with a polystyrene and poly(2-vinylpyridine)- block-poly(tert-butyl acrylate) segments were synthesized. 127 However the number of arms represents an average value, which is only roughly adjustable by the amount of DVB and molecular weight of the starting polymer. 1.2.2 Self-Assembly in Bulk In addition to the self-assembled structures of miktoarm star terpolymers in solution (see 1.1.2.2), their morphology in the bulk state represents an unique property. Already linear triblock terpolymers were shown to produce a wide variety of interesting morphologies due to the linear connection of three immiscible blocks with three different sets of interaction parameters. 27,71,128-131 However, for miktoarm star terpolymers of sufficient incompatibility the constitution of the three polymer segments at a common 1 - Introduction 28 point forces these to align in a one-dimensional fashion. In contrast to that the two different junction points in linear ABC triblock terpolymers are located at two-dimensional polymer interfaces. Consequently, completely segregating miktoarm star terpolymers are supposed to assemble into columnar bulk morphologies (Figure 1-6A). 132 The twodimensional cross-sections of the spatial arrangement of these cylinders can be described by Archimedian tiling patterns. 132-134 Due to the challenging synthesis of miktoarm star terpolymers systematic investigations of the compositional influence on the morphology are quite rare. Besides earlier morphological studies, 135,136 Thomas and co-workers proved the alignment of the junction points in a one-dimensional fashion for a system consisting of polystyrene, polyisoprene and poly(methyl methacrylate). 137 Hückstädt et al. gave the first detailed morphological study on a polystyrene-arm-polybutadiene-arm-poly(2-vinylpyridine) system of various compositions. 94 For another system containing polystyrene, polyisoprene and poly(2-vinylpyridine) arms the variation of the volume fractions yielded manifold structures of complex tiling patterns as exemplarily depicted in Figure 1-6B. 138-141 When miktoarm stars of more asymmetric compositions were investigated, transitions to highly periodic, substructured lamellar morphologies could be observed. 142 Similarly, Ikkala and co-workers reported the hierarchical smectic self-assembly for a miktoarm star terpolymer containing a α-helical poly(ε-tert-butyloxycarbonyl-L-lysine) (PBLL) segment. 143 The morphology composed of an overall lamellar structure with one type of lamella of packed helices of PBLL and a second type formed from alternating rectangular cylinders of polystyrene and polyisoprene (Figure 1-6C). In addition Abetz et al. demonstrated that blending of polystyrene-arm-polybutadiene-arm-poly(2-vinylpyridine) miktoarm stars with diblock copolymers can be utilized to tune the obtained morphology within certain limits. 144 1 – Introduction 29 Figure 1-6. (A) Schematic illustration of the one-dimensional alignment of junction points and the resulting columnar arrangement of the individual blocks of ABC miktoarm star terpolymers. 132 (B) TEM micrographs of four polyisoprene-arm-polystyrene-arm-poly(2vinylpyridine) (µ-ISV) miktoarm star terpolymers (top row) and the corresponding schematic tiling patterns (bottom row): (a) µ-I 1.0 S 1.8 I 1.0 , (b) µ-I 1.0 S 1.8 I 1.6 , (c) µI 1.0 S 1.8 I 2.0 , (d) µ-I 1.0 S 1.8 I 2.9 (the subscripts denote the corresponding volume ratios). The dark, light and gray domains correspond to PI, PS, and P2VP phases. 141 (C) TEM micrograph and schematic illustration of the bulk morphology of a µ-(PS)(PI)(PBLL) miktoarm star. The sample is stained with OsO 4 (PI dark, PS and PBLL light). 143 1.2.3 Application of ABC Miktoarm Star Terpolymers for Functional Materials Due to the broad variety of hierarchies obtained both in solution and bulk (see 1.1.2.2 and 1.2.2), miktoarm star terpolymers are of special interest for the design of novel materials with compartmentalized structures. Up to now only a limited number of publications has been dealing with the application of miktoarm star terpolymers in materials research, most probably due to the complicated synthesis and accessibility of larger amounts of material. However, this situation will change with the advances obtained in miktoarm star synthesis during the last decade. 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W.; Hadjichristidis, N. Macromolecules 2011, 44, 1886-1893. (198) Fleischmann, S.; Komber, H.; Appelhans, D.; Voit, B. I. Macromol. Chem. Phys. 2007, 208, 10501060. (199) Sumerlin, B. S.; Tsarevsky, N. V.; Louche, G.; Lee, R. Y.; Matyjaszewski, K. Macromolecules 2005, 38, 7540-7545. (200) Lang, A. S.; Neubig, A.; Sommer, M.; Thelakkat, M. Macromolecules 2010, 43, 7001-7010. (201) Pfeifer, S.; Lutz, J.-F. Chemistry – Eur. J. 2008, 14, 10949-10957. (202) Barner-Kowollik, C.; Du Prez, F. E.; Espeel, P.; Hawker, C. J.; Junkers, T.; Schlaad, H.; Van Camp, W. Angew. Chem., Int. Ed. 2011, 50, 60-62. 40 2 – Overview of the Thesis 41 2 – Overview of the Thesis This dissertation contains three publications, presented from chapter 3 to 5. Novel miktoarm star terpolymers are the common issue connecting the three publications within this thesis. The main focus of the investigations is based on the synthesis of such materials on the one side and on the characterization of the micellar structures obtained by self-assembly of the polymers in solution on the other side. Therefore, a new approach for the synthesis of various ABC miktoarm star terpolymers was developed. Mid-alkyne functionalized diblock copolymers, which were synthesized by anionic polymerization utilizing 1,1-diphenylethylene (DPE) chemistry, form the key building blocks. These allowed modular ligation with azido-functionalized homopolymers via azide-alkyne Huisgen cycloaddition, which is presented in Chapter 3. For a miktoarm star terpolymer containing a poly(2-vinylpyridine block) (P2VP) the mechanism of hierarchical superstructure formation was investigated. The polymer architecture, quaternization of P2VP and triiodide as counterion were found to be prerequisites for the directed self-assembly into complex “woodlouse”-shaped aggregates. The formation of cylindrical structures from spherical micelles, the meanderlike arrangement of these cylinders and, finally, the fusion into lamellar aggregates of barrel-like shape was shown to be the underlying mechanism. Chapter 4 includes the results of this counterion-mediated hierarchical superstructure formation. With the knowledge of the necessary parameters for such a directed self-assembly, this study was further expanded to other ABC miktoarm star terpolymers and an ABA’ miktoarm star copolymer. Enabled by our modular approach the influence of molecular and chemical composition on the obtained superstructure was evaluated (Chapter 5). Herein, structures similar to the “woodlouse” aggregates were observed, however from a different mechanism starting with vesicles as primary building units instead of micelles. Below, an overview over the most important results of each of the three following chapters is given. The reader is referred to the corresponding chapter for an extensive 2 – Overview of the Thesis 42 discussion on the particular topic of synthesis and self-assembly of the miktoarm star terpolymers. 2.1 Modular Synthesis of Miktoarm Star Terpolymers Besides routes relying exclusively on anionic polymerization, the synthesis of miktoarm star terpolymers is typically accomplished by a combination of different polymerization methods. Therefore, specially designed linking reactions in combination with precisely functionalized diblock copolymers and postfunctionalization/transformation reactions have to be conducted. In this chapter we present a more generalized method, which can be applied to a broad variety of (functional) monomers. The synthetic steps involve sequential anionic polymerization of alkyne mid-functionalized diblock copolymers and subsequent ligation with azido-functionalized diblock copolymers (Scheme 2-1). Scheme 2-1. Synthetic route for ABC miktoarm star terpolymers by the combination of anionic polymerization in THF with DPE chemistry and click chemistry. For the preparation of the alkyne mid-functionalized diblock copolymer we utilized DPE chemistry, as it allows exact incorporation of exactly one DPE unit at the block border under appropriate choice of monomer sequence. For this purpose, we synthesized a novel DPE derivative carrying a protected alkyne-function (click-DPE, 1-[(4-(tertbutyldimethylsilyl)-ethynyl)phenyl]-1-phenylethylene). Due to the conjugation of the πsystem with the alkyne-function in para-position the corresponding living anion exhibits a bathocromic shift in the UV-spectrum as compared to unsubstituted DPE (Figure 2-1A). 2 – Overview of the Thesis 43 As a consequence of this altered electronic configuration click-DPE was shown to copolymerize with living anions of poly(2-vinylpyridine) (P2VP) by MALDI-ToF MS investigation of an appropriate test reaction (Figure 2-1B), in contrast to other DPE derivatives reported in literature. Hence, the living anion of click-DPE can principally both be used to start the anionic polymerization 2-vinylpyridine (2VP) and for endfunctionalization of living anions of P2VP. Figure 2-1. (A) UV-vis absorption spectrum of the adduct of 1,1-diphenylethylene (dotted line) and 1-[(4- (tert-butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (solid line) with sec-BuLi in THF. The inset shows the picture of the cuvette containing the living anion of the unsubstituted DPE (top) and the click-DPE (bottom). (B) MALDI-ToF MS spectra of poly(2-vinylpyridine) before (black line) and after addition of clickDPE (cDPE, red line), recorded in reflectron mode (B) with AgTFA as ionization agent.  Here, we prepared different alkyne mid-functionalized diblock copolymers with polybutadiene as first block and poly(2-vinylpyridine), poly(tert-butyl methacrylate) (PtBMA), or poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) as second block. Except hydrolysis no further transformation reactions are necessary to generate the alkyne-function. We determined the degree of alkyne-functionalization by a test click reaction with an azido-functional perylene bisimide as chromophore in combination with UV measurements. For the diblock copolymers with P2VP as second block only a slight over-incorporation of the click-DPE could be achieved under appropriate reaction conditions (~120% alkyne-functionalization). This is due to the addition of a second DPE unit for some chains. In contrast, for PtBMA as second block near-quantitative functionalization was proven (~93% alkyne functionalization). λ max = 549 nm λ max = 500 nm 4750 4800 4850 4900 4950 VP45 VP42cDPE1 VP41cDPE1 VP44 m/z 4780 4800 4820 VP 44 VP41cDPE1 ~ 4.5 g/mol A B 400 450 500 550 600 650 700 absorbance λ [nm] 2 – Overview of the Thesis 44 By a modular combination of these mid-functional diblock copolymers with azidofunctionalized homopolymers we synthesized different novel ABC miktoarm star terpolymers via copper-catalyzed azide-alkyne Huisgen cycloaddition. For the example of a PB-b-PtBMA diblock copolymer (cBT2) the miktoarm star terpolymers obtained after click reaction with azido-functional polystyrene (PS), poly(ethylene oxide) (PEO) and PDMAEMA homopolymers are listed in Table 2-1. The corresponding SEC eluograms are depicted in Figure 2-2. In all cases narrowly distributed miktoarm star terpolymers were obtained. We further showed for click-reactions with PEO and PDMAEMA homolopolymers that a reaction pathway with an excess of the homopolymer was possible and monomodal miktoarm star terpolymers were achieved after appropriate purification procedures, like e.g. dialysis (Figure 2-2B). Table 2-1. Molecular Characterization of ABC Miktoarm Star Terpolymers Obtained from Click Reactions with Alkyne Mid-Functionalized PB 111 -b-PtBMA 42 Diblock Copolymer cBT2 (Subscripts Denote the Corresponding Degrees of Polymerization) sample ID a DP(3 rd block) b M n,th. c [kg/mol] M n,app d [kg/mol] Ɖ d µ-BT2S1, (µ-B 38 T 38 S 23 15.9 ) 36 15.9 20.5 1.03 µ-BT2S2, (µ-B 33 T 33 S 3318.1 ) 57 18.1 22.4 1.03 µ-BT2E, (µ-B 32 T 32 E 3518.8 ) 150 18.8 25.4 1.03 µ-BT2D, (µ-B 34 T 34 D 3117.6 ) 34 17.6 20.8 1.11 a The superscript denotes the theoretical number average molecular weight of the ABC miktoarm star terpolymer, as calculated from the respective values of the diblock copolymer and homopolymer and the indices the theoretical weight fraction of the respective blocks. b Calculated from the molecular weight of the 3 rd block. c Theoretical molecular weight. d Apparent molecular weight and dispersity determined by SEC with polystyrene calibration. For the miktoarm star terpolymers containing PDMAEMA THF-SEC with additional 0.25 wt% tetrabutylammonium bromide (TBAB) was used. 2 – Overview of the Thesis 45 Figure 2-2. SEC traces of miktoarm star terpolymers from click reactions of a polybutadiene-blockpoly(tert-butyl methacrylate) diblock copolymer (cBT2) with (A) two polystyrene homopolymers (PS1-N 3 , PS2-N 3 ) and a poly(ethylene oxide) homopolymer (PEO-N 3 ) and (B) a poly(N,N-dimethylaminoethyl methacrylate) homopolymer (PDMAEMA-N 3 ). For (B) THF-SEC with additional salt was used and as the click reaction was conducted with 2 equiv of the PDMAEMA-N 3 , excess homopolymer was removed via dialysis. With this modular approach a variety of functional miktoarm star terpolymers are accessible. Furthermore, click-DPE offers novel synthetic advantages in the construction of polymeric architectures via a combination of anionic polymerization and clickchemistry. The miktoarm star terpolymers synthesized herein all carry a polybutadiene block, which can be used for further modification and functionalization via thiol-ene chemistry. Moreover new amphiphilic miktoarm star terpolymers were synthesized, which carry hydrophilic (PEO, PDMAEMA) and stimuli-responsive arms (PDMAEMA, PtBMA after hydrolysis). 2.2 Counterion-Mediated Hierarchichal Self-Assembly of an ABC Miktoarm Star Terpolymer Containing a Poly(N-methyl-2-vinylpyridinium iodide) Segment For a miktoarm star terpolymer containing a polybutadiene, poly(tert-butyl methacrylate) and poly(2-vinylpyridine) segment (µ-BVT), quaternization with methyl iodide and transfer into aqueous solution was observed to yield two structurally completely differing limiting cases of aggregation forms: spherical micelles (d micelle = 24.5 ± 2.0 nm) and particles with a complex lamellar interior (200-500 nm in longitudinal axis). The concentration of triiodide as polarizable counterion for the quaternized P2VP 26 28 30 32 34 36 38 0.0 0.2 0.4 0.6 0.8 1.0 cBT2 PS1$N 3 PS2$N 3 PEO$N 3  $BT2S1  $BT2S2  $BT2E normalized RI signal V e [mL] 14 15 16 17 18 19 0.0 0.2 0.4 0.6 0.8 1.0 V e [mL] cBT2 PDMAEMA$N 3 . $BT2D, undial. . $BT2D, dial. A B 2 – Overview of the Thesis 52 Felix H. Schacher, Hiroshi Jinnai and Axel H. E. Müller were involved in scientific discussions and correction of the manuscript. Chapter 5 This work is published in Polymer 2013, 54, 4528-4537 under the title: “Hierarchical Self-Assembly of Miktoarm Star Polymers Containing a Polycationic Segment: A General Concept” by Andreas Hanisch, André H. Gröschel, Melanie Förtsch, Tina I. Löbling, Felix H. Schacher,* and Axel H. E. Müller* I wrote the publication and conducted most of the experiments. Exceptions are stated below: André H. Gröschel was involved in scientific discussions. Melanie Förtsch performed almost all TEM and cryo-TEM measurements. Tina I. Löbling recorded some of the TEM micrographs. Felix H. Schacher and Axel H. E. Müller were involved in scientific discussions and correction of the manuscript. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 53 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers via a New Alkyne-Substituted Diphenylethylene Derivative Andreas Hanisch, Holger Schmalz, and Axel H. E. Müller ABSTRACT: We introduce a modular route for the synthesis of well-defined ABC miktoarm star terpolymers. To this aim, the synthesis of a 1,1-diphenylethylene derivative bearing a protected alkyne function (1-[(4-(tert-butyldimethylsilyl)ethynyl)phenyl]- 1-phenylethylene) was developed. This compound was for the first time employed in sequential anionic polymerization to readily prepare alkyne mid-functionalized diblock copolymers with polybutadiene as first and a poly(alkyl methacrylate) (poly(tert-butyl methacrylate), poly(N,N-dimethylaminoethyl methacrylate)) as second block. For the third arm controlled radical polymerization methods (polystyrene, poly(tert-butyl methacrylate), poly(N,N-dimethylaminoethyl methacrylate)) and anionic ring-opening polymerization (poly(ethylene oxide)) were used to separately prepare homopolymers with an azido function. Afterward, azide-alkyne Huisgen cycloaddition was successfully employed to synthesize a library of ABC miktoarm star terpolymers with different molecular weights and chemical compositions via modular combination of the functionalized diblock copolymers and homopolymers. The resulting new ABC miktoarm star terpolymers showed narrow, monomodal molecular weight distributions with dispersities typically below 1.10, as determined by size exclusion chromatography. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 54 3.1 Introduction Polymer architecture and composition is a decisive factor for controlling structure formation both in bulk and in solution. Besides the choice of monomer and the number and sequence of polymeric building blocks the way of molecular conjunction of these blocks is of outermost importance. Compared to their linear analogues, miktoarm star terpolymers, where the polymer chains are connected at one common junction point,1 present a much more complex system. A variety of unique bulk morphologies were found,2-4 which are inaccessible by linear triblock terpolymers. This is a direct consequence of the confined geometry of the polymer chains, which forces the common junction points of the three different blocks to be aligned in a 1-dimensional fashion. These miktoarm star terpolymer morphologies were also utilized for generating cylindrical, compartmentalized particles by selective crosslinking of one block.5 In addition, theoretical studies suggest that even more complex morphologies are expected under cylindrical confinement of ABC miktoarm star terpolymers.6 Depending on the length ratio of the respective arms, Hillmyer, Lodge and co-workers7,8 obtained different multicompartment structures in aqueous solution from ABC miktoarm star terpolymers with a fluorinated segment. These ranged from hamburger micelles to segmented wormlike micelles and nanostructured vesicles. The conjunction of three chains at one common linking point leads to special requirements concerning the synthetic strategy. The main difficulties are (1) the exact functionalization of a diblock copolymer with a functional group or polymer block at the junction of the two blocks and (2) the stoichometry of the linking reaction. Four different synthetic strategies have been reported so far for miktoarm star terpolymers. One approach uses the different reactivity of living anionic polymer chains toward the chlorine-silicon groups of trichlorosilanes. Using methyltrichlorosilane as linking agent, Hadjichristidis and coworkers synthesized a miktoarm star terpolymer consisting of polyisoprene, polystyrene, and polybutadiene.9 However, the sequence of introduction of arms is strongly limited to the reactivity and steric demand of the polymeric anions, and adaption to other monomers requires a modification of the method.10,11 The second anionic approach utilizes macromonomers with nonhomopolymerizable endgroups 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 55 which were directly sequentially copolymerized with two sorts of monomers.2,12-14 In another approach, first diblock copolymers were synthesized carrying functional groups at the junction point of the two blocks. Again, nonhomopolymerizable compounds or special polymerization strategies have to be employed to assure that only one functional group is located exactly in between the two polymeric building blocks. Up to now, mainly hydroxyl-functions were used to conjugate the third block, which were introduced via a 1,1-diphenylethylene (DPE) derivative15,16 or 2-methoxymethyloxymethyloxirane17-19 as end-capping molecule. This hydroxyl function serves as anchoring point for the third block, which can be “grafted from” by anionic ring opening polymerization (AROP)15-17 or controlled radical polymerization (CRP) after appropriate modification19 or “grafted to” via direct esterification.18 Hirao and co-workers recently used in-chain-benzyl bromidefunctionalized diblock copolymers for the miktoarm star terpolymer synthesis involving specially designed anionic linking reactions.20 The possibility of constructing complex polymeric architectures with controlled radical polymerization methods increased within the last decade.21,22 This supported the development of new strategies for the synthesis of ABC miktoarm star terpolymers where anionic polymerization is not involved. In addition, click reactions in general, or copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in particular have become powerful tools for highly efficient linking of polymeric building blocks.23-25 These novel synthetic routes take advantage of the orthogonality of the different polymerization/ligation methods, which is a prerequisite for the synthesis of well-defined ABC miktoarm star terpolymers. By designing special core molecules with three different functionalities, the arms can be “grafted onto” or “grafted from” in different steps.26-31 Herein, we present a more general method by combining anionic polymerization, employed for the synthesis of readily alkyne mid-functionalized diblock copolymers, with other polymerization techniques via azide-alkyne Huisgen cycloaddition (Scheme 31). Barner-Kowollik and co-workers already reported the ligation of alkyne midfunctionalized homopolymers with azide-bearing homopolymers.32 However, only A2B miktoarm star terpolymers were accessible. The synthesis of an alkyne midfunctionalized diblock copolymer consisting of polystyrene and poly(tert-butyl acrylate) was also reported.33,34 Nevertheless, multiple polymer analogous reactions had to be 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 56 conducted and/or switching the polymerization system was necessary. Thus, we synthesized 1-[(4-(tert-butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene to directly prepare mid-chain alkyne-substituted diblock copolymers via sequential anionic polymerization. Because of the direct introduction of the alkyne-group into the diblock copolymer by the use of DPE chemistry, a wide variety of functional diblock copolymers are accessible, and except hydrolysis no tedious transformation reactions have to be conducted. The obtained diblock copolymers were afterward ligated with different azide-bearing homopolymers prepared by atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and anionic ring opening polymerization via azide-alkyne click chemistry. Different novel ABC miktoarm star terpolymers were synthesized utilizing this modular combination. Scheme 3-1. Synthetic route for the synthesis of ABC miktoarm star terpolymers for the example of a star consisting of arms of polybutadiene, poly(tert-butyl methacrylate), and polystyrene 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 57 3.2 Experimental Section Materials. For the purification of monomers, reagents and solvent and the synthesis of used compounds see the Supporting Information. Synthesis of 1-[(4-(tert-Butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (ClickDPE). The starting molecule 1-(4-bromophenyl)-1-phenylethylene was synthesized as described elsewhere.35 The synthetic protocol for the coupling reaction with the protected alkyne derivative was adopted from the literature.36 First, 1-(4-bromophenyl)-1phenylethylene was dissolved in piperidine (~50 mg/mL) and bis(triphenylphosphine)palladium(II) dichloride (0.03 equiv) and CuI (0.004 equiv) were added under a nitrogen atmosphere. After degassing with nitrogen for 30 minutes, tertbutyldimethylsilylacetylene (1.2 equiv) was added dropwise at 50 °C, and the solution was stirred overnight. The reaction mixture was filtered, the solvent evaporated, and the crude product was dissolved in THF. After addition of water, the product was extracted with hexane for three times. The organic fractions were dried over sodium sulfate, and the solvent was evaporated to obtain a brown oil. This was further purified by column chromatography with hexane as solvent to obtain a clear oil. Distillation of the product was not possible even under high vacuum conditions. Before the use in anionic polymerization, the compound was freeze-dried from benzene solution on a high vacuum line and subsequently dissolved in dry THF to obtain a stock solution of low viscosity. secBuLi was added dropwise (typically 1-2 drops) under nitrogen flow until a deeply violet color was obtained. Besides proving the absence of impurities, the persistent color also served as indication for the purity of the stock solution. 1H NMR (300 MHz, CDCl3): δ = 7.50-7.27 (m, 9H, Ar), 5.50 (s, 2H, -C=CH2), 1.03 (s, 9H, SiC(CH3)3), 0.22 (s, 6H, SiCH3) Polymerizations. Sequential Living Anionic Polymerization in THF. All polymerizations were carried out at low temperatures in a thermostated laboratory autoclave (Büchi) under dry nitrogen atmosphere using THF as solvent. The day before polymerization, freshly distilled THF (~250 mL for 15 g of polymer) was treated with 1 mL of sec-BuLi per 100 mL of solvent at -20 °C, followed by stirring overnight to form lithium alkoxides. These exhibit stabilizing effects on the living chain end of polybutadiene and furthermore in the case of methacrylate-type monomers no addition of LiCl is necessary to ob- 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 58 tain well-defined polymers.37 For the diblock copolymers, first butadiene was initiated with sec-BuLi at -70 °C and then polymerized at -50 to -15 °C depending on the block length of the different polymerizations. To ensure complete consumption of the butadiene the polymerization was followed by in-line NIR fiber-optic spectroscopy. The living polybutadienyl anion was end-capped with 1-[(4-(tertbutyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (1.1-1.5 equiv) at -50 °C for 2 h. Before and after the addition of the click-DPE samples for SEC and MALDI-ToF MS were withdrawn. In the case of poly(2-vinylpyridine) (P2VP) as second block the monomer was added at -70 °C and polymerized for 5 minutes. After termination with degassed methanol the polymer (cBV) was isolated by precipitation in water. For the diblock copolymers with tert-butyl methacrylate (tBMA) the monomer addition was conducted at -70 °C and the polymerization took place at -45 °C for 1 h before it was terminated with degassed methanol. After the addition of the tBMA the violet color of the DPE end-capped living anion slowly faded away. The final polymer (cBT) was precipitated in a mixture of isopropanol/water 3/1 (v/v). For the third type of diblock the N,N-dimethylaminoethyl methacrylate (DMAEMA) was injected into the reactor at -70 °C and polymerized for 1 h. Immediately after addition of the monomer the color of the DPE end-capped polybutadienyl anion vanished completely. After termination with degassed methanol, the resulting polymer (cBD) was dialyzed against THF and finally freeze-dried from dioxane. The molecular weights of the diblock copolymers with P2VP or PDMAEMA as second block were calculated by a combination of MALD-ToF MS and 1H NMR. Therefore, the Mn of the polybutadiene (PB) precursor polymer was determined by mass spectrometry. By the relative molar ratios of the characteristic signals of PB (5.10-5.60 ppm, 2H 1,2-PB; 5.70-5.15 ppm, 1H 1,2-PB and 2H 1,4-PB) and P2VP (8.50-8.00 ppm, 1H) or PDMAEMA (4.30-3.90 ppm, 2H) the overall molecular weight was calculated using the PB precursor molecular weight as reference. The Mn of the PB-b-PtBMA diblock copolymers was directly measured by MALDI-ToF MS. All the data for the diblock characterization are summarized in Table 3-1. Alkyne Deprotection. For the deprotection procedure, the respective polymer was dissolved in THF (~0.1 g/mL) and degassed for 30 min. Then, 10 equiv of tetrabutylammonium fluoride (1 M solution in THF) relative to alkyne functions were 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 59 added at 0 °C and stirred at this temperature for 2 h. After warming up to room temperature the solution was stirred overnight. The polymer was precipitated in pure water or isopropanol/water 3/1 (v/v) for PB-b-P2VP and PB-b-PtBMA, respectively. Afterward, the polymer was dissolved in THF and dialyzed against THF (MWCO 1 000 g/mol) to remove impurities. Finally, the polymers were freeze-dried from dioxane. In the case of the diblock copolymer with DMAEMA the reaction mixture was directly dialyzed against THF prior to freeze-drying. Click Reactions. For the determination of the degree of functionalization with alkynesubstituted DPE, test click reactions with N-(1-heptyloctyl)-N′-(hexyl-6′-azido)-perylene3,4,9,10-tetracaboxylic acid bisimide were conducted. In a typical run, 100 mg of the deprotected diblock copolymer were dissolved in 5 mL THF in a screw-cap glass. Then, 2 equiv of the azido-functionalized perylene bisimide relative to the alkyne function was added. After purging with nitrogen for 10 min, 1 equiv of CuBr was added, followed by further degassing for 10 min. By addition of 1 equiv of N,N,N′,N′,N′′- pentamethyldiethylenetriamine (PMDETA) as ligand, the click reaction was started and stirred at room temperature for 3 days. After termination by exposure to air, the remaining copper was removed by filtration over a short silica gel column. Preparative SEC with THF as eluent was employed to remove excess perylene. An intense red-colored polymer was obtained after freeze-drying from dioxane. The click reactions for the construction of the miktoarm star terpolymers were conducted in a similar manner. Typically, the molar ratios of alkyne function:azido function:CuBr:PMDETA was set to 1:1:1:1 (if not stated elsewhere), and the polymer concentration was ~20 mg/mL in THF. The reactions were conducted at room temperature and followed by withdrawing samples for SEC measurements. Finally, when no further changes in the SEC eluogramms were observed, the resulting miktoarm star terpolymer was purified by passing through a small silica gel column to remove copper. The polymers were obtained as white powders after freeze-drying from dioxane. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 60 3.3 Results and Discussion 3.3.1 Synthesis of 1-[(4-(tert-Butyldimethylsilyl)ethynyl)phenyl]-1phenylethylene (Click-DPE) The key compound of this modular route toward ABC miktoarm star terpolymers (Scheme 3-1) was directly synthesized by the palladium-catalyzed Sonogashira coupling reaction between 1-(4-bromophenyl)-1-phenylethylene and tert-butyldimethylsilyl protected acetylene. Similar compounds, like 4-(trimethylsilyl)ethynylstyrene36 and methacrylate derivatives as 3-trimethylsilyl-2-propynyl methacrylate,38 have already been synthesized and used in anionic polymerization to obtain polymers with predictable molecular weights and narrow distributions. As known for DPE and its derivatives homopolymerization of such compounds is not possible due to steric hindrance.39 In contrast to methods reported in literature,33,34 this synthetic advantage enables us to use one sequential polymerization process to selectively incorporate exactly one alkyne function between the two polymeric blocks under adequate choice of monomers. The click-DPE was purified via column chromatography to give a clear viscous oil. The chemical structure and the purity were confirmed by 1H NMR spectroscopy (Figure 3-1a). Figure 3-1. 1H NMR spectra of (a) 1-[(4-(tert-butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (clickDPE), (b) the protected alkyne-functionalized diblock cBT2, and (c) the corresponding diblock after hydrolysis of the silyl-protecting group (solvent signals are striked out). 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 61 The UV-vis spectrum of the deeply violet solution of the living anion (Figure 3-2) generated by reaction of 1-[(4-(tert-butyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene with sec-BuLi shows a maximum at 549 nm. In contrast, the living anion of unsubstituted 1,1diphenylethylene has a deep red color, and we determined the absorption maximum at around 500 nm under the same conditions.40 This clear bathocromic shift originates from the conjugation of the π-system with the protected alkyne group. Similarly, Tsuda et al. reported a brownish-red color in the anionic polymerization of 4- (trimthylsilyl)ethylenstyrene,36 in contrast to the orange color of polystyryl anions.41 Figure 3-2. UV-vis absorption spectrum of the adduct of 1,1-diphenylethylene (dotted line) and 1-[(4-(tertbutyldimethylsilyl)ethynyl)phenyl]-1-phenylethylene (solid line) with sec-BuLi in THF. The inset shows the picture of the cuvette containing the living anion of the unsubstituted DPE (top) and the click-DPE (bottom). 3.3.2 Synthesis of Alkyne Mid-Functionalized Diblock Copolymers Butadiene was chosen for the first block and for the second block 2-vinylpyridine (2VP) or methacrylate type monomers as tert-butyl methacrylate (tBMA) and N,Ndimethylaminoethyl methacrylate (DMAEMA). The anionic polymerization of butadiene was conducted in THF at -50 to -15 °C (depending on the respective block length) using sec-BuLi as initiator. After complete conversion – as followed in situ with fiber-optics NIR spectroscopy – 1.1 to 1.5 equiv of the click-DPE stock solution was added via syringe at -50 °C. The addition of the diphenylethylene derivative was clearly visible by the imme- 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 68 weights close to 100 000 g/mol were produced via CuAAC,49,50 we suppose that in our case the alkyne function at the block junction is sterically less amenable than an alkyne function in αor ω-position. Our first trials with higher molecular weight polymers (exceeding 40 000-50 000 g/mol for the individual compounds) were not successful. Therefore, we chose moderate molecular weights of the individual compounds, so that the overall molecular weight did not exceed 25 000 g/mol and the functional groups were not too diluted. The course of the click reactions with cBT diblock copolymers was followed by SEC. The reactions were conducted under equimolar conditions. After 24 h the molecular weight distribution of the diblock copolymers cBT1 and cBT2 shifted completely (see Figure 3-3 and Figure 3-S4), resembling the successful generation of the desired miktoarm star terpolymer. However, despite equimolar reaction conditions in both cases there was still a minor amount of unreacted homopolymer left. Further SEC measurements showed that the peak of the PS1-N3 already disappeared nearly completely after 11.5 h (Figure 3-S5) for the click reaction with cBT2. Increasing the reaction time did not lead to any change in the eluogramm. Therefore, we additionally carried out a reaction between cBT1 and only 0.8 equiv of PS1-N3. Under the same reaction conditions, again, a comparable amount of homopolymer was left (results not shown). We assume that during polymerization and work-up of the homopolymer, a minor part of the bromine group is eliminated, as already reported in literature.51 This leads to a small amount of non-azido-functionalized homopolymer which cannot take part in the click reaction (see discussion of MALDI-TOF spectrum in the Supporting Information). This was also confirmed by the click reaction of cBT2 with PS3-N3 (Figure 3-S6). Under equimolar reaction conditions no further ligation took place after 15 h, leading to a nonnegligible amount of unreacted diblock and homopolymer. In contrast to the click reactions with lower molecular weight polystyrenes the conversion of the click reaction with PS3-N3 was much lower after 3.25 h (where the reaction was more or less finished in the other cases). Further addition of 0.5 equiv of homopolymer resulted in a complete shift of the diblock due to complete click efficiency as depicted in Figure 3-S6. Thus, the degree of unfunctionalized polystyrene increased with increasing molecular weight. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 69 26 28 30 32 34 36 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI signal V e [mL] cBT1 PS1-N3 PS2-N3 µ -BT1S1 µ -BT1S2 Figure 3-3. THF-SEC (RI signal) of alkyne-functionalized diblock cBT1, azido-functionalized PS (PS1-N3, PS2N3) and the corresponding ABC miktoarm star terpolymers (µ-BT1S1 and µ-BT1S2) obtained after equimolar click reaction for 24 h. Nevertheless, complete shifts of the molecular weight distributions of the miktoarm star terpolymers toward lower elution volumes compared to the pristine diblock copolymers were detected in all cases. This demonstrates the successful formation of ABC miktoarm star terpolymers. Additionally, the overall shifts of the resulting ABC miktoarm star terpolymers were not too distinct. This is in accordance with theory, where the hydrodynamic radius of star polymers is expected to be smaller than the hydrodynamic radius of a linear polymer with the same composition and molecular weight due to the higher segmental density of star polymers.52 All synthesized µ-BTS star terpolymers exhibited symmetrical peaks with a narrow molecular weight distribution. The theoretical molecular weights were calculated from the known molecular weights of the ligated diblock copolymers. These are shown together with the apparent molecular weights and the respective dispersities in Table 3-3. Furthermore an exemplarily 1H NMR of µ-BT1S1 is shown in Figure 3-S7. All characteristic signals of the polymeric building blocks are present in the obtained miktoarm star terpolymer. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 70 Table 3-3. Molecular Characterization of ABC Miktoarm Star Terpolymers Obtained from Click Reactions with Alkyne Mid-Functionalized Diblock Copolymers sample IDa DP(PB)b DP(2nd block)c DP(3rd block)d Mn,th. e [kg/mol] Mn,app f [kg/mol] Ɖf µBT1S1 (µ-B 63 T 18 S 18 20.7) 242 27 36 20.7 28.0 1.03 µBT1S2 (µ-B 57 T 17 S 26 22.9) 242 27 57 22.9 30.0 1.03 µBT1E (µ-B 55 T 16 E 28 23.6) 242 27 150 23.6 32.7 1.04 µBT1D (µ-B 58 T 17 D 24 22.4) 242 27 34 22.4 31.5 1.10 µBT2S1 (µ-B 38 T 38 S 23 15.9) 111 42 36 15.9 20.5 1.03 µBT2S2 (µ-B 33 T 33 S 33 18.1) 111 42 57 18.1 22.4 1.03 µ-BT2S3 g (µ-B 29 T 29 S 40 20.5) 111 42 80 20.5 25.0 1.03 µBT2E (µ-B 32 T 32 E 35 18.8) 111 42 150 18.8 25.4 1.03 µBT2D (µ-B 3 4 T 34 D 31 17.6) 111 42 34 17.6 20.8 1.11 µ-BDT g (µ-B 40 D 28 T 31 24.5) 181 44 53 24.5 26.3 1.08 μ-BDE g (µ-B 42 D 29 E 28 23.5) 181 44 150 23.5 32.5 1.06 aThe number-average molecular weight of the ABC miktoarm star terpolymer was calculated from the respective values of diblock copolymers and the homopolymers. Therefore, the superscript denotes the theoretical number-average molecular weight of the ABC miktoarm star terpolymer and the indices the theoretical weight fraction of the respective blocks. bDegree of polymerization (DP) determined from MALDI-ToF MS of the PB-precursor. cCalculated by the difference in Mn determined by the MALDI-ToF MS spectra of diblock and precursor. dCalculated from the molecular weight of the 3rd block. eTheoretical molecular weight. fApparent molecular weight and dispersity determined by SEC with polystyrene calibration. For the miktoarm star terpolymers containing PDMAEMA THF-SEC with additional 0.25 wt% tetrabutylammonium bromide (TBAB) was used. gIn these cases the apparent molecular weight and disperisty were determined excluding the separated homopolymer peak. Click Reactions with ω-Azido-Functionalized Poly(ethylene oxide). These first successful test reactions with azido-functionalized polystyrene prove the feasibility of our approach. To prepare amphiphilic miktoarm star terpolymers we conducted click reactions with PEO-N3. First attempts under equimolar reaction conditions led to a non-negligible amount of unreacted diblock copolymer. Therefore, the click reactions were carried out with an excess of the functionalized PEO-N3. By stepwise addition of PEO-N3 to the reaction mixture, the equivalents necessary for a complete click reaction were determined 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 71 (Figure 3-S8). Therefore, in a first step, the reaction with 1 equiv of PEO-N3 was followed by SEC. As after 24 h no further change in the eluogram was detected, and still a significant amount of unreacted diblock copolymer was left over, 0.5 equiv of PEO-N3 was added subsequently. Finally, after 3.5 h reaction time, further 0.2 equiv of PEO-N3 was added to the click reaction containing already 1.5 equiv. As this did not result in a change compared to the eluogram after reaction with 1.5 equiv of PEO-N3, the equivalents necessary for complete ligation were determined to be 1.5 equiv. The reason for the use of such a huge excess of PEO-N3 still remains unclear. From the amount of coupled product (~6 wt% from SEC) only a slight excess of the azidofunctionalized compound would be reasonable. However, maybe partial elimination of the azido group of PEO or uncomplete functionalization due to side reaction during the end-capping reaction could be a feasible explanation (see discussion of MALDI-ToF spectrum in the Supporting Information). The respective SEC traces for the click reactions with cBT1, cBT2, and cBD (where the ratio of cBX:PEO-N3 was minimum 1:1.5) are shown in Figure 3-4. In case of the diblock copolymers with PtBMA as second block the excess PEO-N3 was easily removed during purification from copper with a short column of silica due to interactions with the column. Using PEO-N3 in excess and subsequent removal of unreacted homopolymer guaranteed the complete conversion of the alkyne function. However, in the case of the diblock cBD containing DMAEMA a higher amount of PEO homopolymer was left over after work-up. Further purification with a column, crystallization from cold THF, or dialysis in aqueous media did not reduce the amount of the undesired PEO homopolymer. A possible explanation could be that PEO forms hydrogen bonds with PDMAEMA, which therefore prevent the complete removal of excess PEO homopolymer. Here, one has to notice that for further applications, where the PEO serves as corona in aqueous solutions, this minor amount of PEO homopolymer is not problematic. In contrast to the previous click reactions, distinct shifts of the molecular weight distributions of the ABC miktoarm star terpolymers compared to the corresponding diblock precursors were detected. The absence of a peak from residual diblock also gives evidence that all diblock copolymer chains carry an alkyne function. Like for the click reac- 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 72 A B 14 15 16 17 18 0.0 0.2 0.4 0.6 0.8 1.0 cBD PEO-N3 µ -BDE, dial. V e [mL] 26 28 30 32 34 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI signal V e [mL] cBT1 cBT2 PEO-N3  BT1E  BT2E tions with PS-N3 low dispersities of the resulting ABC miktoarm star terpolymers were detected (Table 3-3). Figure 3-4. THF-SEC (A) of alkyne-functionalized diblock copolymers cBT1, cBT2, the azido-functionalized PEO-N3, and the corresponding ABC miktoarm star terpolymers (µ-BT1E and µ-BT2E) obtained after azidealkyne click coupling and salt-THF-SEC traces (B) of cBD, PEO-N3, and the resulting ABC miktoarm star terpolymer (µ-BDE) after dialysis. In all cases the RI-signals are shown. Click Reactions with ω-Azido-Functionalized Poly(N,N-dimethylaminoethyl methacrylate). To obtain PDMAEMA as a water-soluble polymer, which responds both to pH and temperature,53 we used an azido-substituted CTA.44 As the general applicability of our approach under equimolar conditions was proven we conducted the click reactions with a 2-fold excess of PDMAEMA-N3 for 5 days, without optimizing the reaction time. With this reaction pathway we were also able to guarantee 100% conversion of the alkynecompound. The SEC traces of the raw product of the click reactions of the azidofunctionalized PDMAEMA-N3 with the two cBT diblock copolymers are shown in Figure 3-5A and 3-5B, respectively. In both cases the molecular weight distributions of the miktoarm star terpolymers shifted completely in the corresponding SEC-traces compared to their diblock copolymer precursors. The excess PDMAEMA-N3 was removed by dialysis in a mixture of methanol/isopropanol (2/1 v/v), where the miktoarm star terpolymer forms micelles with a polybutadiene core. Therefore, a dialysis membrane with a cut-off (50 000 g/mol) much higher than the molecular weight of the homopolymer was used. The SEC traces of the obtained miktoarm star terpolymers are plotted in Figure 3-5A and B. In the case of µ-BT1D the tiny coupling shoulder at lower elution volume became more pronounced after dialysis. Therefore, we assume that this 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 73 A B 14 15 16 17 18 19 0.0 0.2 0.4 0.6 0.8 1.0 V e [mL] cBT2 PDMAEMA-N3 µ -BT2D, undial. µ -BT2D, dial. 14 15 16 17 18 19 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI signal V e [mL] cBT1 PDMAEMA-N3 µ -BT1D, undial. µ -BT1D, dial. shoulder might be the result of some aggregation effects, which occurred for this reaction. Another explanation could be the oxidation of the amine, which leads to the formation of amine oxide. For the dialysis of µ-BT2D even under extensive exchange of the solvent, the small residual peak from unreacted PDMAEMA-N3 could not be removed completely. However, compared to the product peak this peak is negligible. The molecular characterization of the µ-BTD miktoarm star terpolymers is listed in Table 3-3. In a similar way a µ-BDT miktoarm star terpolymer consisting of the same monomer units was synthesized by ligation of cBD with PtBMA-N3 (see Supporting Information and Table 3-3). Figure 3-5. Salt-THF-SEC (RI signal) of µ-BTD miktoarm star terpolymers obtained by click coupling of the alkyne-functionalized diblock copolymers (A) cBT1 and (B) cBT2 with the azido-functionalized PDMAEMAN3. As in both cases 2 equiv of PDMAEMA were used within the click reaction, SEC traces of the raw product and the miktoarm star terpolymer after dialysis are shown. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 74 3.4 Conclusions This general modular route for the synthesis of ABC miktoarm star terpolymers combines anionic polymerization techniques with CRP methods and anionic ring opening polymerization using azide-alkyne Huisgen cycloaddition as ligation method. For this purpose, we successfully employed our alkyne-modified DPE derivative in sequential anionic polymerization. In the case of methacrylate-type monomers well-defined alkyne midfunctional diblock copolymers consisting of a first block of PB and a second block of either PtBMA or PDMAEMA were synthesized. Click reactions with azido-functionalized perylene bisimide verified the successful incorporation of only one DPE unit at the block junction. In contrast to other DPE derivatives, we observed that click-DPE can copolymerize with 2VP, which offers the advantage of producing alkyne mid-functionalized diblock copolymers with P2VP as first block and a methacrylate as second block. Therefore, 2VP and butadiene and styrene and their derivatives can be used as first blocks and methacrylate type monomers as second block to obtain well defined-diblock copolymers bearing an alkyne function at the block junction. By using a toolbox of different azidofunctionalized homopolymers, we demonstrate that a variety of well-defined ABC miktorarm star terpolymers is accessible via this modular approach. These exhibited monomodal molecular weight distributions with small disperisties close to 1.10 or even lower. The obtained miktoarm star terpolymers are interesting new polymers regarding their bulk morphologies and solution-based self-assembly. Results from the selfassembly of these ABC miktoarm star terpolymers will be reported in subsequent publications. A further advantage is that due to the use of anionic polymerization miktoarm star terpolymers with a polybutadiene block are accessible, which can afterward be functionalized through thiol-ene click chemistry54 or used for selective crosslinking.5,55 Furthermore, the ABC miktoarm star terpolymer containing arms of polybutadiene, poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide) is a promising candidate in the field of biotechnological applications like gene delivery.56-58 Our approach enables the upscaling of the synthesis of ABC miktoarm star terpolymers to more than one gram. Both synthetic steps for diblock and homopolymer synthesis are wellestablished polymerization methods, and no further complex synthetic procedures are 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 75 necessary, as compared to other methods. Because of the strong versatility of click chemistry and the increasing amount of publications dealing with it, we expect our clickDPE to offer a variety of new possibilities in the design of novel custom polymer architectures and materials. Supporting Information. Materials section, polymerization procedures, characterization section, characterization of ω-azido homopolymers, click reaction with poly(tert-butyl methacrylate) and discussion of the reactivity of click-DPE toward living P2VP-Li; UV-vis spectra of perylene-labeled diblock copolymers, SEC traces of polybutadiene precursors, poly(ethylene oxide) and click reactions, MALDI-ToF mass spectra of polybutadiene precursors and an exemplarily 1H NMR spectrum of µ-BT1S1. This Information is available free of charge via the Internet at http://pubs.acs.org/. Acknowledgments. This work was supported by the Deutsche Forschungsgemeinschaft within SPP 1165 (Mu896/22). We thank Christo B. Tsvetanov and Markus Müllner for fruitful discussion. Melanie Förtsch, Annika Pfaffenberger, and Christopher V. Synatschke are acknowledged for performing MALDI-ToF MS measurements and Marietta Böhm, Katharina Neumann, and Robin Pettau for conducting SEC measurements. We further thank André H. Gröschel for providing one of the polystyrene homopolymers, Stefan Reinicke for the synthesis of the azidoacetyl chloride, and Julia Ewert for RAFT polymerization of the PDMAEMA homopolymer. Andreas S. Lang is acknowledged for providing the azido-functionalized perylene bisimide. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 76 3.5 References (1) Hadjichristidis, N. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 857-871. (2) Hückstädt, H.; Göpfert, A.; Abetz, V. Macromol. Chem. Phys. 2000, 201, 296-307. (3) Junnila, S.; Houbenov, N.; Hanski, S.; Iatrou, H.; Hirao, A.; Hadjichristidis, N.; Ikkala, O. Macromolecules 2010, 43, 9071-9076. 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Polym. 2010, 70, 1-10. (58) Majewski, A. P.; Schallon, A.; Jérôme, V.; Freitag, R.; Müller, A. H. E.; Schmalz, H. Biomacromolecules 2012, 13, 857-866. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 84 26 28 30 32 34 36 38 0.0 0.2 0.4 0.6 0.8 1.0 normalied RI signal V e [mL] cBT2 PS1-N3 click: 3.25 h click: 11.5 h and 48 h Figure 3-S5. THF-SEC (RI-signal) of samples withdrawn during equimolar click reaction between cBT2 and PS1-N3 after 3.25 h (-), 11.5 h and 48 h (···). 26 28 30 32 34 36 0.0 0.2 0.4 0.6 0.8 1.0 cBT2 PS3-N3 click: 1 eq./3.25 h click: 1 eq./48 h click: 1.5 eq./25 h normalized RI signal V e [mL] Figure 3-S6. THF-SEC (RI signal) of click reaction between cBT2 and PS3-N3 with further addition of 0.5 equiv PS3-N3 48 h after start of the click reaction. 25 h after addition of the second portion of PS3-N3 a final sample was taken for SEC. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 85 Figure 3-S7. 1H NMR spectrum of µ-BT1S1 in CDCl3. The characteristic signals of the respective polymer blocks are highlighted. 26 28 30 32 34 36 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI signal V e [mL] cBT2 PEO-N3 click: 1 eq./1.75 h click: 1. eq./24 h click: 1.5 eq./3.5 h and click: 1.7 eq./1.75 h Figure 3-S8. THF-SEC (RI signal) of click reaction between cBT2 and PEO-N3 with stepwise addition of PEON3. Further 0.5 equiv PEO-N3 were added 24 h after start of the click reaction and 3.5 h later further 0.2 equiv PEO-N3 were added. The corresponding reaction times are relative to the point of addition of a further PEO-N3 portion. The excess of PEO-N3 is not visible as the PEO is adsorbed at the silica column used for removing copper catalyst. 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 86 3.6.3 Test Polymerization with P2VP and Click-DPE In contrast to polybutadienyl and polystyryl lithium, the living anion of poly(2vinylpyridine) is less nucleophilic and hence, the initial assumption was that it is not able to attack excess click-DPE. This would lead to the desired diblock copolymers with only one click-DPE at the block junction. If the 2VP would incorporate more units of click-DPE the diblock would carry more than on alkyne function, which would result in star terpolymers with more than three arms. Hogen-Esch et al. reported that the endcapping of P2VPLi with unsubstituted DPE is an equilibrium reaction9 and that even the addition of a large excess of 1,1-diphenylethylene to living poly(2-vinylpyridine) does not lead to a significant endcapping of the anion.10 Hirao et al. showed that silyl-protected hydroxylfunctionalized DPE, 1-(3-tert-butyldimethylsilyloxymethylphenyl)-1-phenylethylene did not undergo copolymerization with living poly(2-vinylpyridine) anions.11 Also, in previous tests on the endcapping of poly(2-vinylpyridine) with DPE functionalized polybutadiene macromonomers, we found no significant coupling between the two polymeric chains. Therefore, a test reaction was conducted to verify whether the alkyne-substituted clickDPE can be attacked by a living P2VPLi as a result of the altered electronic configuration of the DPE derivative. For this purpose, 2-vinylpyridine was polymerized in THF at -70 °C with sec-BuLi as initiator, yielding a molecular weight of 5 000 g/mol. Then, a 2-fold excess of the alkyne-functionalized click-DPE was added at the same temperature. Directly after injection, the color of the P2VPLi anion changed from red to violet giving a first qualitative indication for the addition of the click-DPE. 1H NMR determined the degree of alkyne functionalization as around 98%. MALDI-ToF MS supported a quantitative end-functionalization (Figure 3-S9A). Before addition the Mn of the P2VP was 4 750 g/mol. After 1 h reaction with click-DPE the Mn increased to 5 230 g/mol. These values indicate that click-DPE addition took place quantitatively, considering the accuracy of the determination method. Moreover, the molecular weight distribution shifted completely and in the case of the reflectron mode a distinct shift of the individual peaks was noticeable (Figure 3-S9B). This proved the presence of only one species after addition of click-DPE. The shift of the peaks with a difference of around 4.5 g/mol is in good agreement with the expected 3 g/mol difference (M(2VP) = 105.14 g/mol, M(click-DPE) = 318.53 g/mol; Δ = 318.53 g/mol - 3 x 105.14 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 87 g/mol = 3.11 g/mol). For example, the peak at m/z = 4791.6 in the P2VP precursor, i.e. before addition of click-DPE, corresponds to 44 repeating units of 2VP with a sec-butyl residue and Ag+ as counter-ion. This is in accordance with the theoretical m/z value of 4792.0. In the spectrum after click-DPE addition, the modified P2VP with the same number of repeating units and an additional click-DPE was detected at m/z = 5111.1. Also here, this value is consistent with the expected 5110.5 (= 4792.0 + 318.5). Hence, clickDPE adds to living poly(2-vinylpyridine) anions quantitatively, in contrast to other DPE derivatives reported in literature. Figure 3-S9. MALDI-ToF MS spectra of poly(2-vinylpyridine) before (black line) and after addition of clickDPE (cDPE, red line), recorded in linear (A) and reflectron mode (B) with AgTFA as ionization agent. In summary, the anion of the alkyne-functionalized DPE has a nucleophilicity comparable to P2VPLi, whereas the anion of unsubstituted DPE is more nucleophilic than P2VPLi. One reason for this might be the electron withdrawing effect of the tertbutyldimethylsilyl group which leads to a reduction of the electron density in the double bond and thus decreases the nucleophilicity of the click-DPE as compared to the unsubstituted DPE.12 As the living anion of the click-DPE is able to start the polymerization of P2VP and click-DPE also adds to P2VPLi, it is possible to copolymerize P2VP and click-DPE. Therefore, if the click-DPE is used in a slight excess and not under equimolar conditions, the DPE derivative might also be incorporated in or most probably after consumption of all 2VP monomer at the end of the P2VP block for the aimed PB-b-P2VP diblock copolymers. In contrast to butadiene and styrene, methacrylates are known to be not nucleophilic enough to attack diphenylethylene and its derivatitives.13,14 Conse4780 4800 4820 VP44 VP41cDPE1 ~ 4.5 g/mol 4750 4800 4850 4900 4950 VP45 VP42cDPE1 VP41cDPE1 VP44 m/z 2000 4000 6000 8000 P2VP-prec P2VP-DPE fit P2VP-prec fit P2VP-DPE m/z 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 88 quently, monomers like tBMA and DMAEMA can be polymerized as second block to obtain well-defined diblock copolymers with exactly one alkyne function at the block junction. Due to the altered reactivity of the click-DPE toward living P2VP-Li compared to other DPE derivatives both αand ω-alkyne modified P2VP homopolymers are accessible. In addition, alkyne mid-functionalized diblock copolymers with poly(2-vinylpyridine) as first and a methacrylate-type monomer as second block can be synthesized. 3.6.4 Characterization of ω-Azido Homopolymers Besides the qualitative proof of the presence of an azido function via IR (Figure 3-S10) the degree of azide-functionalization was additionally tried to be quantified by MALDIToF MS or 1H NMR. 4000 3500 3000 2500 2000 1500 1000 P t BMA-N3 PDMAEMA-N3 PEO-N3 relative transmission wavenumber cm-1 PS-1-N3 Figure 3-S10. IR spectra of the corresponding homopolymers show the characteristic stretching vibration of the azido group at ~ 2098 cm-1. In case of PS1-N3 and PEO-N3 mass spectrometry in reflectron mode was possible. The respective spectra are shown in Figure 3-S11 and 3-S12. In the case of the azidoterminated PS1-N3 only a small peak resembling the desired functional species is present. As already reported in literature formation of postsource metastable ions from azido-functionalized polymers is possible under high laser powers in reflectron mode.15 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 89 Therefore, by comparing the relative intensities of the azido-functionalized species and the corresponding metastable ion with the intensity of the unfunctionalized species, as main population the azido-terminated polystyrene is clearly concluded. We have to mention here that the peak at the left side of the proton-terminated polystyrene could not be assigned clearly. 3320 3360 3400 3440 3480 [PS29H+Ag]+[PS30H+Ag]+ [PS31N3-N2+Ag]+ metastable [PS30N3-N2+Ag]+ metastable [PS31N3+Ag]+ O O N3 x m/z = PSxN3 [PS30N3+Ag]+  ~ 104 Figure 3-S11. MALDI-ToF MS spectrum of PS1-N3 recorded in reflectron mode with AgTFA as ionization agent. In the MALDI spectrum of PEO-N3 (Figure 3-S12) two series of peaks were present. Here, the main population resembles the azido-terminated PEO. The second population could be attributed to azido-terminated PEO with a proton as counter ion. Another possible explanation could be deduced from SEC measurements, where a small second peak at lower elution volume was detected (around 6 wt.%, see Figure 3-S13). This originates from minimal residual amounts of thionyl chloride which was used for the synthesis of the azidoacetyl chloride and is hard to remove completely despite thorough purification.1 This population cannot take part in the click reaction. For the molecular characterization, only the major peak was taken into consideration. The molecular weight determined by MALDI-ToF MS was consistent with the values from SEC. Therefore, the second population in MALDI can be interpreted as the lower molecular weight side of the coupling product with thionyl chloride. Even though the main peak corresponds to the de- 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 90 sired functionalized PEO, one has to notice that chains where HN3 was eliminated (Δ ~43 g/mol) and chains which were terminated with a proton instead of the acid chloride (Δ ~5 g/mol) would not be distinguishable and could possibly be present. 6180 6200 6220 6240 6260 [PEO126SO+Ag]+[PEO127SO+Ag]+ = PEOx+ySO OS O O x y [PEO133N3+H]+[PEO134N3+H]+  ~ 44 = PEOxN3 O O N3 x [PEO131N3+Ag]+ m/z [PEO132N3+Ag]+ Figure 3-S12. MALDI-ToF MS spectrum of PEO-N3 recorded in reflectron mode with AgTFA as ionization agent. 27 28 29 30 31 32 33 34 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI signal V e [mL] PEO-N3 Figure 3-S13. THF-SEC (RI signal) of azido-functionalized poly(ethylene oxide). Both in case of PtBMA-N3 and PDMAEMA-N3 endgroup determination was not possible via MALDI-ToF MS. In contrast to PtBMA-N3 for the DMAEMA homopolymer calculation 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 91 of the degree of azido-functionalization was possible from the characteristic signals in the 1H NMR spectrum (Figure 3-S14). According to the calculated value of approximately 96 %, quantitative azido-functionalization of the polymer was assumed. Figure 3-S14. 1H NMR spectrum of PDMAEMA-N3 in CDCl3. The characteristic signals of the monomer unit and chain transfer agent are highlighted. 3.6.5 Click Reaction with ω-Azido-Functionalized Poly(tert-butyl methacrylate) Since we also synthesized an alkyne-functionalized diblock with DMAEMA as second block (cBD), a µ-BDT miktoarm star terpolymer was achievable by click reaction with the azido-terminated PtBMA. Similar to the click reactions with PDMAEMA-N3, the reaction conditions were not optimized. The ligation was conducted for 3 days with 1.3 equiv of azido-functionalized homopolymer to guarantee 100 % conjugation of the diblock. The SEC of the click-product after dialysis in methanol/isopropanol (2/1 v/v) is shown in Figure 3-S15 and a complete shift of the molecular weight distribution of the miktoarm star terpolymer compared to the diblock terpolymer was detected. This demonstrates the efficiency of our approach. Even though we were not able to remove all excess homopolymer, we showed that formation of a µ-BDT miktoarm star terpolymer is possi- 3 – A Modular Route for the Synthesis of ABC Miktoarm Star Terpolymers 92 ble starting from two different diblock copolymers. The molecular parameters of the obtained ABC miktoarm star terpolymer are summarized in Table 3-3. 14 15 16 17 18 19 0.0 0.2 0.4 0.6 0.8 1.0 cBD P t BMA-N3 µ -BDT, dial. normalized RI signal Ve [mL] Figure 3-S15. Salt-THF-SEC (RI-signal) of the µ-BDT miktoarm star terpolymers obtained after click reaction of alkyne-functionalized diblock cBD with the azido functionalized PtBMA-N3. 3.6.6 References (1) Reinicke, S.; Schmalz, H. Colloid Polym. Sci. 2011, 289, 497-512. (2) Gondi, S. R.; Vogt, A. P.; Sumerlin, B. S. Macromolecules 2007, 40, 474-481. (3) Lang, A. S.; Neubig, A.; Sommer, M.; Thelakkat, M. Macromolecules 2010, 43, 7001-7010. (4) Eßwein, B.; Möller, M. Angew. Chem. 1996, 108, 703-705. (5) Schmalz, H.; Lanzendörfer, M. G.; Abetz, V.; Müller, A. H. E. Macromol. Chem. Phys. 2003, 204, 1056-1071. (6) Schmalz, A.; Hanisch, M.; Schmalz, H.; Müller, A. H. E. Polymer 2010, 51, 1213-1217. (7) Mantovani, G.; Ladmiral, V.; Tao, L.; Haddleton, D. M. Chem. Commun. 2005, 2089-2091. (8) Coessens, V.; Matyjaszewski, K. J. Macromol. Sci. 1999, 36, 667-679. (9) Helary, G.; Fontanille, M.; Khan, I. M.; Hogen-Esch, T. E. Makromol. Chem. 1989, 190, 341-348. (10) Yin, R.; Hogen-Esch, T. E. J. Polym. Sci., Part A: Polym. Chem. 1994, 32, 363-368. (11) Hirao, A.; Murao, K.; Abouelmagd, A.; Uematsu, M.; Ito, S.; Goseki, R.; Ishizone, T. Macromolecules 2011, 44, 3302-3311. (12) Tsuda, K.; Ishizone, T.; Hirao, A.; Nakahama, S.; Kakuchi, T.; Yokota, K. Macromolecules 1993, 26, 6985-6991. (13) Hsieh, H. L.; Quirk, R. P. In Anionic Polymerization: Principles and Practical Applications; Marcel Dekker: New York, 1996. (14) Quirk, R.; Yoo, T.; Lee, Y.; Kim, J.; Lee, B. Adv. Polym. Sci. 2000, 153, 67-162. (15) Li, Y.; Hoskins, J. N.; Sreerama, S. G.; Grayson, S. M. Macromolecules 2010, 43, 6225-6228. 4 – “Woodlouse” Structures from µ-BVqT 93 4 – Counterion-Mediated Hierarchical Self-Assembly of an ABC Miktoarm Star Terpolymer Andreas Hanisch, André H. Gröschel, Melanie Förtsch, Markus Drechsler, Hiroshi Jinnai, Thomas M. Ruhland, Felix H. Schacher, and Axel H. E. Müller ABSTRACT: Directed self-assembly processes of polymeric systems represent a powerful approach for the generation of structural hierarchy in analogy to biological systems. Herein, we utilize triiodide as a strongly polarizable counterion to induce hierarchical self-assembly of an ABC miktoarm star terpolymer comprising a polybutadiene (PB), a poly(tert-butyl methacrylate) (PtBMA), and a poly(N-methyl-2-vinylpyridinium) (P2VPq) segment. Hereby, the miktoarm architecture in conjunction with an increasing ratio of triiodide versus iodide counterions allows for a stepwise assembly of spherical micelles as initial building blocks into cylindrical structures and superstructures thereof. Finally, micrometer-sized multicompartment particles with a periodic lamellar fine structure are observed, for which we introduce the term “woodlouse”. The counterion-mediated decrease in hydrophilicity of the corona-forming P2VPq block is the underlying trigger to induce this hierarchical structure formation. All individual steps and the corresponding intermediates toward these well-defined superstructures were intensively studied by scattering and electron microscopic techniques, including transmission electron microtomography. Keywords: ABC Miktoarm Star Terpolymer, Hierarchical Self-Assembly, Polyelectrolytes, Multicompartment Micelles, Poly(2-vinylpyridine) 4 – “Woodlouse” Structures from µ-BVqT 100 Before studying the self-assembly mechanism in detail, we were interested in the influence of the polymer structure itself on the obtained aggregates. Therefore, materials of similar chemical composition but different architecture were used, more precisely a diblock copolymer (B 109 V 81 ) and a linear triblock terpolymer (B 1108 V 142 T 93 ). However, only ill-defined aggregates were obtained from these linear polymers after quaternization and comparable preparation conditions (see representative micrographs of the triblock terpolymer in Figure 4-S4). These findings demonstrate that the miktoarm architecture itself is an important criterion for the hierarchical self-assembly into substructured particles. Hadjichristidis and co-workers already showed both experimentally and by theoretical investigations that the micellization behaviour of A 2 B miktoarm systems is different compared to linear AB diblock copolymers due to topological differences at the corecorona interface. 45 4.2.2.1 Hierarchical Superstructure Formation According to cryo-TEM (Figure 4-1D), the “woodlouse” aggregates can be preliminarily described as either superstructures from cylindrical micelles or lamellae. However, as the periodicity of these particles is in the size range of the diameter observed for the spherical micelles in Figure 4-1B, we assume these to act as primary building blocks. The different structures observed would then simply correspond to different levels of superstructure formation. Regarding the overall morphology, similar structures, such as laterally structured vesicles or layered structures, were already predicted by simulations of ABC miktoarm star terpolymers with two solvophobic blocks. 46,47 More detailed information about the superstructure formation was gained from investigations of the intermediate structures obtained under comparable preparation conditions (Figure 4-2A). Here, the aggregates clearly consist of several intertwined and partially wrapped-up cylindrical micelles. Again, the dimensions of the cylinders are in good agreement with the diameter of the spherical micelles (d cylinder = 27.5 ± 2.5 nm, as compared to d micelle = 24.5 ± 2.0 nm), which corroborates the assumption that spherical micelles are indeed the underlying building blocks. Staining with OsO 4 reveals a PB (or mixed PB/PtBMA) core of the cylinders (Figure 4-2B). Subsequently, these cylinders align in a parallel, ribbon-like fashion, which we regard as another intermediate level on the way toward highly periodic 4 – “Woodlouse” Structures from µ-BVqT 101 lamellar “woodlice”. Our assumption of a mixed PB/PtBMA core is further supported by the calculated phase segregation parameter, for PB/PtBMA, χN ~1.1. 48 According to the theory of phase separation for diblock copolymers, this is in the disordered regime due to the small degrees of polymerization for our system. 49,50 Furthermore, cast films of PBb-PtBMA diblock copolymers with comparable molecular weights did not show any phase separation in the bulk (results not shown). Cryo-TEM shows highly intertwined and entangled cylindrical micelles (Figure 4-2C). A surprisingly highly regular packing of individual cylinders can be observed in some cases, highlighted in the insets and gray scale analysis of Figure 4-2C. Remarkably, some of the structures show long-range order and dimensions of up to 1 µm in length.  Figure 4-2. TEM (A,B) and cryo-TEM micrographs (C) of intermediate micellar structures from µ-BVqT in aqueous solution. The final polymer concentration was 0.2 g/L for TEM and 0.4 g/L for cryo-TEM. Whereas for (A) no staining was performed, the sample in (B) was stained with OsO 4 . Here, the gray scale analysis of the highlighted area reveals a ribbon-like arrangement of cylinders. With the example of these intermediate structures, we further investigated the influence of two other parameters during the process of structure formation (for details see Supporting Information): (i) the solutions were thermally annealed, and (ii) the time in between quaternization in dioxane and dialysis to water was increased to up to 1 month. Whereas thermal annealing did not result in higher structural perfection at all, the structures were slightly more developed after 1 month in dioxane prior to dialysis (Figure 4-S5). Nonetheless, from all the TEM investigations discussed so far, different 4 – “Woodlouse” Structures from µ-BVqT 102 levels of hierarchy from approximately 20 nm to 1 µm were detected for µ-BVqT, starting from spherical micelles (level 1), which aggregate into cylinders (level 2), and finally into multilamellar superstructures (level 3). 4.2.2.2 Importance of the Nature of the Counterion It is known that methyl iodide can undergo photodecomposition to form free iodine. 51 Based on this fact, one hypothesis for the observed structural differences was the presence of varying amounts of elementary iodine in the respective dioxane solutions. In combination with the iodide counterion, this then forms triiodide, I 3- , which is a strongly polarizable counterion, 52,53 already described for quaternized poly(4-vinylpyridine) (P4VPq). 54 The increased hydrophobicity of P2VPq with triiodide as counterion is clearly demonstrated by DLS measurements of a P2VPq homopolymer in water (Figure 4-S8). Higher amounts of added iodine led to an increase of the hydrodynamic radii, which we assign to hydrophobic interactions. In contrast, chloride or methyl sulfate counterions did not lead to the formation of any hierarchically structured aggregates for the µ-BVqT system (see Supporting Information). However, addition of supplementary iodine to an aqueous micellar solution of µBVqT (Figure 4-1B) did not induce significant structural changes. We attribute this to two different reasons: first, owing to the reduced core dynamics, rearrangement processes are suppressed. Second and most important, iodine itself is not soluble in water but is solubilized by the formation of triiodide. However, the iodide counterions are mainly located within the micellar corona – approximately 90% according to investigations of quaternized poly(N,N-dimethylaminoethyl methacrylate) stars 55 – leading to very slow exchange processes. Consequently, we added different amounts of iodine to the µ-BVqT solution in dioxane prior to dialysis. The samples were allowed to equilibrate for 2 h to guarantee the conversion to triiodide and then dialyzed to water. Already the addition of 0.08 equiv of I 2 induced drastic structural changes (Figure 4-3A), as worm-like micelles and elongated superstructures were observed in contrast to spherical micelles in the absence of iodine (Figure 4-1B). Again, the diameter of these cylindrical aggregates refers to the initially observed spherical micelles (d micelle = 24.5 ± 2.0 nm and d cylinder = 25.0 ± 2.0 nm). The corona-forming P2VPq block can be clearly distinguished in cryo-TEM 4 – “Woodlouse” Structures from µ-BVqT 103 (inset in Figure 4-3A and Figure 4-S9A). For 0.25 equiv, almost exclusively superstructures from aggregated and intertwined cylindrical micelles are found. In addition, fewer protrusions and an increased tendency of the cylinders to form meander-like structures were observed. The inset in Figure 4-3B depicts an area where the cylinders form prestages of ribbons. When 0.42 equiv of I 2 was added, “woodlouse” aggregates with a periodic, multilayered structure were found (d lam = 19.5 ± 1.0 nm, Figure 4-3C). This pattern is also clearly visible in cryo-TEM, accompanied by areas where the lamellae are less densely packed (highlighted area in Figure 4-S9B) or particles which seem to be trapped as cylindrical superstructures (inset in Figure 4-S9B). A minor fraction of micelles and cylinders was found, as well.  Figure 4-3. TEM micrographs from 0.2 g/L aqueous micellar solutions of µ-BVqT after dialysis in the presence of different amounts of iodine. The solutions were prepared with 0.08 (A), 0.25 (B) and 0.42 (C) equiv of iodine with respect to P2VPq monomer units. The inset in (A) shows the corresponding cryo-TEM micrograph. The schematic illustrations represent the dominant aggregate morphology. For the respective sample without additional iodine, see Figure 4-1B. To evaluate this lamellar packing within the particles in more detail, additional SAXS measurements were conducted from freeze-dried powders of the sample depicted in Figure 4-3C. The SAXS pattern (Figure 4-4A) shows peaks with a q ratio of 1:2:3, representing the [100], [200], and [300] reflections, thus confirming the lamellar structure. The long period was calculated to d lam = 20.0 ± 1.5 nm and is in perfect agreement with the values observed in TEM (d lam = 19.5 ± 1.0 nm). In the SAXS pattern for the intermediate structure (as shown in Figure 4-S5), the reflections were less pronounced (Figure 44B). This was already expected from the TEM images (Figure 4-S5), which reveal a less regular arrangement. However, the assumption of an overall lamellar morphology and the presence of the [100] and [200] reflections allow the calculation of d lam = 22.5 ± 2.0 4 – “Woodlouse” Structures from µ-BVqT 104 nm. Comparable long periods from “woodlice” and aggregated cylinders further support our proposed mechanism of superstructure formation. Figure 4-4. SAXS pattern of a freeze-dried powder from (A) the “woodlouse” structure (Figure 4-3C) and (B) the intermediate structure (Figure 4-S5). The integer numbers indicate the relative reflex positions, and the inset in A depicts the scattering pattern observed at the 2D detector. These different levels of hierarchical self-assembly of µ-BVqT into spherical micelles (level 1), followed by cylinders (level 2), superstructures thereof and, finally, the stacking and back-folding of lamellae into compartmentalized micrometer-sized polymer particles (level 3) can be attributed to surface minimization due to decreasing hydrophilicity of the P2VPq corona triggered by the presence of triiodide counterions. Hereby, the superstructure formation of lamellae via folding (or stacking) is supported by simulations on the sphere-cylinder-lamellae transition in diblock copolymer systems. 56 As we are most probably dealing with non-equilibrium structures formed during dialysis, the folded cylinders are supposed to be transient intermediate structures on the way toward lamellae. Similar structures were already reported for linear ABC triblock terpolymers, where either the addition of a diamine in the case of poly(acrylic acid) as solubilizing block 31-33 or changes in solvent quality for the P2VP corona 23 triggered the formation of superstructures. However, in our case, this is clearly caused by changes in the polarizability of the counterion, triggered by the addition of iodine. The amount of triiodide represents the essential trigger here. Both the assembly pathway and a delicate balance of iodide/triiodide regulate structural precision and the overall colloidal stability of the substructured particles. Furthermore, here, the well-defined compact structures 4 – “Woodlouse” Structures from µ-BVqT 105 clearly evolve through a complex aggregation and fusion via cylindrical building units in contrast to the stacking of disk-like structures as reported in literature. 23,31-33 The system presented here is relative simple as no sophisticated preparation pathways utilizing bifunctional additives have to be applied, and the structures are obtained in aqueous solution rather than in solvent mixtures. Also, the directed self-assembly is induced by the monovalent counterion, and the structural integrity is rather unaffected by the pH of the solution (see aggregates within acidic media of pH 3 as depicted in Figure 4-S10) 4.2.3 Structural Characterization of “Woodlouse” Aggregates So far, we have described the triiodide-mediated hierarchical self-assembly of µ-BVqT into compartmentalized particles. The highly periodic internal fine structure is confirmed by gray-scale analysis of the TEM micrographs of individual particles (Figure 4-5A). Here, the darker domains correspond to the P2VPq phase containing iodide/triiodide counterions (d 1 = 8 nm), whereas the contrast is inverted when staining with OsO 4 was performed (selective for the PB phase, d 2 = 11 nm Figure 4-5B). The wider lamellae presumably consist of a mixed PtBMA/PB phase as already discussed above. In cryo-TEM, even three different repeating distances are visible (Figure 4-5C). Here, the observed periodicities can be explained in a similar way. The 8 nm of d 2 represent a mixed phase of PB and PtBMA, which is slightly broadened in TEM (d 2 in Figure 4-5B), as the lamella is flattened and collapsed onto the carbon film in the dried state. On the other hand, both d 1A and d 1B represent P2VPq, serving as the corona for the mixed PB/PtBMA domains. At the interface (d 1A ), the density of P2VPq chains is higher as compared to the periphery (d 1B ), leading to an increased electron density. The proposed chain packing and the corresponding dimensions are illustrated in Figure 4-5D. Hereby, the individual lamellar sheets of the superstructures can either stack (white arrow in Figure 4-5C) or back-fold (black arrows in Figure 4-5C), leading to “open” or “closed” structures at the edge of the particles. 4 – “Woodlouse” Structures from µ-BVqT 106 Figure 4-5. TEM micrographs of “woodlouse” aggregates of µ-BVqT obtained via dialysis from dioxane into water (A,B). The concentration was 0.1 g/L. In (A), the contrast emerges solely from the iodide counterion of the P2VPq phase, whereas (B) was stained with OsO 4 . In cryo-TEM (C), a regular pattern of three distances is visible. The concentration was 0.6 g/L. The corresponding gray scale analyses are shown below the micrographs. The proposed arrangement of the miktoarm star terpolymers is illustrated in (D). We additionally performed cryo-TEM at different tilt angles (Figure 4-S11 and video 4S3). Thereby, the influence of the woodlouse orientation within the vitrified film was examined, and from the projections at different tilt angles, a circular cross section of the aggregates can be clearly deduced. Again, the presence of flat two-dimensional assemblies can be excluded, which is additionally supported by SEM of the dried particles (Figure 4-S12). In the cryo-TEM tilt images, the internal fine structure is only visible if the particles are oriented perpendicular to the beam direction. If the stage is tilted further, the structural features blur and, finally, disappear completely. The fact that such structural features are only visible under specific viewing angles has already been observed. 57 Cross-sectional analysis of the “woodlice” within thin film cuts of epoxy resin embedded particles (Figure 4-6A and 4-S13) clearly showed a periodic fine structure, further confirming our assumption that the particles are not hollow. When the sample was treated with OsO 4 (staining of PB, Figure 4-6A), undulated lamellae are visualized as al- 4 – “Woodlouse” Structures from µ-BVqT 107 ready discussed for the intermediate structures. The rather undulated shape of the PB/PtBMA lamella might be related to partial demixing of PB and PtBMA, despite the rather low χN. This could be a consequence of the longer DP of the PB block (109) as compared to PtBMA (DP = 53), as well as different χ for the PB/P2VP and the PtBMA/P2VP interactions, leading to minimization of the PB/P2VP interface. 48,58 Additionally, from DSC measurements of the miktoarm star terpolymer, the presence of a T g at -2 °C also hints toward the presence of a separated PB phase in the bulk state (Figure 4-S14). This leads to a direct PtBMA/P2VPq interface, accompanied by an interface of P2VPq with the adjacent mixed PB/PtBMA phase and, finally, a pure PB domain without a PB/P2VPq interface. The proposed arrangement of the constituting segments is illustrated in Figure 4-6B. Staining with OsO 4 enhances contrast mainly in the PB phase, but also the mixed phase appears darker (Figure 4-6A), whereas the pure PtBMA domains appear brighter. Similarly, the TEM micrographs of the cylindrical intermediate aggregates under the same sample preparation method also hint toward an undulated phase boundary (Figure 4-S6B). Finally, the volume morphology of the “woodlice” was investigated using TEM tomography (TEMT). We therefore prepared thicker slices (~150 nm) of the resin-embedded sample and performed staining with OsO 4 to provide maximum contrast. In Figure 4-6C, a three-dimensional reconstruction of a slice of a woodlouse particle with view into the lamellar bulk morphology is shown. Additionally, Figure 4-6D shows three different slices of the same reconstruction for a single particle. Both three-dimensional reconstructions clearly confirm the presence of lamellae with an undulated surface throughout the entire sample (also see video 4-S4). Hereby, the formation of well-defined lamellar structures as compared to micellar clusters (observed for linear polymers; see Figure 4-S4) is probably a direct consequence of the miktoarm architecture. In accordance with both theory and experimental work, 59 the increased segmental density has a distinctive effect on the surface curvature and, in the case of our system, facilitates the formation of lamellae with low surface curvature. 4 – “Woodlouse” Structures from µ-BVqT 108 Figure 4-6. (A) TEM micrograph of 50 nm thick cuts from freeze-dried and embedded samples of µ-BVqT aggregates, stained with OsO 4 . (B) Schematic illustration of the block arrangement within the “woodlouse” structure is depicted, with the two possibilities of bent (upper part) and stacked lamellae (lower part). The gray areas resemble the P2VPq phase; PtBMA is red, and PB is blue. The violet domains represent a mixed PB/PtBMA phase. TEM tomography 3D reconstructions of a slice of the “woodlouse” structure (C) and cross-sectional analysis of a single particle (D). The tomography images were obtained from a 150 nm thick cut, which was additionally treated with OsO 4 to selectively stain the mixed PB/PtBMA phase (plotted in green). The approximate length of the long marked edge of the reconstructions is 280 nm in (C) and 220 nm in (D). 4 – “Woodlouse” Structures from µ-BVqT 109 4.3 Conclusions We have demonstrated the hierarchical self-assembly of an ABC miktoarm star terpolymer into substructured particles of up to 1µm in size, which we term “woodlouse” particles. Spherical micelles with a mixed PB/PtBMA core and a P2VPq corona act as the basic building blocks (level 1). Stepwise aggregation of these results in cylindrical micelles (level 2), followed by superstructures thereof, and finally compartmentalized particles of up to 1 µm in size. These particles feature a highly periodic, lamellar fine structure (level 3). The presence and amount of triiodide as a highly polarizable counterion for the P2VPq corona is an essential trigger to induce this superstructure formation into different levels of hierarchy (Figure 4-7). All intermediates of increasing hierarchy involved on the way to the final “woodlice” are visualized by TEM and cryoTEM. The iodide/triiodide system is an elegant approach for directing the hierarchical self-assembly of such materials. The nature of the counterion, the miktoarm star architecture, and the assembly pathway are essential parameters and influence structural perfection and the final morphology. To recapitulate, a detailed understanding of the self-assembly mechanism into complex superstructures was obtained, which bear structural similarities to biological systems such as mitochondria. Applying these results to other miktoarm star terpolymer systems with higher segmental incompatibility might lead to completely phase-separated cores and represents an interesting approach to core-compartmentalized structures of complex shape. Further functionalization and modification of these structures will enable the preparation of a variety of stimuliresponsive highly structured materials with defined internal periodicity. The question arises whether this approach is applicable to other miktoarm star systems containing polycationic segments in general or whether the combination of P2VPq and a “dynamic”, low T g segment like PB is a prerequisite. This will be the subject of future work. The use of iodide/triiodide as a setscrew to direct the self-assembly of different materials into well-defined hierarchical superstructures would be advantageous and desirable. 4 – “Woodlouse” Structures from µ-BVqT 116 4.5 References (1) Whitesides, G. M.; Grzybowski, B. 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London A 1970, 317, 319-340. 4 – “Woodlouse” Structures from µ-BVqT 119 4.6 Supporting Information 4.6.1 Additional Experimental Section Synthesis Quaternization of 2-Vinylpyridine Homopolymers (P2VP) and Preparation of Aqueous Solutions The quaternization procedure was similar to the method described for the miktoarm star terpolymer with slight modifications. After three days of reaction with methyl iodide 10 vol% of water were added to the partially precipitated reaction mixture in dioxane and it was allowed to stir for an additional day. Afterward, it was first dialyzed to a mixture of dioxane:water (90:10) and then the solvent composition was gradually increased to (80:20). The obtained stock solution in the dioxane:water mixture was afterward dialyzed to water. The triiodide complexes were prepared in the same manner as described in the manuscript. Preparation of µ-BVqT with Different Counterions The exchange of the iodide counter ion with chloride was achieved by dialysis of the dioxane stock solution to 50 mM solution of LiCl in THF. Afterward the solution was dialyzed against pure THF to remove excess salt. Before dialysis to water, the solvent was again changed to dioxane through dialysis. The quaternizations with dimethyl sulfate were conducted either in THF or dioxane. A polymer solution was prepared with a concentration of 2 g/L and then degassed for 15 minutes. Afterward, 10 equiv of dimethyl sulfate regarding 2VP units were added and the reaction mixture was allowed to stir at 40 °C for 3 days. Finally the solution was purified from the excess quaternization agent by dialysis with the respective reaction medium. After dilution to a concentration of 1 g/L the solutions were dialyzed to water. Characterization Size Exclusion Chromatography (SEC) SEC measurements were performed on a set of 30 cm SDV-gel columns (5 µm bead size, with pore sizes of 10 5 , 10 4 , 10 3 and 10 2 Å) using refractive index and UV (λ = 254 nm) 4 – “Woodlouse” Structures from µ-BVqT 120 detection. THF was used as eluent at a flow rate of 1 mL/min. Toluene was used as internal standard and the system was calibrated with PS and 1,4-PB standards. 1 H NMR Spectroscopy 1 H NMR spectra were recorded on a Bruker Ultrashield 300 spectrometer at an operating frequency of 300 MHz. CDCl 3 was used as solvent and tetramethylsilane as internal standard. Matrix-Assited Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDIToF MS) MALDI-ToF MS analysis was performed on a Bruker-Reflex III apparatus equipped with a N 2 laser (λ = 337 nm) at an acceleration voltage of 20 kV. trans-2-[3-(4-tert-Butylphenyl)- 2-methyl-2-propenyliden]malonitrile (DCTB, Fluka, 99,0 %) was used as matrix and silver trifluoroacetate (AgTFA, Sigma-Aldrich, 99.99%) as ionization agent. Samples were prepared from THF solution by mixing matrix, polymer and salt in a ratio of 20/5/1 (v/v). Scanning Electron Microscopy (SEM) The particles were analyzed by field emission scanning electron microscopy on a Zeiss LEO 1530 Gemini microscope equipped with a field emission cathode operating at 0.5-5 kV. Specimen preparation was accomplished as follows: silicon wafers were cleaned using isopropanol and acetone in a standard procedure. To obtain areas of different concentration of particles, the wafer was first dip-coated from 0.02 g/L solution and then a further drop of the solution was deposited and allowed to dry. The specimens were coated with a thin platinum layer using a sputter coater (Cressington 208HR) to render the specimen conductive. Dynamic Light Scattering (DLS) DLS measurements were performed on an ALV DLS/SLS-SP 5022F compact goniometer system with an ALV 5000/E cross correlator and a He-Ne laser (λ = 632.8 nm). The measurements were carried out in cylindrical scattering cells (d = 10 mm) at an angle of 90° and a temperature of 20 °C. Prior to the light scattering measurements, the sample solutions were filtered using nylon filters (Magna, Roth) with a pore size of 5 µm. The CON- 4 – “Woodlouse” Structures from µ-BVqT 121 TIN algorithm was applied to analyze the obtained correlation functions. Apparent hydrodynamic radii were calculated according to the Stokes-Einstein equation. DSC Thermal analysis and determination of the glass transition temperatures was performed on a Perkin–Elmer Diamond DSC at a heating rate of 20 K/min. 4.6.2 Synthesis and Characterization of PB-arm-P2VP-arm-PtBMA Miktoarm Star Terpolymer The synthesis of the PB-arm-P2VP-arm-PtBMA miktoarm star terpolymer was accomplished by combining sequential anionic polymerization with azide-alkyne Huisgen cycloaddition. Therefore, an alkyne-functionalized polybutadiene-b-poly(2-vinylpyridine) (PB-b-P2VP) diblock copolymer was directly prepared by anionic polymerization using an alkyne-functionalized DPE derivative (click-DPE). The molecular characterization of the diblock copolymer is listed in Table 4-S1. As already reported, in the case of the clickDPE, incorporation in, or most probably at the end of the poly(2-vinylpyridine) (P2VP) block takes place. This leads to diblock copolymers which bear additional alkynefunctions in or at the end of the P2VP block, as the DPE derivative is used in excess. Nevertheless, under appropriate reaction conditions, i.e. using only a slight excess of clickDPE, low temperatures and short reaction times an overall degree of alkynefunctionalization of 116 % was achieved. 1 Taking into account the errors of the determination method this resembles only a slight over-incorporation of click-DPE into the material. The alkyne group is then used to attach the third block, poly(tert-butyl methacrylate) (PtBMA), via azide-alkyne Huisgen cycloaddition. Thus, in the following click reaction an excess of 10% of azido-functionalized homopolymer chains relative to the diblock chains was used to ensure that all diblock chains undergo conjugation. As a direct consequence the obtained miktoarm star terpolymer contains a minor fraction with more than one chain of PtBMA. Figure 4-S1 displays the SEC traces of the precursor homopolymer and diblock copolymer, and the obtained miktoarm star terpolymer. Table 4-S1 lists the corresponding characterization data. Due to the use of an azido-functionalized ATRP initia- 4 – “Woodlouse” Structures from µ-BVqT 122 tor for the PtBMA polymerization in case of recombination polymers bearing two azido end-groups are formed. Therefore, a small percentage of α,ω-azido-bifunctionalized homopolymer could lead to H-shaped miktoarm star terpolymers and explain the observed higher molecular weight shoulder. From test click reactions with PS homopolymers, where the bromo-function was transformed into an azide after polymerization, no such coupling shoulder was detected, supporting our assumption. Table 4-S1. Molecular Characteristics of Poly(tert-butyl methacrylate) (T), Polybutadiene-block-poly(2-vinylpyridine) (BV), and µ-BVT Polymer a M n b / kg/mol PDI c T 53 7.6 1.17 B 109 V 81 14.7 1.02 µ-B 109 V 81 T 53 22.2 1.07 a The subscripts denote the degrees of polymerization of the corresponding blocks as calculated from the respective number average molecular weights. b For the PtBMA homopolymer the number average molecular weight was determined by SEC using a PtBMA calibration. For the diblock copolymer the molecular weight was calculated from 1 H NMR using the molecular weight of the PB as reference. This was measured directly via MALDI-ToF MS. For the ABC miktoarm star terpolymer the overall molecular weight was calculated from the corresponding precursor polymers. c Determined by SEC in THF calibrated with PS standards or in the case of PtBMA homopolymer with PtBMA standards. Figure 4-S1. SEC eluograms of the PB-arm-P2VP-arm-PtBMA miktoarm star terpolymer (µ-BVT), the precursor diblock copolymer (PB-b-P2VP), and the homopolymer (PtBMA). In all cases the RI signal is shown.  26 28 30 32 34 36 38 0.0 0.2 0.4 0.6 0.8 1.0 normalized RI detection V e [mL] PB b P2VP P t BMA " BVT 4 – “Woodlouse” Structures from µ-BVqT 123 4.6.3 Self-Assembly of µ-BVqT Figure 4-S2. Intensity-weighted DLS CONTIN plot (A) of quaternized µ-BVT (µ-BVqT) in 1 g/L dioxane solution, R h,app = 12.5 nm.  Figure 4-S3. Overview of various cryo-TEM images of different “woodlouse” aggregation forms observed in ~0.6 g/L aqueous solutions of µ-BVqT. 4 – “Woodlouse” Structures from µ-BVqT 124  Figure 4-S4. (A) TEM and (B) cryo-TEM micrographs from aqueous solutions of linear B 1108 Vq 142 T 93 at 0.2 and 0.65 g/L, respectively. After dialysis to water precipitation occurred gradually. Whereas the image in (A) is unstained, the inset in (A) shows an enlarged area after staining with OsO 4 . In both cases micellar clusters can be seen, without any internal fine structure (see staining in the inset in (A) or the brighter areas in the cryo-TEM in (B)). Please note that the molecular weight of the triblock terpolymer was much higher and also the molar fractions were different to the studied miktoarm star terpolymer µ-B 101 V 81 T 53 . Additionally, the sequence was PB-b-P2VP-b-PtBMA due to the restrictions of anionic polymerization and therefore the middle block forms the corona. This leads to a folding of the polymer chain to shield the hydrophobic end blocks. 4.6.3.1 Intermediate Structures of “Woodlouse” Aggregates The dioxane solution of µ-BVqT, which yielded the intermediate structures (Figure 4-2), was allowed to age for an additional month before dialysis to water. TEM analysis showed that the obtained aggregates were of slightly higher structural order and the internal fine structure of the “woodlouse” was already visible for some objects (Figure 4S5A). Nevertheless, the structures seem to be kinetically trapped during the process, as further ageing of aqueous solutions did not lead to any increase in order. As observed by cryo-TEM the superstructures were more densely packed (Figure 4-S5B). Less cylindrical protrusions emanate from the center of the aggregates, and the gray-scale analysis confirms three periodic distances (d 1 = 4.5 ± 1.0 nm, d 2 = 4.5 ± 1.0 nm, d 3 = 9.0 ± 1.0 nm. The structure was further visualized using different tilt angles in cryo-TEM (Supporting videos 4-S1 and 4-S2), confirming a 3-dimensional array of the cylinders.  4 – “Woodlouse” Structures from µ-BVqT 125  Figure 4-S5. TEM (A) and cryo-TEM micrographs (B) of intermediate micellar structures of µ-BVqT obtained by dialysis to water after ageing of the dioxane solution for one month. The final polymer concentration was 0.2 g/L for TEM and 0.4 g/L in case of cryo-TEM. The upper inset in (B) displays a gray-scale analysis of the highlighted area.  We further prepared thin film cuts of this structural intermediate by freeze-drying of the solution and embedding the particles into an epoxy resin. The corresponding TEM micrographs from thin slices (with and without additional OsO 4 staining) are shown in Figure 4-S6 and an internal lamellar structure can be observed (black arrows in Figure 4-S6). Additionally, OsO 4 staining revealed an undulated shape of the lamellae, which can also be interpreted as a dense packing of spheres (Figure 4-S6B). The same observation was made for some of the cylindrical structures (white arrows in Figure 4-S6B). As indicated by the white arrows for the non-stained micrographs in Figure 4-S6A (only P2VPq is visible), cylindrical protrusions with a tubular nature were also identified. These findings confirm our initial assumption of cylindrical micelles as intermediates. Furthermore, the absence of structures with a diffuse core region supports the assumption of compact particles. The presence of both cylindrical protrusions and elongated particles of rather compact shape with a spherical cross section were further confirmed via SEM measurements (Figure 4-S7). 132 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 133 5 – Hierarchical Self-Assembly of Miktoarm Star Polymer Systems Containing a Polycationic Segment: A General Concept Andreas Hanisch, André H. Gröschel, Melanie Förtsch, Tina I. Löbling, Felix H. Schacher, and Axel H. E. Müller ABSTRACT: We recently introduced a concept for the counterion-mediated hierarchical self-assembly of amphiphilic miktoarm star terpolymers in aqueous media into micrometer-sized compartmentalized particles with a highly periodic lamellar fine structure (“woodlice”). Herein, we extend this concept to different miktoarm star polymer systems containing a polycationic segment. The presence of a poly(N-methyl-2vinylpyridinium) (P2VPq) block and its interaction with iodide/triiodide counterions is crucial. In analogy to linear diblock copolymer systems the hydrophilic/hydrophobic balance of polybutadiene-arm-poly(N-methyl-2-vinylpyridinium iodide)-arm-polystyrene miktoarm star terpolymers determines the morphology of the primary building blocks (spherical micelles and cylindrical micelles/vesicles) and the obtained superstructures (stacked lamellar structures and multilamellar vesicles) during this hierarchical process. When an ABA’ miktoarm star copolymer (polystyrene-arm-poly(N-methyl-2vinylpyridinium iodide)-arm-polystyrene) without a dynamic core-froming block was investigated, a different mechanism into “woodlouse”-structured aggregates via aggregation and deformation of intermediate vesicles was found. The individual steps of the 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 134 different self-assembly processes were investigated by transmission electron microscopy and additionally supported by dynamic light scattering, differential scanning calorimetry, and small-angle X-ray scattering. Keywords: Miktoarm Star Polymer, Polyelectrolyte, Hierarchical Self-Assembly 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 135 5.1 Introduction One straightforward method to control the morphology of self-assembled structures from block copolymers is changing the volume fraction of the constituting segments. This led to a diversity of morphologies in the bulk 1-4 and in solution. 5-10 In the latter case, predominantly spherical micelles are formed, but also cylindrical or vesicular structures, as well as the corresponding intermediates have been reported. 11 Despite the plethora of nanostructures being accessible today, the structural diversity and complexity reached in biological systems still represents the ultimate benchmark. Here, self-organization of diverse biomacromolecules induces hierarchy over several length scales through intraand intermolecular interactions by encoding utmost functionality via the individual building blocks. Such processes can be considered as convergent since the cooperativity of a multitude of (different) weak interactions leads to the formation of primary building blocks, which further assemble into structures of increased complexity at a thermodynamic minimum. The accumulation of weak interactions within and between the building blocks compensates for the loss in entropy. 12,13 For example, proteins fold into complex three-dimensional structures guided by the information encoded within the primary structure of the amino acid sequence. 14 Commonly, these structures are monodisperse, as shown for the tobacco mosaic virus (TMV), where 2130 identical protein subunits arrange around a single strand of RNA in a helical fashion to produce a rigid rod of 18 x 300 nm. 15 Even though the dimensions obtained in natural systems are difficult to reach by directed self-assembly of synthetic macromolecules in general, this still provides a straightforward approach for the design of well-defined functional materials. Increasing the transcripted chemical information of the system by an additional block (AB to ABC) already leads to a drastic increase of the mere number of morphologies being accessible. 16 This is a consequence of three unlike polymer-polymer interaction parameters and the possibility of solvents being selective for one or two segments. Depending on the individual solubility, this enables the preparation of coronaor core-compartmentalized structures. 17-19 In this context Hillmyer, Lodge and co-workers gave a detailed investigation of the compartmentalization in micellar systems by using miktoarm star 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 136 terpolymers. Through this unique macromolecular architecture “hamburger” micelles, segmented worm-like micelles, and substructured vesicles could be obtained as corecompartmentalized analogs to spherical, worm-like and vesicular structures. 20-22 Moreover, in the field of triblock terpolymers it is well demonstrated that the complexation with single stranded DNA 23 or the use of diamines as additives 24,25 display additional triggers for hierarchical structure formation. Especially “kinetic control” allowed for the stepwise self-assembly of poly(acrylic acid)-block-poly(methyl acrylate)-blockpolystyrene triblock terpolymers into toroids, stacks of disk-like micelles, and lamellar phase separated droplets. Hereby, the structures were influenced by the polymer sequence, composition, the nature of the divalent counterion, the solvent mixture, and the preparation pathway and the control of these parameters allowed for manipulation of the obtained aggregation forms. 25-30 Recently, we reported the counterion-directed self-assembly of a polybutadiene-armpoly(N-methyl-2-vinylpyridinium iodide)-arm-poly(tert-butyl methacrylate) (µ-BVqT) miktoarm star terpolymer into multilayered “woodlouse” aggregates, taking advantage of both the polymer architecture and the tunability of the counterion. 31 The presence of iodide as counterion for the quaternized poly(2-vinylpyridine) segment was crucial to achieve superstructure formation. Iodine as additive led to the formation of triiodide, a highly polarizable counterion, which induced hierarchical self-organization from spherical micelles into cylindrical structures and well-defined superstructures thereof. Besides the nature of the counterion and the miktoarm star architecture also the preparation pathway was essential to obtain well-defined aggregates. Here, we try to obtain a more detailed insight into the underlying mechanism of this process by expanding it to other miktoarm star polymer systems. We take advantage of our newly developed modular approach for the straightforward synthesis of such materials. 32 First, the influence of the molecular composition on the obtained building units from three different polybutadiene-arm-poly(N-methyl-2-vinylpyridinium iodide)-arm-polystyrene miktoarm star terpolymers (µ-BVqS) and the resulting counterion-directed superstructures will be elaborated. In this context, the P2VPq corona was additionally utilized for the generation and hosting of Au nanoparticles. Second, the triiodide-directed self-assembly of a 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 137 polystyrene-arm-poly(N-methyl-2-vinylpyridinium iodide)-arm-polystyrene miktoarm star copolymer (µ-SVqS’) as a model system without a low T g segment is investigated. 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 138 5.2 Experimental Part Synthesis Materials Butadiene (Rießner-Gase) was passed through columns filled with molecular sieves (4 Å) and basic aluminium oxide and stored over dibutylmagnesium (1 M solution in heptane, Aldrich). 2-Vinylpyridine (2-VP, Aldrich) was degassed, stirred with triethylaluminium (1 M solution in hexanes, Aldrich) and condensed on a high vacuum line. Styrene (BASF) was purified in a similar manner, except that it was stirred with dibutylmagnesium (1 M solution in heptane, Aldrich) instead of triethylaluminium. THF (Sigma-Aldrich) was distilled from CaH 2 and Na/K alloy. sec-Butyllithium (Acros, 1.3 M in cyclohexane/hexane: 92/8) was used without further purification. tert-Butyl methacrylate (tBMA, Sigma-Aldrich) and styrene (Sigma-Aldrich) for ATRP were filtered over basic aluminium oxide before use. N,N,N′,N′,N′′- Pentamethyldiethylenetriamine (PMDETA) and CuBr were purchased from Aldrich and distilled and degassed or treated with pure acetic acid and filtered, respectively. 1-(4-tert-butyldimethylsilyl)ethynylphenyl)-1-phenylethylene (click-DPE) was synthesized from 1-(4bromophenyl)-1-phenylethylene as already reported 32 . The aqueous solutions were prepared with distilled desalinated water. All other chemicals were of analytical grade and used as received. The dialysis membrane used for all steps was purchased from Roth (Spectra Por), with a molecular weight cut-off (MWCO) of 1 000 g/mol. Synthesis and Quaternization of Miktoarm Star Terand Copolymers The detailed procedures for the synthesis of the alkyne mid-functional diblock copolymers (polybutadiene-block-poly(2-vinylpyridine)) and azido-functionalized homopolymers (polystyrene and poly(tert-butyl methacrylate)) have already been reported elsewhere. 32 Similarly, another alkyne mid-functionalized diblock copolymer, polystyrene-block-poly(2vinylpyridine) (PS-b-P2VP), was also synthesized via anionic polymerization in THF utilizing the alkyne-substituted diphenylethylene (click-DPE) derivative. Therefore, styrene was initiated with sec-BuLi at -70 °C and allowed to polymerize at this temperature for 25 minutes before the click-DPE was added (1.1 equiv relative to living chains). After stirring at -50 °C for 2 h the temperature was set to -70 °C and 2VP was added and allowed to polymerize for 5 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 139 minutes before termination with degassed isopropanol. The polymer was purified by precipitation in water. Finally, the ligations of the alkyne-functionalized diblock copolymers with the azidofunctionalized homopolymers were conducted by azide-alkyne Huisgen cycloaddition. 33 Therefore, a mixture of the diblock copolymer and 1.1 equiv of the respective homopolymer were dissolved in THF at a concentration of ~20 g/L and degassed for 10 min. After addition of 1 equiv CuBr, the solution was degassed for further 15 min. PMDETA (1 equiv) was added to complex the copper and start the reaction, which was followed by SEC. After 2 days, the resulting miktoarm star terpolymers were purified by passing the solution through a small column with basic alumina to remove copper and finally freeze-dried from dioxane. Preparation of Aqueous Micellar Solutions The transformation of the P2VP compartment into a strong cationic polyelectrolyte (P2VPq) was conducted using methyl iodide as quaternization agent. The detailed procedure is given in the literature. 31 To simplify matters, in case of all quaternized solutions the given concentrations resemble the concentration of the pristine miktoarm star terpolymer before the modification, thus neglecting the increase of mass due to quaternization. For the preparation of the triiodide complexes an iodine stock solution was prepared with dioxane as solvent at 15 g/L. This iodine solution was then added to the dioxane solution of the quaternized star terpolymers until the desired ratio of iodine to amino-function was achieved. Afterwards, the solutions were stirred for 2 h and then treated with ultrasound for 15 min. Subsequently, the solutions were dialyzed against water, as described above and obtained with concentrations ranging from 0.3 to 0.7 g/L. Some of the solutions were colloidally stable over months, whereas others sedimented with time. Generation of Au Nanoparticles To 1.5 mL of an aqueous solution of µ-BVq1S, which was obtained from dialysis of the corresponding dioxane solution with additional 0.25 equiv I 2 (regarding P2VPq units), was added a highly concentrated aqueous solution of HAuCl 4 until an Au:N ratio of 0.6 was reached. Directly after addition, precipitation occurred, and therefore the sample was treated with ultrasound for 30 minutes. After stirring for 90 minutes the solution was subjected to centrifugation. The supernatant liquid was decanted and the solid content was redispersed in 1.5 mL 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 140 of water. A freshly prepared solution of NaBH 4 was added (5 M equiv with respect to Au) and then the solution was stirred over night. After centrifugation to remove excess reagents, the solid content was again redispersed in 1.5 mL of water to obtain a solution of characteristic red-violet color. Characterization Transmission Electron Microscopy (cryo-TEM and TEM) TEM micrographs were taken with a Zeiss CEM 902 or 922 OMEGA electron microscope operated at 80 kV or 200 kV, respectively. Both machines were equipped with an in column energy filter. For sample preparation 2 µL of the solution (typically 0.1-0.2 g/L) were deposited on a hydrophilized TEM grid (copper, 200 mesh). Afterwards, the remaining solvent was removed with a blotting paper. In another preparation pathway a drop of the solution was deposited on a TEM grid on a piece of parafilm, immediately frozen with liquid nitrogen and afterwards freeze-dried. For investigation of the particle film the freeze-dried polymer was embedded into a resin (EpoTek 301). 50 nm thin cuts were prepared with a Leica EM UC7 microtome equipped with a diamond knife and deposited onto TEM grids (copper, 200 mesh). For cryo-TEM studies, a drop (~2 mL) of the aqueous micellar solution (c ~0.4-0.7 g/L) was placed on a lacey carbon-coated copper TEM grid (200 mesh, Science Services), where most of the liquid was removed with blotting paper, leaving a thin film stretched over the grid holes. The specimens were shock vitrified by rapid immersion into liquid ethane in a temperature-controlled freezing unit (Zeiss Cryobox, Zeiss NTS GmbH) and cooled to approximately 90 K. The temperature was monitored and kept constant in the chamber during all of the preparation steps. After freezing the specimens, they were inserted into a cryotransfer holder (CT3500, Gatan) and transferred to a Zeiss EM922 OMEGA EFTEM instrument. Examinations were carried out at temperatures around 90 K. The microscope was operated at an acceleration voltage of 200 kV. Zero-loss filtered images (ΔE = 0 eV) were taken under reduced dose conditions. All images were registered digitally by a bottom-mounted CCD camera system (Ultrascan 1000, Gatan), combined, and processed with a digital imaging processing system (Gatan Digital Micrograph 3.9 for GMS 1.4). Evaluation of the respective length scales of the structures was achieved by measuring 50-100 different spots within the sample with the UTHSCSA ImageTool V. 3.00. 5 – Triiodide-Directed Self-Assembly of Different Miktoarm Star Polymers 141 UV-Vis Spectroscopy UV-vis spectra were recorded on a Hitachi 3000 spectrophotometer. Dynamic Light Scattering (DLS) DLS measurements were performed on an ALV DLS/SLS-SP5022F compact goniometer system equipped with an ALV 5000/E cross correlator and a He-Ne laser (λ = 632.8 nm). The measurements were carried out in cylindrical scattering cells (d = 10 mm) at an angle of 90 ° and a temperature of 20 °C. The solutions were measured without filtration. The CONTIN algorithm was applied to analyze the obtained correlation functions. Apparent hydrodynamic radii were calculated according to the Stokes-Einstein equation. Small-Angle X-Ray Scattering (SAXS) SAXS measurements of the freeze-dried powders were performed on a Bruker AXS Nanostar (Bruker, Karlsruhe, Germany), equipped with a microfocus X-ray source (Incoatec IµS Cu E025, Incoatec Geesthacht, Germany), operating at λ = 1.54 Å. A pinhole setup with 750, 400, and 1000 µm (in the order from source to sample) was used and the sample-to-detector distance was 107 cm. Samples were mounted on a metal rack and fixed using tape. The scattering patterns were corrected for the beam stop and the background (Scotch tape) prior to evaluations. The measurement time was 12 h. Differential Scanning Calorimetry (DSC) Thermal analyses and determination of the glass transition temperatures were performed on a Perkin-Elmer Diamond DSC at a heating rate of 20 K/min. The description of the SEC, NMR and MALDI-ToF experiments is given in the Supporting Information