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2013 118 Eva Blasco Pomar Photoresponsive materials based on azobenzene: novel macromolecular architectures and applications Departamento Director/es Química Orgánica Oriol Langa, Luis T. Piñol Lacambra, Milagros Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Eva Blasco Pomar PHOTORESPONSIVE MATERIALS BASED ON AZOBENZENE: NOVEL MACROMOLECULAR ARCHITECTURES AND APPLICATIONS Director/es Química Orgánica Oriol Langa, Luis T. Piñol Lacambra, Milagros Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA PHOTORESPONSIVE MATERIALS BASED ON AZOBENZENE: NOVEL MACROMOLECULAR ARCHITECTURES AND APPLICATIONS Eva Blasco Pomar Dpto. Química Orgánica Facultad de Ciencias-ICMA Universidad de Zaragoza-CSIC Zaragoza, July 2013   Los Drs. LUIS T. ORIOL LANGA, Profesor Titular del Departamento de Química Orgánica, y MILAGROS PIÑOL LACAMBRA, Profesora Titular del Departamento de Química Orgánica, pertenecientes a la Facultad de Ciencias y al Instituto de Ciencia de Materiales de Aragón de la Universidad de Zaragoza-CSIC HACEN CONSTAR que el trabajo original titulado “PHOTORESPONSIVE MATERIALS BASED ON AZOBENZENE: NOVEL MACROMOLECULAR ARCHITECTURES AND APPLICATIONS”, ha sido realizado por Dña. EVA BLASCO POMAR bajo nuestra supervisión en el Departamento de Química Orgánica de la Facultad de Ciencias de la Universidad de Zaragoza y reúne las condiciones para su presentación como tesis doctoral. Zaragoza, 9 a de Julio de 2013       Fdo.: Luis T. Oriol Langa Fdo.: Milagros Piñol Lacambra  Resumen Durante estos últimos años, los materiales que responden a uno o varios estímulos externos, conocidos como materiales ‘inteligentes’, han despertado un gran interés en la comunidad científica. Una de las principales razones es la posibilidad de utilizarlos en aplicaciones en campos tan diversos como la electrónica o la medicina, entre otros. De todos los posibles, la luz es probablemente el estímulo más interesante ya que es posible controlar la respuesta del material tanto espacial como temporalmente. Existen numerosos grupos orgánicos en los que la luz puede provocar una variación reversible en sus propiedades físicas y/o químicas pero el azobenceno es sin ninguna duda el grupo fotocrómico más estudiado en la preparación de materiales que respondan a la luz. Las aplicaciones de los materiales basados en azobenceno derivan de la fotoisomerizacion reversible entre los isómeros trans y cis que experimentan. En el grupo de investigación de Cristales Líquidos y Polímeros se han estudiado en profundidad polímeros y copolímeros con unidades azobencenos en la cadena lateral para aplicaciones ópticas, tales como el almacenamiento óptico de información. En los últimos años, el estudio se ha centrado en nuevas arquitecturas poliméricas, en concreto, copolímeros bloque dendrítico-lineales. Partiendo de los resultados previos del trabajo del grupo, en esta tesis doctoral se planteó obtener materiales con fotorrespuesta principalmente basados en azopolímeros con arquitecturas poliméricas alternativas a las convencionales basadas en estructura de cadena lateral. Los objetivos planteados para el desarrollo de esta tesis doctoral son los siguientes - Síntesis y caracterización de copolímeros bloque dendrítico-lineales compuestos por un bloque dendrítico funcionalizado con dieciséis unidades cianoazobenceno y diferentes bloques lineales, poliestireno y poli(metacrilato de etilo) y los análogos con poli(metacrilato de metilo) (Capítulo 2). - Síntesis y estudio del autoensamblaje en agua de nuevos copolímeros bloque dendrítico lineales anfífilos compuestos por un bloque lineal de polietilenglicol y un dendron de tipo poliéster funcionalizado bien con dieciséis unidades 4-isobutiloxiazobenceno o bien codendrones con diferentes proporciones de 4-isobutiloxiazobenceno y cadenas hidrocarbonadas distribuidas aleatoriamente en la periferia. Estudio de la aplicación de estos materiales como nanotransportadores de moléculas orgánicas y liberación fotoestimulada de las mismas (Capítulos 3 y 4). - Síntesis y estudio del autoensamblaje en agua de nuevos copolímeros anfífilos de tipo ‘miktoarm’ AB3, así como su respuesta al irradiar con luz UV. Estos copolímeros están compuestos por un azopolímero y tres ramas idénticas de PEG o un polímero termosensible como la poli(Netilacrilamida). Estudio de la respuesta a la luz, en el caso del polímero con PEG, y de la respuesta dual, luz y temperatura, en el de los polímeros con poli(N-etilacrilamida) de los ensamblados poliméricos (Capítulos 5 y 6). - Preparación de superficies fotosensibles funcionalizadas con unidades azobenceno utilizando luz como estímulo externo tanto para la funcionalización cómo para el control de las propiedades de la superficie (Capítulo 7).  v  7.3 Results and Discussion………………………………………................... 250 7.3.1 Synthesis and Characterisation …….............................................. 250 7.3.2 Premilinary test of the NITEC reaction with azobenzenes ..……… 253 7.3.3 Azobenzene Surface Functionalisation …………………………….. 256 7.3.4 Azobenzene Surface Patterning …………………………………….. 260 7.3.5 Wettability Study………………………………………………………. 262 7.4 Conclusions …………………………………………................................ 265 7.5 Experimental Section ………................................................................. 266 7.5.1 Experimental Details for the Synthesis of the Tetrazole Derivatives…………………………………………………………….. 266 7.5.2 Experimental Details for the Synthesis of the Azobenzene Derivatives……………………………………………………………… 268 7.5.3 General Procedures…………………………………………………… 273 References……………………………………………………………………….. 275 Conclusiones ………………………………………………………………. 281 Appendix Characterisation Techniques …..……………………………………………… 287 Surface Analysis…..…………………………………………………………….. 291 References ……………………………………………………………………… 296  CHAPTER 1 General Introduction  General Introduction 3    1.1 Photoresponsive Materials Stimuli responsive materials have been widely studied in the last years. These materials play an important role in a broad range of fields including biomedicine, microelectronics, optics or sensors among others.1-4 Nevertheless, the design and synthesis of new materials with controlled and predictable properties is still a challenge. Most of the materials reported in literature are polymers due to their versatility and processability.1-3 Photoresponsive polymers have one or more properties that can be significantly changed in a controlled fashion on receiving an external stimulus. The most widely investigated stimuli are pH, temperature, light as well as magnetic fields among others. Light is an especially attractive stimulus allowing temporal and spatial control. The light response of a material can be achieved by incorporation of photochromic moieties, which can reversibly switch between two states with different absorption spectra upon light irradiation. During this process, other properties as refractive index, dielectric constant, redox potential and molecular geometry can also be modified. Some organic molecules in which this photochromic effect has been observed are collected in Scheme 1.1. This effect can be due to either photoinduced reactions or isomerisation.5 Scheme 1.1. Photochromic moieties: a) spiropyrans and spirooxazines, b) fulgides, c) diarylethenes and d) azobenzenes 4 Chapter 1   However, the most studied photoresponsive moiety is the azobenzene.10 Upon irradiation, azobenzene suffers a reversible trans-to-cis isomerisation (Figure 1.1), this photoisomerisation is accompanied by a fast change in the geometric shape and polarity of the molecule. For most azobenzene compounds, the trans isomer is thermodynamically more stable than the cis isomer. The wavelength at which azobenzene isomerisation occurs depends on the particular structure of each azo molecule.11 Usually, azobenzene exhibits a low intensity n-π* absorption in the visible region, and a much higher intensity π-π* absorption in the UV region(see spectra in Figure 1.1). Figure 1.1 Azobenzene isomerisation (left) and a representative UV spectra of the two isomers of an azobenzene (right) The incorporation of these moieties into polymers makes them promising candidates in potential application in different fields ranging from data storage to photomechanical actuators among others (see Section 1.3). 300 400 500 trans cis π-π* n-π* Absorbance (a.u.) Wavelength (nm) Dipolar moment 0D 3D General Introduction 5    1.2 Azopolymers: Structrure & Synthesis 1.2.1 Azobenzene Homopolymers Azopolymers are polymers containing azobenzene moieties. These moieties can be incorporated into a polymeric structure in three different ways: a) hostguest systems, b) main chain azobenzene polymers and c) side chain azobenzene polymers, as it is schematically represented in Figure 1.2. Figure 1.2 Schematic representation of different azobenzene containing polymers: a) host-guest system, b) main chain azobenzene polymer and c) side chain azobenzene polymer Host-guest systems are formed by low molecular weight azobenzene molecules dispersed in a polymer matrix. This approach is the easiest strategy to prepare azobenzene based polymers (Figure 1.2a). It allows to keep the processability and the mechanical stability characteristics of the polymeric host material while the optical properties can be, to some extent, modulated by tuning of the composition of the mixture.12-14 Nevertheless, macroscopic segregation of the chromophore and the matrix might occur, which is the major drawback. An alternative to circumvent this problem is the linkage of azobenzene moieties to a polymer. Azobenzene moieties can be incorporated either in the main chain (Figure 1.2b) or in the side chain as pendant groups (Figure 1.2c). Several main chain azobenzene polymers have been prepared and studied as liquid crystal actuators.15,16 Nevertheless, azobenzene side chain polymers have been widely explored, especially poly(acrylates) and poly(methacrylates) derivatives. It is in the latter type of polymers in which we will focus this section. a) b) c) 6 Chapter 1   1.2.1.1 Synthesis of Azobenzene Homopolymers by Direct Polymerization There are two synthetic approaches for the preparation of azobenzene side chain homopolymers which are direct polymerization of an azomonomer and azobenzene postfunctionalization of a polymer previously synthesised. Scheme 1.2 shows a representation of both possibilities. Scheme 1.2 Different strategies for the preparation of azobenzene homopolymers: a) direct polymerization, b) azobenzene postfunctionalization The main advantage of the direct polymerization of a monomeric azobenzene is that the obtained polymers posses a well controlled composition having an azobenzene moiety per repeating unit. Hvilsted and coworkers have reported the synthesis of different series of liquid crystalline polyesters by step polymerization.17-19 Nevertheless, the vast majority of the reported azopolymers are polyacrylates and polymethacrylates. Traditionally, these azobenzene acrylates and methacrylates derivatives have been polymerized by free radical polymerization in solution using conventional experimental conditions (e.g. AIBN as thermal initiator in dry organic solvents such as DMF, THF or dioxane). The main drawback of this strategy is that the polymerization process of azobenzene (meth)acrylates is limited by the radical transfer reaction promoted by the azo group that seems to be associated to the formation of hydrazyl radicals20,21 and azopolymers can be obtained with uncontrolled and low molecular weights. In the last decades, different controlled radical polymerization (CRP) techniques22,23 have been employed to obtain azopolymers, including atom a) b) General Introduction 7    transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer polymerization (RAFT) among others. ATRP was first reported by the groups of Matyjaszewski and Sawamoto in 1995.24-26 This polymerization process is based on the transfer of an atom (usually an halogen) from a ‘dormant’ initiator or polymeric chain to a transition metal complex. The transition metal is oxidised when the halogen atom is transferred and a free radical is generated. Polymerization is propagated by the addition of monomer molecules to the thus generated free radicals (Scheme 1.3).Since the dormant state of the polymer is preferred in this equilibrium, side reactions including undesired termination are suppressed and a well control in the molecular weight and polydispersity of the polymers is achieved. ATRP can be mediated by a variety of transition metals from which copper is the most widely employed. Scheme 1.3 ATRP polymerization mechanism. X = Halide, L = Ligand Alkyl bromides such as 2-bromoisobutyrate derivatives (R-X) as the initiator and Cu(I) metal salts (CuBr or CuCl) in combination with nitrogen ligands such as N,N,N’,N’’,N’’-hexamethyltriethylenetetramine (HMTETA), N,N,N',N’’,N’’-penta methyldiethylenetriamine PMDETA or bipyridine ligands are the most commonly catalytic systems used for the ATRP polymerization of azobenzene (meth)acrylates. Keller and coworkers obtained the first azopolymer by ATRP27 and since then, the technique has been large described for the preparation of azopolymers for different purposes (Scheme 1.4). 8 Chapter 1   Scheme 1.4 Examples of azopolymers obtained by ATRP27,28 RAFT polymerization was discovered at Commonwealth Scientific and Industrial Research Organisation (CSIRO) in 1998.29,30 In this polymerization, thiocarbonylthio compounds (RAFT agents), such as dithioesters, thiocarbamates, and xanthates, are employed to mediate the polymerization via a reversible chain-transfer process. The accepted mechanism of the RAFT process consists of a sequence of addition-fragmentation equilibria as it is shown in Scheme 1.5. Initiation is achieved by decomposition of an initiator and subsequent propagation. In the early stages of the polymerization, addition of a propagating radical to the thiocarbonylthio compound is followed by fragmentation of the intermediate radical into a polymeric thiocarbonylthio compound and a new radical (R·). Addition of R· to the monomer forms a new propagating radical (Pm·). A rapid equilibrium, i.e. main equilibrium, between the propagating radicals (Pn· and Pm·) and the dormant species results in an General Introduction 15    continues phases. These microstructures can be tuned by adjusting the relative volume fraction of each block (f), Flory-Huggins interaction parameter (χ), and the degree of polymerization (N). Figure 1.5 shows a typical phase diagram of a coil-coil diblock copolymer. Furthemore, amphiphilic BC are able to selfassemble in solution forming different nanostructures such as micelles, nanospheres, vesicles among others (see Section 1.3.2).53-58 Figure 1.5 Schematic diblock copolymer phase diagram: f= volume, χ= Flory-Huggins interaction parameter and N= degree of polymerization (top). Different nanostructures formed by BCs: S= spheres, C=cylinders, G= gyroid and L=lamellar. (Image adapted from ref.57) Azobenzene containing BCs can combine in the same material light responsive properties with self-assembly abilities making the resulting nanostructures of interest in nanotechnology (see Section 1.3). Linear-linear azobenzene BCs can be approached by several general strategies as it is collected in Scheme 1.10. Direct polymerization of azomonomers by using a macroinitiator composed of a non azopolymer is the strategy most widely used for the preparation of these BCs (Scheme 1.10a). More specifically, ATRP macroinitiators based on poly(ethylene glycol) (PEG)59,60, 16 Chapter 1   poly(methyl methacrylate) (PMMA)61-63, poly(n-butyl methacrylate) (PBA)64 as well as polystyrene (PS)65-67 among others, have been employed for the polymerization of azomonomers (Figure 1.6). Besides, the alternative strategy, i.e the use of an azopolymer as the macroinitiator for the polymerization of conventional monomers, usually lead to poor results as it was reported by our research group.61 More recently studies have employed RAFT polymerization for the preparation of these BCs by using a macromolecular chain transfer agent composed either poly(acrylic acid)68 or poly(N-isoporopylacrylamide) (PNIPAM)69 to obtain the BCs shown in Figure 1.7. Scheme 1.10 General synthetic approaches for the synthesis of azobenzene containing BC: a) direct polymerization by using a macroinitiator b) postfunctionalization of a conventional BC and c) coupling of preformed building blocks a) b) c) + General Introduction 17    Figure 1.6 Examples of azobenzene BCs prepared by ATRP polymerization42-50  Figure 1.7 Examples of azobenzene BCs prepared by RAFT polymerization68-69 18 Chapter 1   Azobenzene units can also be also introduced in a BC architecture by a postfunctionalisation reaction (Scheme 1.10b). For this purpose, the previously synthesised BC should contain one block with reactive groups. By this approach, Gronski et al. prepared the first liquid crystal BC.70 Some years later, the same strategy was used by Schmidt and coworkers for the preparation of azobenzene containing block copolymers (Scheme 1.11).66 Firstly, the polybutadiene block was converted in a polyalcohol by hydroboration and finally the hydroxyl groups were functionalised with azobenzene units by an esterification reaction. Scheme 1.11 Synthesis of an azobenezene BC by postfunctionalization66 The last possibility consists of the coupling of two blocks previously prepared (Scheme 1.10c). This strategy requires the synthesis of polymers containing complementary end-chain group allowing the subsequent coupling. Although this is the most versatile synthetic approach, it relies on the availability of highly General Introduction 19    efficient and selective chemistry under mild conditions, which are the main features of the ‘click chemistry’ reactions. Combination of controlled radical polymerization that allows the synthesis of polymers with reactive ending groups, and ‘click chemistry’ is the best option for this approach as was recently demonstrated in the example collected in Scheme 1.12. On one hand, PMMA was synthesised by using an ATRP initiator containing an azide group, and on the other hand an azopolymer was also prepared by ATRP but using an initiator containing a complementary alkyne group. Both blocks were finally coupled by CuAAC reaction. O ON3 Br O O Br O O O 4 NN CN O O + CuBr/PMDETA O O Br O OO O 4 NN CN NBr N N O O Scheme 1.12 Synthesis of an azobenzene BC by CuAAC71 20 Chapter 1   1.2.3 Other Azobenzene Macromolecular Architectures Most of the reported azopolymers possess a linear structure. Nevertheless, other azobenzene macromolecular architectures have also been studied (Figure 1.8). In the next sections, a general overview about dendritic structures, linear-dendritic BC (LDBC) and miktoarm star polymers will be presented since they are connected (in particular linear-dendritic BC and miktoarm) with the materials aimed in this thesis. Figure 1.8 Azobenzene containing macromolecular structures: a) dendrimer, b) lineardendritic BC and c) miktoarm star polymer 1.2.3.1 Dendritic Structures Dendritic structures like dendrimers and dendrons are one of the most promising polymeric structures and have been the object of a growing number of publications. 72-75 Dendrimers are highly branched monodisperse molecules with a nanometric size. Their unique nature, shape and size make them ideal as for interesting applications in different field as catalysis, biology and materials science.72,73 Numerous dendritic structures have been synthesised and studied, including poly(amidoamine), poly(amide), poly(phenyl ether), and carbosilanes.74,75 a) b) c) General Introduction 21    During the last years, photoresponsive dendrimers have also been studied as an alternative to conventional linear azopolymers due to their potential applications. There are several reviews focused in these materials, including azobenzene functionalised dendrimers.76-78 The azobenzene moieties can be located in different positions of the dendritic structure. However, dendrimers having azobenzene moieties linked to periphery is the most frequent case. Poly(propilenimine) (PPI) is the most employed dendrimer for the preparation of azodendrimers (Figure 1.9).79-82 Due to the presence of amino groups at the periphery of these dendrimers, azobenzene moeites can be incorporated via amide linkages in most of the cases. Bifunctional codendrimers containing alkyl chains as well as other functional moieties as biphenyl or naphtyl have also been prepared.80,82 Figure 1.9 Examples of azobenzene functionalizated PPI dendrimers79-82 Other families of dendrimers such as poly(amidoamine) (PAMAM) have also been used for the preparation of azobenzene containing dendrimers.83,84 Similarly to PPI dendrimers, azobenzene units were incorporated into the dendrons via amide bond formation. In some cases, the resulting azobenene containing dendrimer was not fully functionalised.84 22 Chapter 1   In general, one of the main advantage of dendrimers is that the number of functional units introduced in the dendritic structure is better controlled than in the case of linear polymers. Dendritic and linear macromolecules can be combined in BCs containing both architectures. These new materials will be briefly reviewed below. 1.2.3.2 Linear-Dendritic Block Copolymers Linear-dendritic BCs (LDBC) are hybrid structures composed of a linear polymer block and a dendritic block. This new architecture was first introduced by Gitsov and Fréchet85-87 and might leads to substantial changes in some properties, such as solubility, intrinsic viscosity or microphase segregation among others, in comparison with the conventional linear-linear BC.89 There are three strategies used for the synthesis of these copolymers:87 ‘chainfirst’ route, ‘dendron-first’ route and the coupling of the preformed blocks, as it is collected in Scheme 1.13. Scheme 1.13 Synthetic approaches for the preparation of LDBC: a) ’chain-first’ strategy, b) ’dendron-first’ strategy and c) coupling strategy + a) b) c) General Introduction 23    The ‘chain-first’ route consists in the synthesis of a linear polymer having a reactive end group polymer that can be used for a divergent dendron construction (Scheme 1.13a). One of the first examples was reported by Meijer et al. by combining anionic polymerization and the divergent synthesis of PPI dendrimers.88 Similarly, Hammond et al. prepared LDBC by using the amino group terminated methoxy-poly(ethyleneglycol) to grow Tomalia type dendrons on the linear chain.89 Although the first examples were prepared following this strategy, it is not the most common strategy employed for the synthesis of these copolymers. The ‘dendron-first’ route implies that the dendron acts as the initiator for the polymerization of the linear block (Scheme 1.13b) and it is the most widely employed strategy so far. This concept was developed by Matyjaszewski as well as Hawker and Fréchet by using polyether dendrons as macromolecular initiators for the controlled free radical polymerization of vinyl monomers. In particular, polyether dendrons containing a benzylic 2,2,6,6-tetramethyl-1piperidinyloxy (TEMPO) group at their focal point have been used for the nitroxide mediated polymerization of styrene (Scheme 1.14).90 The ring opening polymerization (ROP) of lactones initiated by dendrons was also explored by several groups.91,92 Scheme 1.14 Example of the synthesis of a LDBC by employing the ‘dendron-first’ strategy90 24 Chapter 1   The coupling strategy requires the previous synthesis of the linear and dendron segments, followed by their coupling through complementary functional groups located at the end position of the linear chain and the focal point of the dendron (Scheme 1.13c). Although this seems to be the most versatile synthetic approach for the preparation of LDBCs, it relies on the efficiency of the coupling reaction. Initially, Williamson as well as palladium catalysed reactions were used for the coupling of different preformed blocks.93,94 The first reported example was based on the reaction of ‘living’ poly(styrene) dianion with aryl ether dendrons having a benzyl bromide group at the focal point.95 During the last years, the intense research in ‘click chemistry’ reactions rendered more effective coupling reactions for the preparation of these LDBCs.96,97 Scheme 1.15 shows an example of the synthesis of LDBC following this strategy. A dendritic block with a clickable alkyne group was first synthesised by ROP of εcaprolactone monomer using a propargyl focal point dendrons and coupled by a click reaction with azide functionalised PEG. Scheme 1.15 Example of the synthesis of a LDBC by employing the coupling strategy96 OON3 n G= dendron generation N N H H N O O NH G O OH m NH O OH m CuBr/PMDETA DMF OO n N N H H N O O NH G O OH m NH O OH m N NN General Introduction 31    Figure 1.13 a) Statistical copolymers containing azobenzene and a non absorbing monomer or mesogenic group and b) block copolymers Statistical polymers composed of azobenzene and non absorbing monomers like methyl methacrylate have been prepared.62,121 However, the main drawback of this approach is the lack of stability of the photoinduced birefringence, due to the lack of cooperative interactions between the azobenzene units. An alternative strategy, also based in statistical copolymerization, is the use of non absorbing mesogenic comonomers (Figure 1.13a). Bieringer and coworkers prepared a series statistical copolymers from azobenzene and mesogenic phenyl ester monomers (Figure 1.14).122 Other examples using different mesogenic moieties like tolane (Figure 1.4) and biphenyl (Figure 1.14) have been also employed for the exploration of this approach.49,50 The presence of these mesogenic groups does not contribute to the absorption at the same wavelength as azobenzene, but they can be oriented because of cooperative motions helping to increase the stability of photoresponse. Thick films with low absorption and good optical response have been prepared although the liquid crystalline character of the materials can give problems associated to light scattering. a) b) azobenzene group mesogenic group 32 Chapter 1   R= OC2H5,CN,NO 2 R1=Br,H R3=OMe,OEt O O O O O O rr O O O O r O O NN R CN 44 O O NN O O R2 R1 44 Figure 1.14 Examples of random copolymers containing azobenzene units for optical data storage50,122 A promising strategy is the use of BCs, in which one of the the block contains azobenzene units while the other one contains units that do not absorb at the recording/writing wavelength (Figure 1.13b). As it was noted above, BCs are able to undergo phase segregation. The advantage of this segregation is that azobenzene moieties are confined in nanometric regions preserving cooperative interactions between chromophore units. These domains are smaller than the recording wavelength and light scattering can be avoided. The expected behaviour of the chromophores in the BCs should be similar to the photoresponse of the corresponding homopolymers as it was demonstrated by our research group.61 Most of the reported azobenzene BCs are copolymers where the non absorbing block is either PMMA61,63 or PS (Figure 1.15).66,67 The photoresponsive block can be composed of an azobenzene hompolymer or a random copolymer containing azobenzene unit and non absorbing groups.61,63,66,123 The influence of the morphology of the microdomains as well as the length of the block in the photoinduced response has been investigated in these materials as optical storage media.114,115 Volume holograms with high efficiency and good stability have been recorded in this materials or blends from these BCs in an attempt to achieve photoresponsive materials with very low contents of photochromic units.66,71 General Introduction 33     Figure 1.15 Examples of linear-linear BC with azobenzene and mesogenic monomers 61,63,66 Recently, our research group explored a novel architecture, i.e. LDBCs allowing to combine the segregation ability of BCs and an exact control of the number of azobenzene units introduced per macromolecule. Several photoresponsive LDBCs have been prepared composed of dendritic aliphatic polyesters based on bis-MPA functionalised at the periphery with 4-cyanoazobenzene moieties and PEG or PMMA (Figure 1.10).97,98 1.3.1.2 Photomechanical Actuators Another application of interest in azopolymers is the preparation of phomechanical actuators. It is well known that nematic elastomers are able to change their shape due to the nematic to isotropic transition in the material. By incorporating azobenzene moieties into an elastomer, photoinduced contractions/expansions have been observed.124 Upon UV irradiation, azobenzene nematic elastomers suffer a reduction in alignment order as a result of the trans-to-cis isomerisation. While the rodlike trans-azobenzene moieties stabilise the liquid crystalline alignment, the bent cis forms can provoke a nematic to isotropic transition being the motor of the macroscopic contraction (Scheme 1.17). This deformation is reversible upon cis-to-trans back isomerisation. 34 Chapter 1   Scheme 1.17 Schematic representation of a photoinduced deformation in an azobenzene elastomeric film Pioonering studies of Finkelmann and coworkers demonstrated experimentally and theoretically that large shape changes in azobenzene containing polysiloxanes based elastomers can be generated by UV irradiation.125 Keller and coworkers first reported the synthesis of nematic azobenzene side chains elastomers by photopolymerization.126 The polymeric films showed a fast (less than 1 min) photoinduced contraction up to 18% by irradiation with UV light (Figure 1.16). Figure 1.16 Schematic representation of the elastomer prepared from a mixture of an azobenzene monomer, a liquid crystal monomer and 1,6-hexanediol diacrylate as crosslinker (left). Photographic frames (right) of the film (25%azo) a) before UV irradiation b) under UV irradiation (130s)126 Ikeda and coworkers have also been intensively working in this field.124,127,128 As an example, in a pioneering work they reported the preparation of films by LIGHT a) b) General Introduction 35    thermal polymerization of a liquid crystalline monomer and a diacrylate crosslinker both of which possessed azobenzene moieties.129 Upon UV irradiation (366 nm), the film bent towards the direction of light with the bending occurring parallel to the direction of light polarisation. When the bent film were exposed to visible light (540 nm), the film was able to recovered its initial flat state (Figure 1.17). Figure 1.17 Schematic representation of the azobenzene containing elastomer employed for preparation of the film (top) and photographic frames of the film bending in different directions (down) in response to irradiation by LPL of different angles of polarisation (white arrows) at 366 nm, and being flattened again by visible light at 540 nm128 36 Chapter 1   1.3.1.3 Photopatterning of Nanostructures in Block Copolymers As mentioned above, one of the most important properties of BCs is their ability to segregate and give rise to different nanostructures (see section 1.2.2). By incorporation of azobenzene moieties in one of the blocks, photoalignment of the microdomains has been achieved.129 Ikeda and coworkers demonstrated this phenomenon by using a liquid crystalline BC composed of PEG and an azobenzene containing block, which is able to self-assemble in a nanostructure composed of PEG cylinders into an azobenzene containing matrix.130 BC films of about 100 nm thickness were prepared on a glass substrate and were irradiated with LPL and annealed at 140ºC, temperature at which the BC posses a liquid crystalline behaviour. The PEG cylinders were perfectly aligned orthogonal to the polarisation of the light by the supramolecular cooperative motions of the ordered azobenzene block (Figure 1.18). Figure 1.18 Chemical structure of the azobenzene BC employed by Ikeda and coworkers (left). AFM images of the photooriented nanostructures (right): a) before irradiation, PEO cylinders are perpendicular to the substrate and b) after irradiation with LPL, the cylinders are aligned perpendicularly to the polarization direction of light (Image adapted from ref.130) LPL a) b) General Introduction 37    Similarly, Seki and coworkers studied the photoaligment of a BC composed of PS and azopolymer which self-assembly into a PS nanocylinder structure.131 Again, films with a thickness of about 100 nm were prepared on a glass substrate and were irradiated with LPL with different angles. Annealed non irradiated films provided PS cylinders in the upright orientation after annealing, while after irradiation with LPL cylinders were perfectly oriented in the orthogonal direction to the light. The initial situation was recovered by irradiation with non polarised light (Figure 1.19). Figure 1.19 Chemical structure of the BC employed by Seki and coworkers and AFM images of the photooriented nanostructures after irradiation with LPL (436 nm) with different angles followed by annealing (Image adapted from ref.131) 1.3.2 Photoresponsive Properties in Solution Photoinduced isomerisation of azobenzenes can also be used to promote changes in macromolecular self-assemblies dispersed in a liquid media, such as micelle dissociation or vesicle deformation. These photoresponsive properties are promising for applications in different areas as it will be discuss below. 1.3.2.1 Amphiphilic Block Copolymers: Self-assembly and their Applications as Controlled Delivery Systems One of the most interesting properties of amphiphilic BCs is their ability to form in water different nanostructures like micelles and vesicles among others. In the 38 Chapter 1   last years, amphiphilic BCs have received considerable attention due to the variety of applications in different fields ranging from biomedicine to catalysis.5358 Morphology and size of the self-assemblies can be modulated by controlling the hydrophilic/hydrophobic balance, which can be tuned by adjusting the length of the blocks and their chemical nature. On increasing volume fraction of the hydrophobic blocks, it has been observed a general evolution from spherical micelles to vesicles according to Figure 1.20. Figure 1.20 Different morphologies found for amphiphilic BCs in aqueous media Polymeric micelles consist of a core formed by the hydrophobic blocks and a corona or shell formed by the hydrophilic blocks.56 These micelles can be used as nanocarriers since hydrophobic drugs can be encapsulated in the core and transported at concentrations that can exceed their intrinsic water solubility. On the other hand, polymeric vesicles, also known as polymersomes, contains an inner volume enclosed by a thin membrane composed of a polymeric bilayer.58 Vesicles are of particular interest as drug nanocontainers because of their internal hydrophilic cavities and robust hydrophobic membranes which can encapsulate both hydrophobic and hydrophilic molecules. The incorporation of stimuli responsive moieties in amphiphilic BC makes them potentially useful as controlled delivery systems.132-135 The majority of the reported stimuli responsive materials are sensitive to a few common triggers, including pH, temperature and light.1-4 As mentioned before, the advantage of using light as external stimulus is the possibility to apply a temporal and spatial VesicleMicelle Cylindrical Micelle Hydrophilic block Hydrophobic block General Introduction 39    control in the material response. Light responsiveness can be introduced in amphiphilic BCs in different ways. The most common strategy for the preparation of photocontrolled delivery systems is the incorporation of photochromic moieties in the one of the blocks of the BC (Scheme 1.18a). Upon UV irradiation, an alteration of the hydrophobic/hydrophobic balance due to the photoinduced reaction takes place leading to a deformation or even disruption of the self-assemblies and subsequent release of encapsulated substances. Several photochromic systems including azobenzene,68,69,99,101,136143 spiropyran,144-147 dithienylethene and diazonaphthoquinone148,149 have been study for this purpose. As an example, Mezzenga and coworkers reported the first spiropyrane functionalised amphiphilic BCs (Figure 1.21) which were able to form micellar aggregates in a mixture of water and ethanol. They demonstrated that the self-assemblies were able to undergo a reversible aggregation-dissolution-aggregation process in water in response to irradiation with a suitable wavelength. In the next section, several examples of amphiphilic azobenzene containing BCs will be detailed. Scheme 1.18 Schematic representation of photoresponsive BCs: a) photochromic containing BCs and b) photodegradable BCs UV UV a) b) Photochromic group Photocleavage group before UV After UV 40 Chapter 1   Figure 1.21 Chemical structure of the spyropyrane functionalised amphiphilic BCs studied (left). Schematic representation of the photoresponsive micellization/dissolution process of the BCs upon irradiation (right) (Image adapted from ref.147) On the other hand, incorporation of photocleavage groups into the BC is also an interesting strategy to prepare controlled delivery systems (Scheme 1.18b). The most popular photocleavable moieties are o-nitrobenzyl-based derivatives. Burdick and coworkers prepared a BC composed of PEG and poly(caprolactone) and the photolabile 2-nitrophenylalanine as the linker of the two blocks (Figure 1.22).150 This BC self-assembled into vesicles in water. Upon irradiation, a gradual collapse of the vesicles membrane took place due to the photoinduced cleavage of the linker. In comparison with photochromic containing systems, irreversibility of this light induced process is one of the disadvantages Figure 1.22 Chemical structure of a BC composed of PEG and poly(caprolactone) and the photolabile 2-nitrophenylalanine as the linker of the two blocks (left) and Cryo-TEM images (right) of the vesicles before a) and after b) irradiation150 a) b) General Introduction 47    Closely related amphiphilic LDBCs were also reported by Shi et al. by combining PEG and different generations of 4-octyloxyazobenzene poly(amido amine) (PPI) dendrons where this generation dependent self-assembling behaviour was also observed (Figure 1.30).100 These authors reported on the reversible photoinduced trans-to-cis isomerisation in solution but not on the photoresponse of the aqueous self-assemblies. Figure 1.30 Chemical structure of the amphiphilic LDBCs reported by Shi et al. (top) and TEM images of the different self-assemblies in a 17:83 dioxane:water mixture(bottom): a) nanofibers, b) nanospheres in coexistence with nanosheets (indicated by the white arrow) c) polymeric vesicles and d) large micelles100 a) b) c) d) GPhobic/Philic Ratio (wt%) 0 33/67 1 52/48 2 70/30 3 82/18 48 Chapter 1   1.4 Photoresponsive Surfaces Responsive smart surfaces have recently attracted significant attention because of their associated interesting applications such as biosensors, intelligent membranes or microfluidic devices.154-156 During the last years, the study of surfaces with controllable wettability has emerged as a major focus of interest in responsive surface field,155 especially photoresponsive surfaces prepared from inorganic oxides and/or photoresponsive organic molecules.157-160 Photoresponsive organic surfaces are based on photochromic moieties such as azobenzene, spiropyrans, fulgides among others.157,161 The photoresponsive moieties are usually incorporated in a suitable platform, usually a small molecule or a polymer, to form self-assembled monolayers (SAMs) or polymer based surfaces. SAMs are spontaneously formed by adsorption of an active surfactant into solid surfaces. Thiol and silane derivates are examples of two widely used organic groups to functionalise inorganic surfaces. The photoswitching of SAM modified surfaces is normally based on chemical or conformational changes of the photoresponsive group. As an example, Rosario et al. reported photoresponsive surfaces by covalently bound spiropyran to a glass surface.162 The surface modification was carried out by reaction of the corresponding organic silanes with silicate surfaces to form Si-O-Si bonds. The relatively nonpolar spiropyran can be reversibly switched to a polar, zwitterionic merocyanine isomer that has a much larger dipole moment by UV light, and back again by visible light (Figure 1.31). The light induced changes observed in the surface energy were correlated to the switching of the surface bound spiropyran molecule between polar and nonpolar forms by means of fluorescence spectroscopy. General Introduction 49    Figure 1.31 Spiropyran functionalised surface and the behaviour of a water droplet a) under Vis irradiation and b) under UV irradiation162 On the other hand, polymer films can be prepared on substrate surfaces using several deposition techniques as well as chemical reactions. One of the simplest techniques of applying thin films onto substrates is either casting or spin coating of a polymer solution. As an example, spiropyrane photoresponsive polymeric films were prepared by Sumaru and coworkers.163 A polymer blend of an spiropyran containing polymer and PMMA was dissolved in 1,2dichloroethane and poured onto a glass substrate, which had been hydrophobicised with dichlorodimethylsilane and dried in air for 3 days. Due to the photocontrolled change in the polarity of the surface, a reversible cell adhesion control was achieved. 1.4.1 Azobenzene Functionalised Surfaces Azobenzene functionalised surfaces have also attracted much attention. As mentioned, the modification the dipole moment of the molecule due to trans-tocis isomerisation gives the possibility to prepare surfaces with photocontrolled wettability (Scheme 1.19). Si OOH Si NH N O O OO Si O ONO2 a) b) 50 Chapter 1   Scheme 1.19 Photoinduced isomerisation of azobenzene moieties in a surface Pioneering studies on photoresponsive azobenzene surfaces were reported by Ichimura and coworkers using a flat surface modified with a calix[4]resorcinarene containing four pendant azobenzene units that was irradiated with a gradient in light intensity achieving light driven motion of liquids (Figure 1.32).164,165 The asymmetrical irradiation caused a gradient in the surface free energy because of the photosiomerisation azobenzene moieties generating CA hysteresis on both edges of the droplet. This induced tension led to a directional motion of the droplet. UV Vis Dipolar moment 0D 3D General Introduction 51    Figure 1.32 Chemical structure of the azobenzene containing calix[4]resorcinareno reported by Ichimura and coworkers and light driven motion of an olive oil droplet under asymmetrical irradiation164 Selected examples in compact monolayers containing azobenzene moieties prepared on silicon substrates were described by Delorme et al. as well as Hamelmann et al. (Figure 1.33).166,167 These photoresponsive surfaces were prepared either by covalent grafting of azobenzene moieties onto a surface previously functionalised with an isocyanate monolayer or direct grafting of silane containing azobenzene. These studies provided evidence of controlled photoisomerisation of the azobenzene moieties in the surface and subsequently a photocontrolled change in the CA of the surface. Figure 1.33 Azobenzene functionalised surfaces prepared by covalent grafting of azobenzene moieties166,167 t=0 t=35s t=80s UV light blue lightOlive oil droplet 52 Chapter 1   Recently, rough surfaces with a good photoresponsiveness fabricated via layerby-layer deposition were also reported. For example, Zhou et al. prepared a switchable surface changing from a slippery to a sticky state when the azocompound assumes trans or cis conformation, respectively (Figure 1.34).168 The coating consists of a siloxane elastomer containing trifluoromethoxy azobenzene moieties. Cho et al. also prepared fluorinated azobenzene modified nanoporous substrates.169 Upon UV irradiation, the surface was reversibly switched between superhydrophobic and superhydrophilic states. These studies evidenced that the presence of nanostructures strongly enhanced the wettability changes resulting from azobenzene isomerisation in comparison with monolayers. 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Journal of the American Chemical Society 2006, 128,14458-14459.  CHAPTER 2 Linear-Dendritic Block Copolymers for Optical Applications Published in Polymer 2012, 53, 4604‐4613  Linear-Dendritic Block Copolymers for Optical Applications 79     Scheme 2.2 Synthesis of the fourth generation polyester dendron bearing an azide group in the focal point: a) 2,2-dimethoxypropane, TsOH, acetone, b) BnBr, KOH, DMF, c) DCC/DPTS, CH2Cl2, d) Dowex®, CH3OH and e) Pd(C) 20%, AcOEt For the preparation of the alkyne functionalised linear chains, ATRP was selected as polymerization method (Scheme 2.4). Alkyne functionality can be introduced in either by utilizing functionalised initiators or a postpolymerization end group modification (bromine substitution in the case of ATRP). While the first procedure ensures complete functionalisation of all polymer chains, in the latter incomplete functionalisation due to the lost of the bromine group in the ATRP may occur. Following the strategy reported by van Hest and coworkers,16 80 Chapter 2   an initiator with a trimethylsilyl protected alkyne group was prepared by esterification of α-bromoisobutyryl bromide with trimethylsilyl propargyl alcohol and employed for ATRP. Scheme 2.3 Synthesis of the azobenzene containing dendron (d16AZO) Linear blocks of PMMA, PEMA and PS with two different average molecular weights of approx. 10000 and 20000 g/mol were prepared. The polymerizations were performed in bulk at 90ºC (PMMA and PEMA) or 110ºC (PS) and employing CuBr and N,N,N',N’’,N’’- pentamethyldiethylenetriamine (PMDETA) as the catalyst system, according to procedures reported in the literature for PMMA and PS.13,17 Polymerization times were adjusted to obtain different molecular weights. Number average molecular weights, Mn, of the linear blocks were determined by end group analysis of the TMS ended polymers by 1H-NMR using the relative integral of the –Si(CH3)3 and –COOCH3 of PMMA, –COOCH2– of PEMA or aromatic protons of PS. Figure 2.3 shows the 1H-NMR of PEMA2-TMS indicating the signals used for Mn calculation. All data are gathered in Table 2.1. Molecular weight distribution were also determined by size exclusion chromatography (SEC) using PMMA standars in the case of PMMA and PEMA and PS standards for PS. SEC traces of linear blocks showed monomodal molar mass distributions. Low polydispersities (ĐM Linear-Dendritic Block Copolymers for Optical Applications 81    <1.1) were determined for PMMA and PS homopolymers and slightly higher (ĐM ≈1.2) for PEMA ones. This increment in the molecular weight distribution of PEMA could arise from uncontrolled termination processes.18 In general, average molecular weights obtained by 1H-NMR and SEC are very similar except for PEMA, although it should be taken account that data are referenced to PMMA standards. Finally, the trimethylsilyl protected alkyne functionalised linear blocks were deprotected with tetrabutylammonium fluoride (TBAF). Molecular masses of the deprotected polymers were also studied by SEC and the results are, as expected, very similar to those for the protected precursors.  Scheme 2.4 Synthesis of the alkyne terminated linear homopolymers (see Table 2.1 for the corresponding Mn) Figure 2.3 1H-RMN spectrum of the PEMA2-TMS in CDCl3 (400 MHz) showing the signals used for Mn calculation 54321 Chemical shift (ppm) a b c de f g a b c d e f g x 82 Chapter 2   Table 2.1. Molecular weight of the synthesised polymers Polymer Mn Mn [ c ] ĐM [ c ] PMMA1-TMS 10300 [ a ] 11800 1.05 PMMA1 - 12100 1.04 PMMA2-TMS 19800 [ a ] 19100 1.05 PMMA2 - 20200 1.04 PEMA1-TMS 9120 [ a ] 11500 1.26 PEMA1 - 11800 1.22 PEMA2-TMS 18240 [ a ] 23600 1.16 PEMA2 - 22800 1.20 PS1-TMS 10504 [ a ] 10900 1.04 PS1 - 11100 1.04 PS2-TMS 20488 [ a ] 19100 1.05 PS2 - 19500 1.05 PMMA1-b-d16AZO 18417 [b] 16200 1.08 PMMA2-b-d16AZO 27917 [b] 21600 1.09 PEMA1-b-d16AZO 17237 [b] 20900 1.14 PEMA2-b-d16AZO 26357 [b] 32400 1.19 PS1-b-d16AZO 18621 [b] 16700 1.08 PS2-b-d16AZO 28695 [b] 23500 1.08 [a] Number average molecular weight (Mn) calculated by 1H-NMR (see text). [b] Calculated by the sum of the linear block Mn calculated by 1HNMR and the molecular weight of d16AZO. [c]Mn and ĐM of PMMA and PEMA homopolymers and their corresponding BCs were determined by SEC using PMMA standars. Mn and ĐM of PS homopolymers and their corresponding BCs were determined by SEC using PS standars. Linear-Dendritic Block Copolymers for Optical Applications 83    In the final synthetic step, the azido functionalised dendritic block and the alkyne functionalised polymers were coupled by CuAAC using DMF as solvent and CuBr and PMDETA as the catalytic system (Scheme 2.1). A slight excess of the alkyne ended linear block was employed to ensure the completeness of the reaction and was eventually removed using an azido functionalised polystyrene resin. The efficiency of the coupling was asserted by SEC analysis. For PMMA and PEMA containing LDBCs, evidence of residual azodendron was not observed in SEC traces. This was not the case of PS containing LDBCs, where a very small peak corresponding to residual azodendron was detected in the SEC curve that indicates a less effective coupling (Figure 2.4a). Therefore, preparative SEC was used in order to purify completely the LDBC. Figure 2.4b collects the SEC curves corresponding to the precursor blocks and the PS2-b-d16AZO once purified. As can be observed, CuAAC coupling of the precursor blocks gives rise to a shift of the molar mass distribution peak towards lower retention times that indicates LDBC formation. Further evidence for the formation of the BCs was gained from the IR spectra, as can be seen for PS2-b-d16AZO in Figure 2.5 as a representative example, where the band at 2100 cm-1 due to the azido group of the azodendron has disappeared upon coupling. The 1H-NMR spectra of the LDBCs also confirm the coupling, as is shown in Figure 2.6 for PEMA1-bd16AZO as an example. Relative integration of azobenzene aromatic protons signals and the corresponding ones to the linear block protons (–COOCH3 of PMMA at 3.60 ppm, –COOCH2 of PEMA at 4.02 ppm or aromatic protons of the PS at 6.50 ppm) is in good agreement with the LDBCs structure, and confirms that there is not excess of any of the blocks. Furthermore, new peaks corresponding to the formed triazol ring appeared at 8.56 ppm (see peak labelled as ‘o’ in Figure 2.6), and at 5.15 and 4.10 ppm corresponding to the methylenic protons linked to it (see protons ‘n’ and ‘p’ labelled in Figure 2.6). 84 Chapter 2   Figure 2.4 SEC traces of d16AZO (black line) and PS2 (dashed line) and PS2-bd16AZO (grey line): a) before and b) after purification Figure 2.5 FT-IR spectra in KBr of the LDBC PS2-b-d16AZO and the corresponding azodendron d16AZO and linear block PS2 (bottom to top)  10 12 14 16 18 20 0.0 0.5 1.0 Normalized signal Retention time (min) 12 16 20 0.0 0.5 1.0 Normalized Signal Retention Time (min) a) b) 4000 3500 3000 2500 2000 1500 1000 500 Transmittance Wavenumber (cm -1 ) Linear-Dendritic Block Copolymers for Optical Applications 85     Figure 2.6 1H-RMN spectrum of the PEMA1-b-d16AZO in CDCl3 (400 MHz) showing the signals used for Mn calculation 2.3.2 Thermal Characterisation and Morphological Study Thermal stability of the LDBCs as well as of the isolated blocks was studied by thermogravimetric analysis (TGA) under nitrogen atmosphere up to 600ºC using powdered samples. Weight losses associated to the presence of residual solvents or water were not detected. From the TGA curves, significant differences were observed for PMMA, PEMA and PS containing LDBCs (Table 2.2). PMMA and PEMA LDBCs showed major weight losses associated to sample decomposition above 315ºC. PS imparted superior thermal stability with major weight losses associated to sample decomposition above 390ºC. 87654321 Chemical shift (ppm) b,c ad pn j,l e,t i q r h m s g,k,u abcd e f g h i j k l m nop q rs t u f o 86 Chapter 2   Thermal transitions were studied by combining differential scanning calorimetry (DSC) and polarised optical microscopy (POM). Relevant data are collected in Table 2.2. The azodendron d16AZO is a vitreous material that exhibits a mesomorphic phase above glass transition, Tg. The DSC curve of the azodendron d16AZO presented a glass transition at 22ºC and a peak at 141ºC corresponding to a mesophase-to-isotropic transition (Figure 2.7). POM images of the azodendron showed fan shaped textures characteristic of a smectic A mesophase as can be seen in Figure 2.8a. The linear blocks were essentially amorphous materials. DSC curves showed a clear baseline jump corresponding to the glass transition with Tg values of around 115ºC for PMMA, 70ºC for PEMA and 100ºC for PS. The investigated LDBCs exhibited DSC curves where two glass transitions were detected indicating microphase segregation of blocks. The lowest Tg, at 33-34ºC, corresponds to the glass transition of the azodendron block even if the calculated values are slightly higher (about 10ºC) than that of d16AZO. The highest Tg corresponds to the linear block and calculated values are also slightly higher than those of the corresponding homopolymers. All the LDBCs showed a peak corresponding to the mesophase-to-isotropic transition. The comparison between DSC curves of the azodendron, a linear block PS-2 and the corresponding LDBC PS2-b-d16AZO is shown in Figure 2.7. For PMMA LDBCs, the higher Tg (at around 115ºC) overlaps the mesophase-to-isotropic transition. PEMA containing LDBCs circumvent this problem due to the lower Tg (at around 70ºC) of the linear block. All these LDBCs show liquid crystalline behaviour, although under POM they exhibited poorly defined textures which do not allow a clear identification of the mesophase (Figure 2.8b). Linear-Dendritic Block Copolymers for Optical Applications 87    Table 2.2. Thermal properties of the LDBCs and their building blocks TGA [ a ] DSC [b] Polymer Td Tg(1) Tg(2) Ti ∆Hi d16AZO 313 22 - 141 78.8 PMMA1 342 - 115 - - PMMA2 361 - 113 - - PEMA1 252 - 69 - - PEMA2 271 - 66 - - PS1 390 - 97 - - PS2 390 - 98 - - PMMA1-b-d16AZO 341 32 116 [ c ] 135 [ c ] 63.4 [ c ] PMMA2-b-d16AZO 354 36 116 [ c ] 134 [ c ] 67.4 [ c ] PEMA1-b-d16AZO 326 33 70 134 64.9 PEMA2-b-d16AZO 317 33 76 133 69.4 PS1-b-d16AZO 392 34 102 134 34.6 PS2-b-d16AZO 393 34 102 141 39.7 [a] Td (in ºC): decomposition temperature associated to mass lost calculated by TGA at the onset point in the weight loss curve. [b] Transition temperatures and enthalpies were determined by DSC from the second heating scan (10ºC/min): Tg = glass transition; Ti = isotropisation; ∆Hi= enthalpy associated to isotropisation. [c] Data cannot be calculated accurately. Mesophase-to-isotropic transition was overlapped with Tg(2)  88 Chapter 2   Figure 2.7 DSC traces recorded at 10ºC/min corresponding to the second heating of PS2, d16AZO and the corresponding LDBC PS2-b-d16AZO (from top to bottom) Figure 2.8 POM images of d16AZO and PEMA1-b-d16AZO taken at 75ºC on cooling from the isotropic state The microphase segregation pointed by the DSC study was also confirmed by TEM in the study carried by the group of Prof. Alcalá. Small pellets of the LDBCs were prepared by heating the powdery polymers at 180ºC for about 2 min and subsequent fast cooling to room temperature. Pellets were then annealed for 1h at 140ºC and fast cooled again to room temperature. It was corroborated that longer annealing times at 140ºC does not introduce any significant change in the nanostructure. Then, thin slices (of about 100 nm thick) were cut from the pellets using a ultramicrotome, put on copper grids and 0 40 80 120 160 Heat Flow (Exo Down) Temperature (؛C) a) b) Linear-Dendritic Block Copolymers for Optical Applications 95    Synthesis and Characterisation of Benzyl 2,2-di(hidroxyethyl)propanoate (7) Bis-MPA (10.00 g, 74.55 mmol), and KOH (4.81 g, 85.73 mmol) were dissolved in DMF (50 mL). The mixture was heated at 100 °C for 1 h and benzyl bromide (10.6 mL, 89.46 mmol) was added then. After stirring for 15 h at 100 °C, DMF was evaporated off using a rotary evaporator. The residue was dissolved in DCM (200mL) and washed with water. Organic solvent was evaporated and the crude product was recrystallised from hexane/dicholoromethane (1:1). Yield: 60 %. IR (KBr), v (cm-1): 3360, 1706, 1606, 1499, 1226. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.45-7.28 (m, 5H), 5.20 (s, 2H), 3.93 (d, J= 11.3 Hz, 2H), 3.73 (d, J=11.3 Hz, 2H), 2.98 (s, 2H), 1.08 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 175.7, 135.6, 128.6, 128.3, 127.8, 68.1, 66.6, 49.2, 17.1. Synthesis and Characterisation of Compound (8) Compound (8) was prepared according to the described general esterification procedure by employing compound (1) (10.11 g, 58.06 mmol), compound (7) (6.20 g, 27.64 mmol), DPTS (3.25 g, 11.06 mmol) and DCC (14.26 g, 69.12 mmol) in dry DCM (80 mL). The crude product was purified by flash column chromatography on silica gel, eluted with hexane, gradually increasing the polarity to ethyl acetate/hexane (8:2). Compound (8) was obtained as a colourless viscous oil. Yield: 63%. IR (NaCl),  (cm-1): 1738, 1259. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.42-7.32 (m, 5H), 5.12 (s, 2H), 4.44 – 4.25 (m, 4H), 4.11 (d, J = 11.9 Hz, 4H), 3.58 (d, J = 11.6 Hz, 4H), 1.41 (s, 6H), 1.34 (s, 6H), 1.30 (s, 3H), 1.09 (s, 6H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 173.5, 171.8, 96 Chapter 2   135.5, 128.6, 128.5, 128.3, 98.1, 66.0, 65.9, 64.8, 46.9, 46.7, 42.1, 25.2, 22.1, 18.5, 17.7. MALDI-TOF MS (matrix: dithranol, m/z): 559.3 [M+Na]+. Synthesis and Characterisation of Compound (9) The compound (8) (4.00 g, 7.52 mmol) was dissolved in ethyl acetate and Pd/C (10%) (0,40 g) was added. Then the flask was evacuated from air and filled with H2. After 4 h of stirring at room temperature, the catalyst was filtered off using Celite® and carefully washed with ethyl acetate. The solvent was evaporated and the product was obtained as a viscous oil. Yield: 98%. IR (NaCl),  (cm-1): 3300, 1742, 1258. 1H-NMR (CDCl3, 400MHz) δ (ppm): 4.35 (s, 4H), 4.17 (d, J=11.1 Hz, 4H), 3.63 (d, J= 11.9 Hz, 4H), 1.42 (s, 6H), 1.36 (s, 6H), 1.32 (s, 3H), 1.15 (s, 6H).13C-RMN (CDCl3, 100 MHz) δ (ppm): 173.6, 175.43, 98.2, 66.9, 66.0, 65.4, 46.8, 42.1, 25.1, 22.1, 18.5, 17.7. MALDI-TOF MS (matrix: α-cyano-4-hydroxycinnamic acid, m/z): 469.2 [M+Na]+. Linear-Dendritic Block Copolymers for Optical Applications 97    Synthesis and Characterisation of Compound (10) O O O ON3 6 O O O O O O O O O O O O O O O O OO O O O O O O O O O O OO O O O O O O O O O O O O (10)  Compound (10) was prepared according to the general esterification procedure described from compound (6) (2.20 g, 4.48 mmol), compound (9) (12.00 g, 26.88 mmol), DPTS (5.27 g, 17.92 mmol) and DCC (6.01 g, 29.12 mmol) dissolved in dry DCM (80 mL). The crude product was purified by liquid chromatography on silica gel, eluted with hexane, gradually increasing to ethyl acetate/hexane (8:2). The product was obtained as colourless viscous oil. Yield: 63%. IR (NaCl),  (cm-1): 2097, 1725, 1259. 1H-NMR (CDCl3, 400MHz) δ (ppm): 4.37-4.20 (m, 30H) 4.14 (d, J=11.9 Hz, 16H), 4.11 (t, J=6.8 Hz, 2H), 3.62 (d, J=11.9 Hz, 16H), 3.28 (t, J=6.8Hz, 2H), 1.70-1.52 (m, 8H), 1.41 (s, 24H), 1.35 (s, 24H), 1.28-1.21 (m, 21H), 1.14 (s, 24H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 173.5, 171.8, 98.1, 66.0, 65.9, 64.8, 46.9, 46.7, 42.1, 25.2, 22.1, 18.5, 17.7. MALDI-TOF MS (matrix: α-cyano-4-hydroxycinnamic acid, m/z): 2205.1 [M-H]+. 98 Chapter 2   Synthesis and Characterisation of d16OH DOWEX-50-X2 resin (0.20 g) was added to a solution of compound (10) (1.00 g, 0.46 mmol) in methanol (10 mL). The mixture was stirred for 18 h at room temperature. Then the resin was filtered off and the solvent eliminated under vacuum to give d16OH as a colourless viscous oil. Yield: 97 %. IR (KBr),  (cm-1): 3400, 2099, 1729, 1239. 1H-NMR (DMSO-d6, 400MHz) δ (ppm): 4.65 (t, J=5.3Hz, 16H), 4.30-4.02 (m, 30H), 3.50-3.27 (m, 34H), 1.61-1.55 (m, 2H), 1.55-1.47 (m, 2H), 1.36-1.32 (m, 4H), 1.20 (s, 3H). 1.17 (s, 6H), 1.15 (s, 12H), 1.00 (s, 24H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 173.9, 171.7, 63.7, 63.6, 50.6, 50.2, 46.2, 28.0, 27.7, 25.7, 24.8, 17.1, 16.6. MALDI-TOF MS (matrix: dithranol, m/z): 1884.1 [M+H]+. Linear-Dendritic Block Copolymers for Optical Applications 99    2.5.2 Experimental Details for the Synthesis of 11-[4-(4’-cyanophenylazo) phenyloxy]undecanoic acid (AZO) Synthesis and Characterisation of 4-(4’-hydroxyphenyazo)benzonitrile (11)  A mixture of 4-aminobenzonitrile (10.00 g, 84.60 mmol) and HCl 6M (40 mL) was cooled into an ice bath. A 2.5 M NaNO2 solution (50 mL, 84.60 mmol) was added dropwise to the mixture and it was kept stirring in the ice bath. Then, a solution of phenol (7.10 g, 84.60 mmol) in 2 M NaOH (75 mL) was carefully added. The product was precipitated upon addition of HCl until neutral pH and it was purified by flash column chromatography on silica gel using DCM as an eluent. The product was obtained as a yellow powder. Yield: 65%. IR (KBr),  (cm-1): 3300, 2240, 1606, 1586, 1503, 1219, 844. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.95-7.91 (m, 4H), 7.81-7.79 (m, 2H), 6.98-6.96 (m, 2H), 5.33 (s, 1H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 133.1, 125.6, 123.0, 115.9. Synthesis and Characterisation of Methyl 11-[4-(4’-cyanophenylazo) phenyloxy]undecanoate (12) A solution of 4-(4’-hydroxyphenyazo)benzonitrile (11) (6.90 g, 30.90 mmol), methyl 11-bromoundecanoate (9.50 g, 34.05 mmol) in butanone (80 mL) was prepared. 18-Crown-6 ether (0.05 g) and potassium carbonate (5.10 g, 37.11 mol) were added. The suspension was stirred and heated under reflux for 24 h. Then, it was filtered and concentrated. The crude product was purified by flash column chromatography on silica gel using DCM as eluent. The product was obtained as a yellow powder. Yield: 65%. IR (KBr),  (cm-1): 2233, 1730, 1602, 1583, 1500, 1251, 863. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.95-7.93 (m, 4H), 100 Chapter 2   7.80-7.72 (m, 2H), 7.02-7.00 (m, 2H), 4.06 (t, 2H, J=6.6 Hz), 3.67 (s, 3H), 2.31 (t, 2H, J=7.6 Hz), 1.85-1.78 (m, 2H), 1.69-1,53 (m, 4H), 1.51-1.41 (m, 2H), 1.39-1.17 (m, 8H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 174.8, 161.3, 146.9, 133.1, 125.5, 123.1, 118.6, 114.8, 68.1, 51.1, 33.8, 29.4, 28.8, 25.6, 24.6. Synthesis and Characterisation of 11-[4-(4’-cyanophenylazo)phenyloxy] undecanoic acid (AZO) An aqueous solution of KOH (1.5 g, 15 mL) was added to a solution of methyl 11-[4-(4’-cyanophenylazo)phenyloxy]undecanoate (12) (8.00 g, 19.03 mmol) in ethanol and butanone (120 mL and 40 mL, respectively). The mixture was stirred and heated under reflux for 1 h. Then, the crude product was precipitated by addition of HCl until pH 2 and it was recovered by filtration. The product was recrystallised from ethanol. Yield: 70%. IR (KBr),  (cm-1): 3300, 2242, 1714, 1600, 1580, 1499, 1255, 851. 1H-NMR (DMSO-d6, 400MHz) δ (ppm): 8.05-8.03 (m, 2H), 7.97-7.92 (m, 4H), 7.16-7.13 (m, 2H), 4.08 (t, J=6.4 Hz, 2H), 2.18 (t, J=7.2 Hz, 2H), 1.78-1.69 (m, 2H), 1.52-1.44 (m, 2H), 1.44-1.36 (m, 2H), 1.34-1.17 (m, 10H). 13C-NMR (DMSO-d6, 100 MHz) δ (ppm): 174.5, 162.4, 154.1, 145.9, 133.7, 125.2, 122.8, 118.5, 115.2, 112.4, 68.1, 33.6, 28.9, 28.8, 28.7, 28.5, 28.4, 25.4, 24.4. Linear-Dendritic Block Copolymers for Optical Applications 101    2.5.3 Synthesis and Characterisation of the Azodendron d16AZO d16OH (0.75 g, 0.70 mmol), 11-[4-(4’-cyanophenylazo) phenyloxy]undecanoic acid (AZO) (3.11 g, 7.64 mmol) and DPTS (1.87 g, 6.36 mmol), were dissolved in a mixture of DCM (40 mL) and DMF (15 mL). The reaction flask was flushed with argon, and DCC (1.73 g, 8.40 mmol) was added. The mixture was stirred at room temperature for 48 h under argon atmosphere. The white precipitate formed was filtered off, and the solvent was evaporated. The crude product was purified by flash column chromatography on silica gel and eluted with DCM, gradually increasing the polarity to ethyl acetate:DCM (1:10). The target azodendron was obtained as a red powdery solid. Yield: 55%. IR (KBr),  (cm1): 2227, 2096, 1741, 1600, 1582, 1501, 1257, 859. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.93-7.91 (m, 64H), 7.79-7.77 (m, 32H), 7.00-6.98 (m, 32H), 4.36-4.11 (m, 62H), 4.02 (t, J=6.5 Hz, 32H), 3.29 (t, J=6.7 Hz, 2H), 2.31 (t, J=7.5 Hz 32H), 1.81-1.78 (m, 32H), 1.64-1.56 (m, 32H), 1.50-1.40 (m, 36H), 1.39-1.24 (m, 209H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 173.1, 172.0, 162.6, 154.7, 146.6, 133.1, 125.4, 123.0, 118.6, 114.8, 113.2, 68.4, 64.7, 46.3, 34.0, 29.6, 29.5, 29.4, 29.3, 29.2, 26.0, 24.8, 17.8. MS (MALDI+, dithranol ) m/z: 8116.9 102 Chapter 2   [M-H]+. Anal. Calc for C465H565N51O78: C, 68.82; H, 6.97; N, 8.81. Found: C, 68.34; H, 7.22; N, 8.64. 2.5.4 Experimental Details for the Synthesis of the Linear Blocks PMMA, PEMA and PS were synthesised by ATRP using 3-(trimethylsilyl)prop2-ynyl 2-bromo-2-methylpropanoate,16 an initiator with a protected alkyne function that was subsequently deprotected (refer to Scheme 2.4). PMMA Polymerization Methyl methacrylate (18.70 g, 0.19 mol), PMDETA (200 µL, 0.9 mmol), CuBr (134.3 mg, 0.9 mmol) and the initiator (260.2 mg, 0.9 mmol) were added to a Schlenk tube. The reaction mixture was degassed by three freeze-pump-thaw cycles and flushed with argon. The polymerization was carried out in a thermostated oil bath at 90ºC. After 5 min for PMMA1-TMS or 10 min for PMMA2-TMS the polymerization mixture was diluted with THF, passed through a column of neutral alumina to remove the catalyst and precipitated into methanol. The polymer was dried in a vacuum oven at 40ºC. PEMA Polymerization Ethyl methacrylate (13.76 g, 0.12 mol), PMDETA (144.1 µL, 0.7 mmol), CuBr (99.1 mg, 0.7 mmol) and the initiator (190.2 mg, 0.7 mmol) were added to a Schlenk tube. The reaction mixture was degassed by three freeze-pump-thaw cycles and flushed with argon. The polymerization was carried out in a thermostated oil bath at 90ºC. After 3 min for PEMA1-TMS or 8 min for PEMA2-TMS the polymerization mixture was diluted with THF, passed through a column of neutral alumina to remove the catalyst and precipitated into hexane. The polymer was dried in a vacuum oven at 40ºC. PS Polymerization Styrene (13.59 g, 0.14 mol), PMDETA (and 21.3 µL, 0.1 mmol), CuBr (14.6 mg, 0.1 mmol) and the initiator (28.3 mg, 0.1 mmol) were added to a Schlenk tube. Linear-Dendritic Block Copolymers for Optical Applications 103    The reaction mixture was degassed by three freeze-pump-thaw cycles and flushed with argon. The polymerization was carried out in a thermostated oil bath at 110ºC. After 45 min for PS1-TMS or 4 h for PS2-TMS the polymerization mixture was diluted with THF, passed through a column of neutral alumina to remove the catalyst and precipitated into methanol. The polymer was dried in a vacuum oven at 40ºC. General Procedure of Alkyne Deprotection A 0.01 M solution of the protected alkyne-terminated polymer in THF was prepared and a five-fold excess of 1.0 M solution of TBAF in THF with respect to trimethylsilyl group (TMS) was added dropwise. The reaction mixture was stirred overnight at room temperature and the product was precipitated into cold methanol. The alkyne-ended linear polymer was dried at 40ºC under vacuum for 48 h. Characterisation Data for PMMA1: IR (KBr),  (cm-1): 1728, 1240, 1150. 1HNMR (CDCl3, 400MHz) δ (ppm): 4.68-4.58 (m), 3.59 (s), 2.06-1.75 (m), 1.481.38 (m), 1.26-1.13 (m), 1.10-0.80 (m). Anal. Calc: C, 59.98%; H, 8.05% Found: C, 60.30%; H, 7.89. SEC: Mn= 12100, ĐM =1.04 (PMMA standars). Characterisation Data for PMMA2: IR (KBr),  (cm-1): 1729, 1242, 1147. 1HNMR (CDCl3, 400MHz) δ (ppm): 4.65-4-52(m), 3.60 (s), 2.07-1.72 (m), 1.501.35 (m), 1.26-1.13 (m), 1.09-0.70 (m). Anal. Calc: C, 59.98%; H, 8.05% Found: C, 59.20%; H, 7.80%. SEC: Mn= 20200, ĐM =1.04 (PMMA standars) Characterisation Data for PEMA1: IR (KBr),  (cm-1): 1728, 1269, 1146. 1HNMR (CDCl3, 400MHz) δ (ppm): 4.60-4.36 (m), 4.02 (q, J=6.7 Hz), 2.10-1.70 (m), 1.34-1.10 (m), 1.10-0.80 (m). Anal. Calc: C, 63.14%; H, 8.83% Found: C, 63.34 %; H, 9.13 %. SEC: Mn= 11800, ĐM =1.22 (PMMA standars). Characterisation Data for PEMA2: IR (KBr),  (cm-1): 1729,1272, 1147. 1HNMR (CDCl3, 400MHz) δ (ppm): 4.60-4.36 (m), 4.02 (q, J=6.7 Hz), 2.10-1.70 (m), 1.24-1.10 (m), 1.10-0.80 (m). Anal. Calc: C, 63.14%; H, 8.83% Found: C, 63.42 %; H, 9.08 %. SEC: Mn = 22800, ĐM =1.20 (PMMA standars). 104 Chapter 2   Characterisation Data for PS1: IR (KBr),  (cm-1): 1601, 1493, 756, 698. 1HNMR (CDCl3, 400MHz) δ (ppm): 7.36-6.89 (m), 6.85-6.30 (m), 4.60-4.36 (m), 4.08-4.01 (m) , 2.29 (s), 2.05-1.65 (m) 1.62-0.85 (m). Anal. Calc: C, 92.26%; H, 7.74% Found: C, 91.99 %; H, 7.89 %. SEC: Mn = 11100, ĐM =1.04 (PS standars). Characterisation Data for PS2: IR (KBr),  (cm-1): 1601, 1492, 756, 697. 1HNMR (CDCl3, 400MHz) δ (ppm): 7.36-6.89 (m), 6.85-6.30 (m), 4.60-4.36 (m), 4.08-3.98 (m), 2.29 (s), 2.03-1.65 (m) 1.63-0.85 (m). Anal. Calc: C, 92.26%; H, 7.74% Found: C, 91.81 %; H, 7.98 %. SEC: Mn = 19500, ĐM =1.05 (PS standars). CHAPTER 3 Amphiphilic Linear-Dendritic Block Copolymers: Self-assembly and Photoresponse Published in Polym. Chem. 2013, 4, 2246-2254   Amphiphilic Linear-Dendritic Block Copolymer 113    3.1 Introduction and Aims As it was described in Chapter 1, one of the most important features of amphiphilic BCs is their ability to undergo spontaneous phase separation in solution forming different supramolecular structures of nanoscale dimensions, such as spheres, rods, lamellae or vesicles.1-3 Among all the different morphologies, polymeric vesicles, also known as polymersomes, are of particular interest as drug nanocontainers. Polymer vesicles originate from closing bilayers forming a central aqueous compartment which is enclosed by an amphiphilic copolymer bilayer membrane.4 The hydrophobic chains create the wall membrane stabilised by the hydrophilic chains forming internal and external coronas (Figure 3.1). Figure 3.1 Schematic representation of a polymeric vesicle Because of their internal hydrophilic cavities and robust hydrophobic membranes, vesicles can physically store both hydrophobic and hydrophilic compounds. The hydrophilic molecules will be encapsulated within the aqueous interior and hydrophobic molecules will be integrated within the membrane. The methods of loading the molecules into the polymersomes are diverse.5 Hydrophobic compounds can be solubilised into the vesicle bilayer by diffusion on stirring together with the vesicles suspension or by cooperative encapsulation during the self-assembly process. Hydrophilic molecules can be directly encapsulated during the vesicle formation. Depending on the properties of the hydrophobic block, vesicles might retain loaded molecules very long periods of time, from days to weeks. hydrophobic block hydrophilic block 114 Chapter 3   The membrane regulates transport of molecules between the inside and outside of the vesicle and its properties can be easily designed and tailored on varying the structural and chemical features of the BCs to include a range of desirable functions which make them useful in various technologies.6 For instance, modification of the coronas might determine the surface characteristics of the vesicles and hence their interactions with the environment. But also, the incorporation of stimuli sensitive groups into the membrane wall might activate the delivery of cargo molecules on demand.5 In this context, our research group studied a diversity of aqueous assemblies (cylindrical micelles, sheet-like micelles, tubular micelles, as well as polymer vesicles) exhibited by a series of amphiphilic LDBCs composed of PEG of different molecular weights and dendrons based on bisMPA functionalised at the periphery with 4-cyanoazobenzene moieties. In particular, vesicles were observed for the LDBC consisting of a fourth generation dendron with sixteen 4cyanoazobenzene units and a 2000 g/mol linear PEG.7 The proposed model for these vesicles consists of a bilayer organisation for the azobenzene groups packing in the hydrophobic domains with internal and external PEG coronas. In order to check the photoresponse of these vesicles, they were irradiated with UV light to induce trans-to-cis isomerisation of the azobenzene located in the inner part of the bilayer. Nevertheless, morphological changes of the vesicles were only achieved by irradiation with intense UV light (Figure 3.2). It is well recognised the strong tendency of 4-cyanoazobenzenes towards antiparallel dipolar interactions8,9 and it has been described that for highly and densely packed azobenzene moieties the fast and highly efficient trans-to-cis isomerisation is hindered.10,11 Consequently, disruption of the membrane formed by such densely packed arrangements of 4-cyanoazobenzene might be restricted. Amphiphilic Linear-Dendritic Block Copolymer 115    Figure 3.2 Cryo-TEM images of cyanoazobenzene containing vesicles before (a) and after (b) irradiation for 35 min at 360 nm and 150 mW/cm2 Due to the potential of vesicles as controlled delivery systems, the aim of this work is the preparation of new LDBCs able to self-assembly into polymeric vesicles which can act as light controlled delivery nanocarriers upon irradiation with low intensity UV light. The use of low intensity UV irradiation limits possible undesired side photochemical process as well as the damage of the organic structures when exposed to UV irradiation. For that purpose, the fourth generation of azodendrons derived from bisMPA and PEG of 2000 g/mol average molecular weight as the linear block has been selected (Figure 3.3). The cyano group at the paraposition of the azobenzene moiety of the previously mentioned materials has been substituted by an alkoxy one. This substituents should have a lower tendency to antiparallel and dense arrangements. Furthermore, alkoxy para-substituents increase the difference in polarity between the trans and the cis isomers compared to the cyano one and, as it was above recognised, this facilitates the disruption of self-assemblies under UV irradiation.12 a) b) 116 Chapter 3   Figure 3.3 Chemical structure of the proposed LDBCs Amphiphilic Linear-Dendritic Block Copolymer 117    3.2 Tasks and Methods - Synthesis of the blocks consisting of a fourth generation polyester dendron based on the bisMPA acid functionalised with sixteen 4-alkoxyazobenzene moieties linked through different spacers and an azido functional group at the focal point. - Synthesis of alkyne functionalised PEG 2000 g/mol as linear block. - Synthesis of the target LDBCs approached by a coupling the preformed blocks using the CuAAC reaction (Figure 3.4). Figure 3.4 Synthetic approach for the synthesis of the LDBCs - Structural characterisation of the building blocks and derived LDBCs by FTIR, NMR, MS as well as elemental analysis. Thermal characterisation using POM, TGA and DSC. - Self-assembly of the LDBCs in water. - Morphological study of the self-assemblies in water by electron microscopy: TEM and Cryo-TEM. - Study of the photoresponsive behaviour of the self-assemblies in water. - Study of the encapsulation and photoinduced release of hydrophobic and hydrophilic fluorescent probes. + 118 Chapter 3   3.3. Results and Discussion 3.3.1 Synthesis and Characterisation of the Amphiphilic Block Copolymers The synthesis of the amphiphilic LDBCs was carried out by using the same coupling strategy presented in Chapter 2 where a fourth generation of bisMPA based dendron with an azido group at the focal point and functionalised at the periphery with azobenzene units was ‘click’ coupled to a previously synthesised alkyne terminated PEG chain (Scheme 3.1). DCC/DPTS CH2Cl2/DMF O O O O O O O O O O O OH OH O OH OH O O O OOH OH O OH OH O O O O OH OH OOH OH O O O O OH OH O OH OH O O d16OH O O O O O O O O O O O OR OR O OR OR O O O OOR OR O OR OR O O O O OR OR OOR OR O O O O OR OR O OR OR O O d16isoAZO (n=10) d16isoAZOb (n=5) O O O O O O O O O O O OR OR O OR OR O O O OOR OR O OR OR O O O O OR OR OOR OR O O O O OR OR O OR OR O O CuBr/PMDETA DMF O O O 45 isoAZO (n=10) isoAZOb (n=5) N N OO CH2 n=10,5 O R= n N N OO CH2 O HO n O N O O N N O 45 6 O 6 O PEG-b-d16isoAZO (n=10) PEG-b-d16isoAZOb (n=5) N3 6 N3 Scheme 3.1 Synthesis of the aimed LDBCs starting from d16OH described in Chapter 2 Amphiphilic Linear-Dendritic Block Copolymer 119    The synthesis of the azido functionalised dendron having sixteen hydroxyl groups (d16OH) was described in the Experimental section in Chapter 2. The 4alkoxyazobenzene unit was attached to the periphery of the dendron d16OH by an esterification reaction between its hydroxyl groups and the appropriate acids using the DCC/DPTS system. In a first attempt, esterification of d16OH with 11-[4-(4’-methyloxy-phenylazo)phenyloxy] undecanoic acid was approached but the sequential incorporation of 4-methyloxyazobenzene units during the course of the reaction decreased the solubility of the resulting dendron causing its precipitation from the reaction medium and preventing the complete functionalisation of the hydroxyl groups at dendron periphery. This result contrast with the solubility exhibited by dendrons functionalised with analogous cyanobenzene moieties. Therefore, the 4-methyloxy substituent was replaced by the 4-isobutyloxy one while keeping the decamethylenic spacer. Nevertheless, a shorter flexible chain was also introduced that should decrease the hydrophobicity of the dendrons and influence the self-assembly process. For this purpose, two acids having the 4-isobutyloxyazobenzene photoactive unit and a decamethylenic (isoAZO) or a pentamethylenic (isoAZOb) flexible spacer were synthesised according to Scheme 3.2 using previously reported synthetic methods.13 Esterification of d16OH with 4-isobutyloxyazobenzene derivates, isoAZO and isoAZOb, rendered the corresponding dendrons, d16isoAZO and d16isoAZOb in 30-40% yield. Evidence for the complete functionalisation of the periphery of the dendrons was provided by several techniques. The MALDI-TOF mass spectra showed the expected ion peaks (see MALDI spectrum of d16isoAZOb in Figure 3.7b as an example).The 1HNMR spectra of the azodendrons are fully consistent with the proposed chemical structures. As an example, the 1H-NMR spectrum of d16isoAZO is shown in Figure 3.5. Relative integration between the signals corresponding to the aromatic protons of the azobenzene units and the signal corresponding to the methylene unit linked to the azide group, CH2-N3 at 3.27 ppm (labelled as ‘a’ in Figure 3.5) also confirmed the complete functionalisation of the periphery of the dendron. 120 Chapter 3   Scheme 3.2 Synthesis of 4-isobutyloxyazobenzene derivates isoAZO and isoAZOb Figure 3.5 1H-NMR spectrum of d16isoAZO in CDCl3 (400MHz) 87654321 Chemical shift (ppm) t,u s,v h,f rw a a w i sl j,e b,p y k,l,m n,o,c, d,g Amphiphilic Linear-Dendritic Block Copolymer 127    3.3.2 Self-assembly of the Linear-Dendritic Azobenzene Block Copolymers in Water Self-assembled structures of the LDBCs were prepared by the solvent switch (or co-solvent) method using THF-water. The process was followed by measuring the loss of intensity of transmitted light due to scattering (turbidimetry) when pass from a solution to a micellar dispersion. The two LDBCs were first dissolved in THF which is a good a solvent for both blocks. Then water, which is non solvent for the hydrophobic block, was slowly added to the solution while the turbidity of the mixture was monitored as a function of the water content (Figure 3.11). When a critical water content was reached a sudden increase in turbidity occurred indicating that polymer self-assembly starts. At this point the hydrophilic block tends to shield the hydrophobic block apart from the solvent. The self-assembly process sacrifices the entropy of the single chains, but prevents a larger enthalpy penalty resulting from energetically unfavourable hydrophobe-water interactions.4 Once turbidity reached an almost constant value, the resulting dispersion was dialyzed against water to remove the organic solvent as it is described in the Experimental Section. Figure 3.11 Turbidity plot of the LDBCs THF solution versus amount of water added Unfortunately, for PEG-b-d16isoAZO a precipitate was obtained by removing the organic solvent through dialysis which points to the collapse of the self0 5 10 15 20 25 Turbidity Water wt (%) PEGb -d16isoAZOb PEGb -d16isoAZO 128 Chapter 3   assemblies in water. In the case of PEG-b-d16isoAZOb, a stable water suspension of self-assemblies was eventually obtained using the same procedure. The hydrophilic/hydrophobic balance is responsible of this behaviour and put in evidence the influence of the structural design on the preparation of polymeric vesicles. The morphology of the stable self-assemblies of PEG-b-d16isoAZOb was first investigated by TEM on dried samples stained with uranyl acetate (see Experimental Section for further details). In fact, the TEM images confirmed the formation of vesicular self-assemblies with a deflated appearance because of dehydration in the procedure of the sample preparation (Figure 3.12a). Figure 3.12 a) TEM image of PEG-b-d16isoAZOb non-irradiated vesicles. Cryo-TEM images of PEG-b-d16isoAZOb vesicles before b) and after c) irradiation for 1 h at 365 nm and 2.6 mW/cm2. The length of the scale bar corresponds to 200 nm in a) and 100 nm in b) and c) The aqueous suspension PEG-b-d16isoAZOb vesicles were also analyzed by Cryo-TEM (Figure 3.12b). In this case, the sample was vitrified in liquid ethane at –170ºC, and images were obtained with liquid nitrogen cooling without the need of staining. Spherical vesicles were observed with dark regions corresponding to the aromatic azobenzene moieties that form the hydrophobic membrane. The vesicle diameter was in the range 250-450 nm and the thickness of the membrane around 8 nm, which fits well with a bilayer arrangement of the azodendrons as was previously reported for analogous azobenzene containing LDBCs.7 Size of the vesicles was also evaluated by dynamic light scattering (DLS) (see below Figure 3.16 before irradation). The a) b) c) Amphiphilic Linear-Dendritic Block Copolymer 129    mean hydrodynamic diameter (Dh) was found to be 365 nm, which is in good agreement with Cryo-TEM observations. The critical aggregation concentration (CAC) of PEG-b-d16isoAZOb in water was determined by fluorescence spectroscopy using Nile Red as a polarity sensitive probe.17-20 In water, Nile Red exhibits a weak emission at 660 nm (with excitation at 550 nm) due to excimer formation but if the dye is located in a more hydrophobic environment its emission is blue shifted and the intensity increases dramatically.20 The self-assembly of this LDBCs produces a hydrophobic environment into which Nile Red can enter and the CAC can be determined by recording the fluorescence intensity as a function of the LDBC concentration. It has to be emphasise that Nile Red was also chosen because the excitation/emission wavelengths of this particular probe are separated from the wavelengths required to induce the trans-to-cis photoisomerisation of the azobenzene. Besides, the intrinsic fluorescence emission of the vesicles with excitation at 365 nm (maximum absorption wavelength of azobenzene), was discarded.21 Samples of PEG-b-d16isoAZOb were stirred together with Nile Red at room temperature overnight and the emission spectra of Nile Red were registered from 560 to 700 nm (see Experimental Section for further details). As expected, the fluorescence spectra show that the emission intensity increases on increasing the concentration of PEG-b-d16isoAZOb. At low concentrations of PEG-b-d16isoAZOb, the weak fluorescence intensity indicates that Nile Red is preferentially in water and, therefore, few micellar self-assemblies are present. At higher concentrations, the emission intensity increases indicating that Nile Red is located in a more hydrophobic environment as a consequence of being encapsulated by the polymer self-assemblies. The relationship between fluorescence intensity (maximum) and logarithm of the PEG-b-d16isoAZOb concentration is non-linear and the onset point corresponds to the CAC (Figure 3.13). The observed onset point corresponded to a concentration of 35 µg/mL, which is comparable with CAC values reported for other LDBC selfassemblies.19,22 130 Chapter 3   Figure 3.13 Fluorescence intensity of Nile Red at 606 nm (λexc = 550 nm) versus PEGb-d16isoAZOb concentration (mg/mL) 3.3.3 Light Responsive Behaviour of the Vesicles In order to study the photoresponse of the PEG-b-d16isoAZOb vesicles, the UV-vis spectra of both a solution of the LDBC in chloroform and the vesicles suspension in water were first recorded (Figure 3.14a). The spectra of PEG-bd16isoAZOb in solution was characterised by two absorption bands corresponding to the trans-isomer, a strong one centred at 360 nm attributed to the π-π* transition and a weak one at about 450 nm corresponding to n-π* transition. The spectrum of the vesicles showed a large broadening and a hypsochromic shift of the π-π* band (Figure 3.14a). The absorption maximum shifted down to 320 nm which indicates the dominant formation of H-aggregates of azobenzene units. Furthermore two shoulders at higher wavelengths were observed, one at 360 nm which corresponds to the value determined for the non-aggregated trans-azobenzene and the other at 375 nm which is characteristic of the formation of J-aggregates. Exposure of the vesicles to UV irradiation, 365 nm and 2.6 mW/cm2, caused significant spectral changes (Figure 3.14b). A remarkably decreasing of π-π* absorbance was observed accompanied by a notably increase of the 1E-4 1E-3 0.01 0.1 1 0 20 40 60 80 100 Relative Fluorescence Emmision (606 nm) Conc (mg/mL) Amphiphilic Linear-Dendritic Block Copolymer 131    absorbance at 450 nm that can be attributed to the photo-isomerisation of the transto the cis-azobenzene. After 30 min of light exposure only slight changes were further detected in the UV-vis spectrum. When the irradiated suspension of the vesicles was kept in the dark, after 2 h the spectrum gradually started to recover its initial shape due to the slow thermal back isomerisation cis-to-trans. Although the thermal isomerisation is slow (hours) it can be readily accelerated by exposure to visible light. Thus, the vesicles (previously irradiated at 365 nm) were irradiated at 450 nm (Figure 3.15). After 10 min, absorbance at around 360 nm increased which can be attributed to the back cis-to-trans photoisomerisation. After 1 h, the spectrum almost recovered the initial shape. Figure 3.14 a)UV-Vis spectra of PEG-b-d16isoAZOb in a 5x10-6 M solution in CHCl3 (straight line) and a water suspension of PEG-b-d16isoAZOb vesicles (dashed line). b) UV-Vis spectra of PEG-b-d16isoAZOb irradiated vesicles (concentration of 1 mg/mL) for different times at 365 nm and 2.6 mW/cm2 Figure 3.15 UV-Vis spectra of PEG-b-d16isoAZOb irradiated vesicles (concentration of 1 mg/mL) at different times at 450 nm (0 min correspond to vesicles previously irradiated at 365 nm for 30 min) 280 320 360 400 440 480 520 560 0.0 0.2 0.4 0.6 0.8 1.0 Absorbance Wavelenght (nm) 0 min 10 min 30 min 60 min 15 h after irradiation b) 280 320 360 400 440 480 520 560 0.0 0.2 0.4 0.6 0.8 1.0 Absorbance Wavelenght (nm) a) 280 320 360 400 440 480 520 560 600 0.0 0.1 0.2 0.3 0.4 Absobance Wavelengh (nm) 0 min 10 min 30 min 60 min 132 Chapter 3   The irradiated samples were also studied by DLS and Cryo-TEM in order to study the modification on the particle dimension and morphology. For these studies the vesicles were irradiated for 1h at 365 nm and then measured. By DLS measurements a permanent change of the vesicles size upon irradiation was observed with a mean Dh of 270 nm determined after irradiation (Figure 3.16). The Dh was evaluated immediately upon irradiation and after 24 h of irradiation and no evolution of the Dh was found evidencing an irreversible morphological change. The Cryo-TEM image of the irradiated sample after 15 h shows deformed vesicles with a distorted membrane in contrast to the nonirradiated samples (Figure 3.12c). Therefore, the experiments suggest remarkable changes in the morphology of PEG-b-d16isoAZOb vesicles before and after UV irradiation as a consecuence of the azobenzene photoisomerisation. Figure 3.16 DLS measurements of a water suspension of PEG-b-d16isoAZOb vesicles before and after UV light irradiation at 365 nm and 2.6 mW/cm2 For the sake of comparison, vesicles of LDBC functionalised with 4cyanoazobenzene were irradiated under the same conditions of low intensity used in this work. Recording of the UV data at different irradiation times indicated that trans-to-cis isomerisation took place to a lesser extent (Figure 3.17a). Substantial changes were not observed in the mean Dh determined by DLS upon irradiation (Figure 3.17b). Therefore, under the same experimental 100 1000 10000 0.0 0.5 1.0 Normalized signal Diameter (nm) Before irradiation After irradiation Amphiphilic Linear-Dendritic Block Copolymer 133    conditions, changes on vesicles containing 4-cyanoazobenzene units were only moderate. We can assume that the higher tendency towards aggregation and the higher polarity of the trans 4-cyanoazobenzene might hinder an effective disruption of the photochromic bilayer shell. Figure 3.17 a) UV-Vis spectra and b) dynamic light scattering measurements of the 4cyanoazobenene-containing vesicles (concentration of 1 mg/mL) irradiated for different times at 365 nm and 2.6 mW/cm2 3.3.4 Encapsulation and Photoinduced Release of Molecular Probes The potential of the vesicles as light responsive nanocontainers was investigated by encapsulation and subsequent release of fluorescent probes. Since molecules of interest can be trapped either in the hydrophilic hollow cavity or in the hydrophobic membrane of the vesicle, the ability to encapsulate both Nile Red and Rhodamine B, which are respectively of hydrophobic and hydrophilic nature, was tested. Encapsulation of Nile Red was already demonstrated in the determination of the CAC. Because of its hydrophobic nature, Nile Red should be retained in the hydrophobic region of the vesicle, i.e. in the membrane formed by the photoresponsive azobenzene block, rather than in the internal compartment of the vesicle.23 Thereof, Red Nile was encapsulated by difussion stirring an aqueous vesicle suspension of 1 mg/mL concentration in Nile Red 10–6 M. The suspension was stirred overnight to reach the equilibrium before fluorescence was measured. The emission spectra of Nile Red were registered from 560 to 280 320 360 400 440 480 520 560 0.0 0.2 0.4 0.6 Absorbance Wavelenght (nm) 0 min 10 min 30 min 60 min 100 1000 10000 0.0 0.5 1.0 Normalized signal Diameter (nm) Before irradiation After irradiation a) b) 134 Chapter 3   700 nm while exciting at 550 nm. Nile Red exhibits an strong fluorescence revealing that the probe has been encapsulated. The suspension of the loaded vesicles was irradiated with 365 nm UV light, 2.6 mW/cm2, and the fluorescence of Nile Red was recorded at different exposure times. Upon irradiation, an abruptly decrease on the initial fluorescence intensity at 606 nm was observed (Figure 3.18). This change in fluorescence upon UV light exposure indicates that the environment of the probe becomes less hydrophobic. A priori this can be reasonably related to the disruption of the vesicle membrane and subsequent release of Nile Red into water due to transto-cis isomerisation of azobenzene. But also, the trans-to-cis isomerisation of the azobenzene brings about a change in the polarity of membrane which becomes more hydrophilic and this can also explain the decrease on the fluorescence intensity of the Nile Red without its complete releasing into water. When the irradiated vesicles were kept in the dark the fluorescence intensity was slowly and partially recovered. Therefore, because the slow back thermal cis-to-trans relaxation of azobenzene takes place the fluorescence intensity should increases again because the recovery of a more hydrophobic environment within the membrane.24 Figure 3.18 Emission spectra of the Nile Red encapsulated vesicles of PEG-bd16isoAZOb recorded after irradiating at 365 nm and 2.6 mW/cm2 for different time intervals 580 600 620 640 660 680 700 Fluorescence (a.u) Wavelenght (nm) 0 min 10 min 30 min 15 h after irradiation Amphiphilic Linear-Dendritic Block Copolymer 135    To gain more information on whether or not the photoinduced increase of membrane permeation takes place, vesicles were loaded with Rhodamine B dye and confocal microscopy was used for monitoring the process. Rhodamine B exhibits emission at 575 nm when excitation with 554 nm light which do not overlap with absorption bands of azobenzene. Due to its hydrophilic nature, Rhodamine B should be loaded in the hydrophilic internal cavity of the vesicle. In this case, vesicles were formed in THF by slowly addition of an aqueous solution of Rhodamine B following the self-assembling process by turbidity analysis (see experimental section for further details).25 The vesicles were dialysed against water to remove THF as well as the non-encapsulated Rhodamine B. Dye encapsulation was confirmed by confocal microscopy images where fluorescence dots in a dark background were observed (Figure 3.19a). Figure 3.19 Fluorescence microscopy images of the water supension of loaded PEGb-d16isoAZOb vesicles before a) and after b) irradiation for 1 h at 365 nm and 2.6 mW/cm2. The length of the scale bar corresponds to 5 µm After irradiation at 365 nm for 1h, fluorescent dots were still visible by fluorescence microscopy but also a fluorescent background was observed due to Rhodamine B release from the interior of the vesicles to the aqueous media (Figure 3.19b). These experiments indicate that under UV illumination the vesicle membrane became permeable to the loaded fluorescent probe. The persistence of fluorescent dots after 1 h irradiation might be due to the fact that some of the vesicles are still unaffected by irradiation but more likely it can be due to only partial release of the encapsulated dye. A continuous permeation a) b) 136 Chapter 3   through the membrane of the encapsulated Rhodamine B occurs on exposure due to its deformation but, reassembly (at least partially) of the vesicle membrane on the dark due to thermal cis-to-trans back isomerisation limits the release. Recent simulation studies pointed out that despite the release of hydrophilic substance starts as soon as the vesicles are exposed to light, the membrane permeation does not enhance suddenly.26 Dual Responsive Miktoarm Star Polymers 239    References 1 Savic, R.; Luo, L. B.; Eisenberg, A.; Maysinger, D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003, 300, 615-618. 2 Riess, G. Micellization of block copolymers. Progress in Polymer Science 2003, 28, 1107-1170. 3 Gohy, J.-F. in Block Copolymers II Vol. 190 Advances in Polymer Science (ed Volker Abetz) Ch. 48, 65-136 (Springer Berlin Heidelberg, 2005). 4 Lavasanifar, A.; Samuel, J.; Kwon, G. S. Poly(ethylene oxide)-block-poly(l-amino acid) micelles for drug delivery. Advanced Drug Delivery Reviews 2002, 54, 169190. 5 Adams, M. L.; Lavasanifar, A.; Kwon, G. S. Amphiphilic block copolymers for drug delivery. Journal of Pharmaceutical Sciences 2003, 92, 1343-1355. 6 Schacher, F. H.; Rupar, P. A.; Manners, I. Functional Block Copolymers: Nanostructured Materials with Emerging Applications. Angewandte Chemie International Edition 2012, 51, 7898-7921. 7 Gil, E. S.; Hudson, S. M. Stimuli-reponsive polymers and their bioconjugates. Progress in Polymer Science 2004, 29, 1173-1222. 8 Stuart, M. A.; Huck, W. T.; Genzer, J.; Müller, M.; Ober, C.; Stamm, M.; Sukhorukov G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Emerging applications of stimuli-responsive polymer materials. Nature Materials 2010, 9, 101-113. 9 Liu, F.; Urban, M. W. Recent advances and challenges in designing stimuliresponsive polymers. Progress in Polymer Science 2010, 35, 3-23. 10 Idziak, I.; Avoce, D.; Lessard, D.; Gravel, D.; Zhu, X. X. Thermosensitivity of Aqueous Solutions of Poly(N,N-diethylacrylamide). Macromolecules 1999, 32, 1260-1263. 11 Abulateefeh, S. R.; Spain, S. G.; Aylott, J. W.; Chan, W.C.; Garnett, M.C.; Alexander C. Thermoresponsive Polymer Colloids for Drug Delivery and Cancer Therapy. Macromolecular Bioscience 2011, 11, 1722-1734. 240 Chapter 6   12 Gaucher, G.; Dufresne, M. H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J. C. Block copolymer micelles: preparation, characterization and application in drug delivery. Journal of Controlled Release 2005, 109, 169-188. 13 Xu, J.; Liu, S. Polymeric nanocarriers possessing thermoresponsive coronas. Soft Matter 2008, 4, 1745-1749. 14 Hu, Z.; Cai, T.; Chi, C. Thermoresponsive oligo(ethylene glycol)-methacrylatebased polymers and microgels. Soft Matter 2010, 6, 2115-2123. 15 Pasparakis, G.; Vamvakaki, M. Multiresponsive polymers: nano-sized assemblies, stimuli-sensitive gels and smart surfaces. Polymer Chemistry 2011, 2, 1234-1248. 16 Jochum, F. D.; Theato, P. Temperatureand light-responsive smart polymer materials. Chemical Society Reviews 2013. DOI: 10.1039/C2CS35191A 17 Feng, Z.; Lin, L.; Yan, Z.; Yu, Y. L. Dual Responsive Block Copolymer Micelles Functionalized by NIPAM and Azobenzene. Macromolecular Rapid Communications 2010, 31, 640-644. 18 Jochum, F. D.; Theato, P. Thermoand light responsive micellation of azobenzene containing block copolymers. Chemical Communications 2010, 46, 6717-6719. 19 Boissiere, O.; Han, D.; Tremblay, L.; Zhao, Y. Flower micelles of poly(Nisopropylacrylamide) with azobenzene moieties regularly inserted into the main chain. Soft Matter 2011, 7, 9410-9415. 20 Schmidt, B. V. K. J.; Hetzer, M.; Ritter, H.; Barner-Kowollik, C. Miktoarm star polymers via cyclodextrin-driven supramolecular self-assembly. Polymer Chemistry 2012, 3, 3064-3067. 21 Goodwin, A. P.; Mynar, J. L.; Ma, Y. Z.; Fleming, G. R.; Frechet, J. M. J. Synthetic micelle sensitive to IR light via a two-photon process. Journal of the American Chemical Society 2005, 127, 9952-9953. 22 Mynar, J. L.;Goodwin, A. P.; Cohen, J. A.; Ma, Y.; Fleming J. R.; Fréchet J.M.J. Two-photon degradable supramolecular assemblies of linear-dendritic copolymers. Chemical Communications 2007, 2081-2082. 23 Ferreira, S. A.; Coutinho, P. J. G.; Gama, F. M. Self-Assembled Nanogel Made of Mannan: Synthesis and Characterization. Langmuir 2010, 26, 11413-11420. Dual Responsive Miktoarm Star Polymers 241    24 Dong, J.; Wang, Y.; Zhang, J.; Zhan, X.; Zhu, S.; Yang, H.; Wang, G. Multiple stimuli-responsive polymeric micelles for controlled release. Soft Matter 2013, 9, 370-373. 25 Jin, Q.; Liu, G.; Ji, J. Micelles and reverse micelles with a photo and thermo double-responsive block copolymer. Journal of Polymer Science Part A: Polymer Chemistry 2010, 48, 2855-2861. 26 Díaz, D. D.; Punna, S.; Holzer, P.; McPherson, A. K.;Sharpless, K. B.;Fokin, V. V.; Finn, M. G. Click chemistry in materials synthesis. 1. Adhesive polymers from copper-catalyzed azide-alkyne cycloaddition. Journal of Polymer Science Part A: Polymer Chemistry 2004, 42, 4392-4403.  CHAPTER 7 Light Responsive Surfaces     Published in Adv. Funct. Mat. 2013. DOI: 10.1002/adfm.201203602   Light Responsive Surfaces 245    7.1 Introduction and Aims Engineering surface chemistry and topography affords technological advancements for a variety of applications ranging from biosensors to microelectronics.1,2 Surface functionalisation is an essential process for the construction of patterned surfaces and microarrays, surface immobilisation of biological molecules or just to tune or confer new properties to substrates. It can be completed by physical deposition (physisorption), but covalent immobilisation (chemisorption) is preferable because of the added stability of the coating. Smart functional surfaces can be created by covalent inmmobilisation of stimuli responsive molecules to tailor-made properties and generate substrates with switchable properties such as pH or wetability.3-5 In particular, the attachment of photoresponsive molecules onto surfaces is very attractive as the properties of the surfaces can be controlled by light as an external and non contact stimulus (see section 1.4). One critical aspect of the immobilisation is retaining the activity of the molecule once it is immobilised onto the surface. To cover demands, the development of fabrication methods for soft material surfaces with precise control over functionality, architecture, reactivity and domain size is required. During the last years, development of the ‘click chemistry’ methods has had an enormous impact on surface functionalisation.6,7 These reactions provide an efficient strategy because of the functional group versatility, high yields with no side products and simple reaction conditions. Nevertheless, in some applications an efficient reaction is not enough since patterning of the surface with spatial control of chemical functionality might also be required. The utilisation of light initiated ‘click reactions’ represents a powerful ligation protocol. These UV induced reactions include thiol-ene/thiolyne coupling,8 1,3-dipolar cycloaddition reactions9,10 and Diels–Alder reactions11 among others (Scheme 7.1). 246 Chapter 7   Scheme 7.1 Examples of light induced reactions employed for the preparation of patterning surfaces: a) CuAAC,9,12 b) photoenol chemistry,13 c) NITEC reaction14 Bowman and coworkers have recently developed a new photochemical protocol for the in situ generation of Cu(I) from a Cu(II) complex using light to catalyze a CuAAC reaction between azides and alkynes. Patterned material fabrication was achieved with this reaction by using standard photolithographic techniques (Figure 7.1).9,12 Barner-Kowollik and coworkers have introduced a novel procedure for click conjugations based on a Diels–Alder reaction of hydroxy-oquinodimethanes (photoenols) generated by photoisomerisation of omethylphenyl ketones or aldehydes.15,16 Photoenols are highly reactive dienes that can react with activated alkenes. This chemistry has been successfully applied to polymer conjugation as well as to surface patterning using different maleimide derivatives.17 The nitrile imine-mediated 1,3-dipolar cycloaddition of a tetrazole and an alkene derivative (NITEC reaction) was firstly reported by Huisgen and Sustmann in 196718 and recently significantly expanded by Lin and coworkers.19 The NITEC reaction proceeds via the generation of a nitrile imine dipole by irradiation with UV light of a tetrazole compound. The nitrile imine intermediate is able to react spontaneously with a large variety of alkenes forming a pyrazoline cycloadduct in near quantitative yields.19-21 This strategy have also been successfully employed for room temperature grafting of Light Responsive Surfaces 247    polymers onto variable surfaces such as silicon or cellulose by Barner-Kowollik and coworkers.22 Figure 7.1 Photopatterning of an azide functionalised polypropylene using a photomask (Image adapted from ref. 9) In the current chapter, the preparation and study of responsive surfaces using exclusively light as a stimulus for both the preparation of azobenzene modified surfaces and the subsequent control of the surface properties is approached by using chromophores analogues to those described in previous chapters. The preparation of these spatially controlled photoresponsive surfaces is addressed by use of the NITEC reaction using azobenzene dipolarophiles as it is collected in Scheme 7.2. In this strategy, the previously activated surfaces were modified with a silane derivative containing a tetrazole group. Then, a NITEC reaction between tetrazole and dipolarophiles was employed to obtain the photoresponsive surfaces. The dipolarophiles consist of a maleimide containing either a single azobenzene (AZO1) moiety or a first-generation dendron carrying two azobenzene units (AZO2). To the best of our knowledge, this reaction has not been used before in the presence of species exhibiting strong absorption in the UV−Vis region. Again, 4-isobutyloxyazobenzene unit was chosen due to the increment in polarity difference between the trans and the cis isomers. PEG-alkyne CuAAC EDTA PHOTOPATTERNED SURFACE 248 Chapter 7   Scheme 7.2 Azobenzene functionalisation of surfaces via the NITEC reaction N 2 OHOH OH OH OH OHOH OH OH OH OHOH OH OH OH OHOH OH OH OH Light Responsive Surfaces 255    Figure 7.4 UV spectra of the tetrazole functionalised silane TET (grey line) and maleimide-containing azobenzene AZO1 (black line) solution in acetonitrile (10-4 M) Figure 7.5 ESI-MS spectra of the NITEC reaction depicted in Scheme 7.5 at different reaction times 300 400 500 0.0 0.2 0.4 0.6 Absorbance Wavelenght (nm) 400 500 600 700 800 900 1000 1100 Relative Abundance m/z 1 h 40 min 20 min 0 min [TET-Na] + [AZO1-H] + [AZO1-Na] + [AD-H] + [AD-Na] + 256 Chapter 7   7.3.3 Azobenzene Surface Functionalisation After evidencing the efficiency of the NITEC reaction in the presence of azobenzene and in solution, the following step was to carry out the same reaction with tetrazole functionalised silicon wafers (Scheme 7.6). The silicon after were cleaned and hydroxylated with Piranha solution and subsequently the covalent binding of the tetrazole containing silane was performed by heating an activated silicon wafer with TET in toluene at 50 ºC (see Experimental Section). The tetrazole functionalised silicon wafer (Si-TET) was thoroughly rinsed with fresh solvent and sonicated to ensure no physisorbed tetrazole was present onto the surface. XPS was employed to prove the functionalisation of the surface. In the XPS spectra (Figure 7.6a) it is possible to observe intense peaks around 285-290 eV corresponding to C 1s and around 400-402 eV attributed to N 1s. As it was reported, peaks at 286.6 eV and 288.5 eV can be assigned to carbon atoms single bonded with oxygen and nitrogen (C-O, C-N) and to carboxylic groups (-N-C=O, -O-C=O) respectively .32,33 The N 1s spectrum presents a strong peak at 400.2 eV that can be assigned to the tetrazole species34 and a weak one at 402.7 eV that probably correspond to positively charged nitrogen.35 Then, the NITEC reaction was employed to graph AZO1 onto the surface by using the optimum conditions identified in the solution tests (290-315 nm). The Si-TET silicon wafer was placed in a quartz flask containing a maleimide AZO1 solution in DCM (7 mM) and exposed to UV light (9W, 290-315 nm) The azobenzene functionalised wafers Si-AZO1 were analysed by XPS and compared with the tetrazole functionalised one (Si-TET). As in the case of SiTET (Figure 7.6a), XPS spectra of Si-AZO1 shows peaks at 285.0 at 286.6 and 288.5 eV corresponding to C 1s and around 400.2 and 402.7 eV attributed to N 1s (Figure 7.6b). In order to establish comparison, the relative peak areas were calculated by using (C-O, C-N) signal at 286.6 eV as reference (Figure 7.7). As expected, in comparison with Si-TET the N / (C-O, C-N) ratio decreased from 1.12 to 0.50 on the functionalised surface Si-AZO1 evidencing the presence of azobenzene on the surface (Figure 7.6b). Light Responsive Surfaces 257    N N NNR O N O O NH Si O O O N NNO O O NH Si O O O hv O N NO O R R= O OO O O O N N N N O O O or Si-TET AZO1 AZO2 -N 2 Si-AZO1 Si-AZO2 R= Scheme 7.6 NITEC reaction between a tetrazole-functionalised surface and the azobenzene derivatives AZO1 and AZO2. To simplify, only one Si-anchoring has been considered Figure 7.6 Comparison of the C 1s (left) and N 1s (right) normalised regions of the XPS spectra of functionalised silicon wafers: a) Si-TET, b) Si-AZO1 and c) Si-AZO2 397398399400401402403404405 Normalized Intensity (a.u.) Binding Energy (eV) a) b) c) 282283284285286287288289290291 Normalized Intensity (a.u.) Binding Energy (eV) c) b) a) 258 Chapter 7   Figure 7.7 Chemical structures and theoretical C 1s and N 1s XPS peak abundances (top) for the modified silicon wafers Si-TET, AZO1 and AZO2. Bar chart comparing theoretical and experimental C 1s and N 1s XPS peak abundances for the silicon wafers Si-TET, Si-AZO1 and Si-AZO2 (bottom). For wafer Si-AZO2 a theoretical reaction yield of 50% is assumed, the experimental data are based on 6 h reaction time. The high experimental intensity of all C-H components is due to adventitious carbon. 0 1 2 3 4 5 6 7 C -H N /O - C = O N atomic concentration normalized to C-O content Si-TET theo Si-TET exp Si-AZO1 theo Si-AZO1 exp Si-AZO2 theo Si-AZO2 exp Si-AZO1 Si-TET Si-AZO2 Surface C-C, C-H C-O, C-N N-C=O, O-C=O NC-C, C-H)/ C-O, C-N N-C=O, OC=O/ C-O, C-N N/ C-O,C-N Si-TET 13 3 1 5 4.33 0.33 1.67 Si-AZO1 29 12 4 6 2.42 0.33 0.50 Si-AZO2 46 20 6 8 2.33 0.30 0.40 Light Responsive Surfaces 259    The photoligation reaction was also carried out with the first-generation azodendron AZO2. The silicon wafer Si-TET was immersed into a solution of AZO2 in DCM (3.5 mM) in a quartz flask and irradiated at 290-315 nm. The functionalised silicon wafer Si-AZO2 was analysed by XPS (Figure 7.6c). The relative areas of the signals were again compared using C-O, C-N as reference and it was observed that the N/(C-O, C-N) ratio decreased from 1.12 in for SiTET to 0.57 for Si-AZO2 (Figure 7.7). Nevertheless, in this case the experimental result is not in agreement with the theoretical value (N/(C-O, C-N) ratio of 0.40) evidencing incomplete functionalisation of the surface. In order to optimise the efficiency of the photoconjugation, the progress of the reaction with AZO2 was followed by XPS from the changes in N/(C-O, C-N) ratio (Figure 7.8) The best result was achieved with 6 h of reaction for which a functionalisation close to 50% was reached according to the XPS data. The lower efficiency might be attributed to a higher steric hindrance in the case of the azodendron AZO2 in comparison with the single molecule AZO1. Figure 7.8 Time dependent evolution of the XPS N / (C-O, C-N) ratio of Si-AZO2. Dashed line indicates the assumed max. 50% reaction yield, 0 h reaction time represents pure wafer Si-TET. Error bars represent standard deviations of the measurements. 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 0123456789 N/C-O, C-N ratio reaction time (h) 50% 260 Chapter 7   7.3.4 Azobenzene Surface Patterning To prove the spatial control in azobenzene functionalisation, the concept was extended to the formation of a micropatterned substrate by using a photomask. Tetrazole functionalised surfaces, Si-TET, were covered with a shadow mask containing a micropattern, immersed in an azobenzene solution of AZO1 or AZO2 and UV illuminated in the same conditions as before (Scheme 7.7). After removing the mask and washing the surfaces, the patterns were revealed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is a surfacesensitive analytical method providing chemical images generated by collecting mass spectra at a high lateral resolution (see Appendix). Scheme 7.7 Azobenzene functionalisation of the surfaces with spatial control employing a micropatterned shadow mask. Figure 7.9a and Figure 7.9b depicts the ToF-SIMS images of the patterned surfaces. Two azobenzene fragments, C16H17N2O2- (m/z=269.2) and C12H8N2O2at 212.1 m/z, were exclusively detected in the UV exposed areas, and not in the non irradiated regions. Further, the [M-Na]- ions (m/z=530.4 for AZO1 and 1012.5 for AZO2) cannot be detected after the photografting step discarding physisorption of the precursor molecules AZO1 and AZO2 and consequently unambiguously evidencing a covalent attachment. Figure 7.10 shows as an example the SIMS data in the region of the molecular ion of AZO2, where [M-Na]- was not detected in the case of the covalently functionalised surface. N 2 Light Responsive Surfaces 261    Figure 7.9 ToF-SIMS images of a) the azobenzene AZO1 and b) azobenzence AZO2 immobilised in a zigzag pattern defined by the applied photomask. Negative polarity SIMS, 269.1 u and 212.1 u, assigned to C16H17N2O2- (left) and C12H8N2O2- (right) Figure 7.10 SIMS spectrum of the azobenzene AZO2 physically adsorbed by solventcasting deposition (red) and grafted (black) onto silicon wafers a) b) C 12 H 8 N 2 O 2a) b) C 16 H 17 N 2 O 21010 1012 1014 1016 1018 1020 0.0 0.5 1.0 1.5 2.0 2.5 3.0 kcts Mass (u) grafted bulk 262 Chapter 7   7.3.5 Wettability Study After evidencing the presence of azobenzene on the surface by XPS and ToFSIMS, azobenzene trans-to-cis photoisomerisation was provoked by UV irradiation of the surface. For this photoisomerisation, a lamp with the maximum emission wavelength close to 355 nm was chosen (the strongest absorption band of trans-azobenzene is centered about 360 nm). Functionalised surfaces Si-AZO1 and Si-AZO2 were illuminated through a mask covering half of the surface as a way to generate two regions having different polarities due to the azobenzene isomerisation in the selectively exposed areas (Scheme 7.8). This would allow fine tuning of the surface wettability. A simple and effective technique employed to macroscopically monitor the photoisomerisation is the contact angle (CA) measurement. Advancing and receding CAs were measured in non irradiated (transazobenzene rich areas) and irradiated (cis-azobenzene rich areas) regions of both surfaces (Table 7.1). On an ideal surface, the advancing and the receding angles will be identical.36 It is well known that roughness or chemical heterogeneity can cause CA hysteresis, yet it has also been reported that even surfaces – which are initially smooth and homogeneous – can exhibit CA hysteresis because of a reorganisation of surface molecules.37,38 In the present case, no significant modification in the advancing CA can be observed. However, a significant change of 15º in receding CA occurred on Si-AZO1 surface evidencing that the photoisomerisation occurs and has influence on the wettability of the surface. In the case of Si-AZO2 surface, smaller differences were detected between the non-irradiated and the irradiated zone were detected, probably due to a more heterogeneous and less azobenzene functionalised surface being produced. As expected, the contact angle in the cis-azobenzene region in both cases decreased as a consequence of the increase on the dipole moment. Reported differences in CA on trans and cis azobenzene functionalised smooth surfaces did not exceed 10º39,40 whereas higher differences were achieved in the azobenzene functionalised surface SiAZO1. Light Responsive Surfaces 263    Scheme 7.8. Spatially controlled photoisomerisation of an azobenzene functionalised surface by using a mask to cover half of the surface. Table 7.1 Contact angle measurements of the azofunctionalised surfaces Surface Advancing angle Receding angle AZO1 (non-irradiated) 87.0º + 1.0 56.7º + 3.5 AZO1 (irradiated) 86.7º + 1.5 41.7º + 1.5 AZO2 (non-irradiated) 83.0º + 2.5 52.3º + 2.5 AZO2 (irradiated) 83.5º + 3.0 42.3º + 4.5 Visual experiments by using a water droplet were performed to demonstrate the photoswitchable wettability of the functionalised surface. It was evidenced that when azobenzenes at the surface adopt the trans configuration a water droplet can slips the surface whereas the water droplet is sticky if azobenzenes are in the cis configuration. Such a different behaviour is more pronounced in the case of surface modified with azobenzene Si-AZO1. For a visual demonstration of the switching effect, please refer to the movies (http://onlinelibrary.wiley.com /doi/10.1002/adfm.201203602/suppinfo). In these experiments, a water droplet was placed in each region of the surface, irradiated as well as non-irradiated, and it was forced to move over the surface. Reversible cis to trans thermal isomerisation was checked by keeping the surface in the dark for 24 hours before evaluating the water droplet behavior again. After 24 h, the water droplet slipped over the entire surface proving that azobenzene adopted trans 264 Chapter 7   configuration in both regions. Although the thermal isomerisation is slow (hours) it can be readily accelerated by heating or by exposure to visible light. Light Responsive Surfaces 271    1247. 1H-NMR (CDCl3, 400MHz) δ (ppm): 6.51 (t, J = 1.0 Hz, 2H), 5.26 (t, J = 1.0 Hz, 2H), 4.32-4.27 (m, 2H), 4.13 (d, J = 11.8 Hz, 2H), 3.83 - 3.73 (m, 2H), 3.58 (d, J = 11.8 Hz, 2H), 2.86 (s, 2H), 1.40 (s, 3H), 1.37 (s, 3H), 1.18 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 175.8, 173.8, 136.4, 97.9, 80.7, 65.7, 61.1, 47.4, 41.6, 37.7, 23.8, 23.2, 18.4. Synthesis and Characterisation of (23) DOWEX-50-X2 resin (0.10 g) was added to a solution of compound (22) (0.50 g, 1.50 mmol) in methanol (15 mL). The mixture was stirred for 3 h at room temperature. Subsequently, the resin was filtered off and the solvent removed under vacuum to give (23) as a colourless viscous oil. Yield: 90%. IR (KBr),  (cm-1): 3500, 1772, 1721, 1699, 1279, 1246. 1H-NMR (CDCl3, 400MHz) δ (ppm): 6.45 (t, J = 1.0 Hz, 2H), 5.22 (t, J = 1.0 Hz, 2H), 4.32 – 4.19 (m, 2H), 3.75-3.69 (m, 4H), 3.64-3.60 (m, 2H), 2.83 (s, 2H), 2.81 (t, J=6.8 Hz ,1H), 0.97 (s, 3H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 175.8, 173.8, 136.4, 80.7, 65.7, 61.1, 47.4, 41.6, 37.7, 18.4. Synthesis and Characterisation of (24)  The acid chloride derivate of 6-[4-(4’-isobutyloxyphenylazo)phenyloxy] hexanoic acid (isoAZOb) was prepared by reaction of isoAZOb (0.93 g, 2.43 mmol) with oxalyl chloride (0.4 mL, 4.86 mmol) in DCM (20mL). After stirring at room temperature for 4h, the solvent was distilled. The acid chloride derivated was directly added to a solution of compound 23 (0.40 g, 1.12 mmol) and 272 Chapter 7   triethylamine (0.23 g, 2.40 mmol) in DCM (20 mL). The mixture was stirred for 3 h at room temperature under argon atmosphere. After this time, the white precipitate formed was filtered off, and the solvent was removed under vacuum. The crude product was purified by flash column chromatography on silica gel using 7:3 ethyl acetate:DCM as eluent. Yield: 65%. IR (KBr),  (cm-1): 1739, 1703, 1601, 1581, 1498, 1243, 1149, 1024, 843. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.90-7.86 (m, 8H), 6.96-6.90 (m, 8H), 6.48 (t, J = 1.0 Hz, 2H), 5.29 (t, J = 1.0 Hz), 4.26-4.08 (m, 6H), 4.01 (t, J = 6.4 Hz, 4H), 3.82-3.73 (m, 6H), 2.86 (s, 2H), 2.35 (t, J = 7.4 Hz, 4H), 2.10-2.03 (m, 2H), 1.86-1.75 (m, 4H), 1.73-1.64 (m, 4H), 1.54-1.44 (m, 4H), 1.22 (s, 3H), 1.05 (d, J = 6.7 Hz, 12H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 175.9, 172.9, 172.3, 161.9, 160.9, 146.8, 146.8, 136.4, 124.1, 114.6, 114.5, 80.7, 74.6, 67.8 , 64.9, 61.4, 47.4, 46.2, 37.6, 33.8, 28.8, 28.2, 25.5, 24.5, 19.2, 17.5. Synthesis and Characterisation of AZO2 The protected maleimide (24) was suspended in toluene (150 mL) and heated to reflux. The reaction was monitored by thin layer chromatography After 4 hours, the solvent was removed under reduced pressure to give AZO2 as a yellow powder. Yield: 100%. IR (KBr),  (cm-1): 1731, 1713, 1601, 1582, 1498, 1243, 1149, 1034, 845. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.90-7.86 (m, 8H), 6.96-6.90 (m, 8H,), 6.70 (s, 2H), 4.27-4.12 (m, 6H), 4.01 (t, J = 6.4 Hz, 4H), 3.82-3.73 (m, 6H), 2.35 (t, J = 7.4 Hz, 4H), 2.10-2.03 (m, 2H), 1.86-1.75 (m, 4H), 1.73-1.64 (m, 4H), 1.54-1.44 (m, 4H), 1.22 (s, 3H), 1.05 (d, J = 6.7 Hz, 12H). 13C-NMR (CDCl3, 100 MHz) δ (ppm): 172.9, 172.3, 170.3, 161.9, 160.9, 146.8, 146.8, 134.2, 124.1, 114.6, 114.5, 74.6, 67.8, 64.9, 61.4, 46.2, 37.6, 33.8, 28.8, 28.2 (CH), 25.5, 24.5, 19.2, 17.5. MALDI-TOF MS (matrix: dithranol, m/z): 990.6 [M-H]+, 1012.5 [M-Na]+. Anal. Calc. for C55H67N5O12: C, 66.72 %; H, 6.82 %; N, 7.07 %. Found: C, 66.53 %; H, 7.01 %; N 7.05 %. Light Responsive Surfaces 273    7.5.3 General Procedures Solution Tests Solution tests were performed in a quartz cuvette by employing a hand-held UV lamp and low pressure mercury lamp OSRAM Puritec HNS L 36 W (dominant wavelength 254 nm). The photoreaction was carried out in DCM (7mM AZO1) at room temperature. Activation of Silicon Wafers Prior to surface activation, the silicon wafers (p-type, boron doped (100) from Si-Mat Silicon Materials, Landsberg, Germany) were cleaned with chloroform, acetone and ethanol. The wafers were rinsed thoroughly with fresh solvent and sonicated 5 min several times with each solvent. After cleaning, the silicon wafers were activated by immersion in Piranha solution (H2SO4 95%/H2O2 35% 3:1 vol/vol) at 90 °C for 1h. After extensive rinsing with deionised water, they were dried under a stream of argon. Functionalisation of Silicon Wafers with Tetrazole (Si-TET) The activated silicon wafers were placed in a flask containing a solution of silane functionalised tetrazole (TET) in dry toluene (4.8 mg in 1 mL). The flask was heated to 50 °C overnight. Subsequently, the wafers were rinsed thoroughly with fresh toluene and chloroform and sonicated for 5 min. The wafers were finally dried in a stream of argon. Functionalisation of Silicon Wafers with Azobenzene (Si-AZO1 and SiAZO2) The tetrazole functionalised silicon wafers were placed in a quartz flask containing an azobenzene solution in DCM (7mM for AZO1 and 3.5 mM for AZO2). The flask was introduced into a photoreactor with two lamps and irradiated for a pre-set time interval. Subsequently the wafers were rinsed thoroughly with fresh chloroform and sonicated for 5 min. The wafers were finally dried in a stream of argon. The experiments were carried out with 274 Chapter 7   compact low-pressure fluorescent lamps Philips PL-S 9W/12 emitting UV irradiation between 290 and 315 nm. Photoisomerisation of Azobenzene Functionalised Silicon Wafers The azobenzene functionalised silicon wafers were introduced in a photoreactor fixed with two lamps and irradiated for 30 min. After this time, the wafers were kept in the dark. The experiments were performed using compact low-pressure fluorescent lamps Philips CLEO PL-L 36W emitting between 310 and 400 nm (λmax=355 nm). Light Responsive Surfaces 275    References 1 Shipway, A. N.; Katz, E.; Willner, I. Nanoparticle arrays on surfaces for electronic, optical, and sensor applications. Chemphyschem 200, 1, 18-52. 2 Falconnet, D.; Csucs, G.; Grandin, H. M.; Textor, M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006, 27, 3044-3063. 3 Russell, T. P. Surface-responsive materials. Science 2002, 297, 964-967. 4 Sun, T. L.; Feng, L.; Gao, X. F.; Jiang, L. Bioinspired surfaces with special wettability. Accounts of Chemical Research 2005, 38, 644-652. 5 Mendes, P. M. Stimuli-responsive surfaces for bio-applications. Chemical Society Reviews 2008, 37, 2512-2529. 6 Binder, W. H.; Sachsenhofer, R. 'Click' chemistry in polymer and materials science. Macromolecular Rapid Communications 2007, 28, 15-54. 7 Binder, W. H.; Sachsenhofer, R. 'Click' chemistry in polymer and material science: An update. Macromolecular Rapid Communications 2008, 29, 952-981. 8 Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chemical Society Reviews 2010, 39, 1355-1387. 9 Adzima, B. J.; Tao, Y.; Kloxin, C. J.; Forest, C. A.; Anseth, K.S.; Bowman, C. N. Spatial and temporal control of the alkyne-azide cycloaddition by photoinitiated Cu(II) reduction. Nature Chemistry 2011, 3, 256-259. 10 Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons, G.-J.; Popik, V. V. Selective Labeling of Living Cells by a Photo-Triggered Click Reaction. Journal of the American Chemical Society 2009, 131, 15769-15776. 11 Arumugam, S.; Popik, V. V. Light-Induced Hetero-Diels−Alder Cycloaddition: A Facile and Selective Photoclick Reaction. Journal of the American Chemical Society 2011, 133, 5573-5579. 12 Chen, R. T.; Marchesan, S.; Evans, R. A.; Styan, K. E.; Such, G. K.; Postma, A.; McLean, K.M.; Muir, B.W.; Caruso, F. Photoinitiated Alkyne–Azide Click and 276 Chapter 7   Radical Cross-Linking Reactions for the Patterning of PEG Hydrogels. Biomacromolecules 2012, 13, 889-895. 13 Pauloehrl, T.; Delaittre, G.; Winkler, W.; Welle, A.; Bruns, M.; Börner, H. G.; Greiner, A. M.; Bastmeyer, M.; Barner-Kowollik, C. Adding Spatial Control to Click Chemistry: Phototriggered Diels-Alder Surface (Bio)functionalization at Ambient Temperature. Angewandte Chemie-International Edition 2012, 51, 1071-1074. 14 Dietrich, M.; Delaittre, G.; Blinco, J. P-; Inglis, A.J.; Bruns, M.; Barner-Kowollik, C. Photoclickable Surfaces for Profluorescent Covalent Polymer Coatings. Advanced Functional Materials 2012, 22, 304-312. 15 Gruendling, T. Oehlenschlaeger, K. K.; Frick, E.; Glassner, M.; Schmid, C. BarnerKowollik, C. Rapid UV Light-Triggered Macromolecular Click Conjugations via the Use of o-Quinodimethanes. Macromolecular Rapid Communications 2011, 32, 807-812. 16 Glassner, M., Oehlenschlaeger, K. K., Gruendling, T. & Barner-Kowollik, C. Ambient Temperature Synthesis of Triblock Copolymers via Orthogonal Photochemically and Thermally Induced Modular Conjugation. Macromolecules 2011, 44, 4681-4689. 17 Pauloehrl, T.; Delaittre, G.; Bruns, M.; Meissler, M.; Börner, H. G.; Greiner, A. M.; Bastmeyer, M.; Barner-Kowollik, C. (Bio)Molecular Surface Patterning by Phototriggered Oxime Ligation. Angewandte Chemie International Edition 2012, 51, 9181 (2012). 18 Clovis, J. S.; Eckell, A.; Huisgen, R.; Sustmann, R. 1.3-Dipolare Cycloadditionen, XXV. Der Nachweis des freien Diphenylnitrilimins als Zwischenstufe bei Cycloadditionen. Chemische Berichte 1967, 100, 60-70. 19 Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. A Photoinducible 1,3-Dipolar Cycloaddition Reaction for Rapid, Selective Modification of Tetrazole-Containing Proteins. Angewandte Chemie International Edition 2008, 47, 2832-2835. 20 Wang, Y. Z.; Hu, W. J.; Song, W. J.; Lint, R. K. V.; Lin, Q. Discovery of longwavelength photoactivatable diaryltetrazoles for bioorthogonal 1,3-dipolar cycloaddition reactions. Organic Letters. 2008, 10, 3725-3728. Light Responsive Surfaces 277    21 Wang, Y. Z.; Vera, C. I. R.; Lin, Q. Convenient synthesis of highly functionalized pyrazolines via mild, photoactivated 1,3-dipolar cycloaddition. Organic Letters 2007, 9, 4155-4158. 22 Dietrich, M.; Delaittre, G.; Blinco, J. P-; Inglis, A.J.; Bruns, M.; Barner-Kowollik, C. Photoclickable Surfaces for Profluorescent Covalent Polymer Coatings. Advanced Functional Materials 2012, 22, 304-312. 23 Meier, H.; Heimgartner, H. Intramolekulare 1,3-dipolare Cycloadditionen von Diarylnitriliminen aus 2,5-Diaryltetrazolen. Helv. Chim. Acta 1985, 68, 1283-1300. 24 Lohse, V.; Leihkauf, P.; Csongar, C.; Tomaschewski, G. Photochemie diarylsubstituierter 2H-Tetrazole. VI. Quantenausbeuten der Photolyse diarylsubstituierter 2H-Tetrazole. J. Prakt. Chem. 1988, 330, 406-414. 25 Darkow, R.; Yoshikawa, M.; Kitao, T.; Tomaschewski, G.; Schellenberg, J. Photomodification of a poly(acrylonitrile-co-butadiene-co-styrene) containing diaryltetrazolyl groups. Journal of Polymer Science Part A: Polymer Chemistry 1994, 22, 1657-1664. 26 Bertrand, G.; Wentrup, C. Nitrile Imines: From Matrix Characterization to Stable Compounds. Angewandte Chemie-International Edition 1994, 33, 527-545. 27 Moderhack, D. Ring Transformations in Tetrazole Chemistry. Journal für Praktische Chemie/Chemiker-Zeitung 1998, 340, 687-709. 28 Wang, J.; Zhang, W.; Song, W.; Wang, Y.; Yu, Z.; Li, J.; Wu, M.; Wang, L.; Zang, J.; Lin, Q. A Biosynthetic Route to Photoclick Chemistry on Proteins. Journal of the American Chemical Society 132, 14812-14818, doi:10.1021/ja104350y (2010). 29 Song, W.; Wang, Y.; Qu, J.; Lin, Q. Selective Functionalization of a Genetically Encoded Alkene-Containing Protein via “Photoclick Chemistry” in Bacterial Cells. Journal of the American Chemical Society 2008, 130, 9654-9655. 30 Wang, Y.; Song, W.; Hu, W. J.; Lin, Q. Fast Alkene Functionalization In Vivo by Photoclick Chemistry: HOMO Lifting of Nitrile Imine Dipoles. Angewandte Chemie, International Edition 2009, 48, 5330-5333. 278 Chapter 7   31 Madden, M. M.; Rivera Vera, C. I.; Song, W.; Lin, Q. Facile synthesis of stapled, structurally reinforced peptide helices via a photoinduced intramolecular 1,3-dipolar cycloaddition reaction. Chemical Communications 2009, 5588-5590. 32 De Marco, C.; Eaton, S.M; Suriano, R.; Turri, S.; Levi, M.; Ramponi, R.; Cerullo, G.; Osellame, R. Surface Properties of Femtosecond Laser Ablated PMMA. ACS Applied Materials & Interfaces 2010, 2, 2377-2384. 33 Lock, E. H.; Petrovykh, D. Y., Mack, P.; Carney, T.; White, R. G.; Walton, S. G.; Fernsler, R. F. Surface Composition, Chemistry, and Structure of Polystyrene Modified by Electron-Beam-Generated Plasma. Langmuir 2010, 26, 8857-8868. 34 Szocs, E.; Bakó, I.; Kosztolányi, T.; Bertóti, I.; Kálmán, Electrochimica Acta 2004, 49, 1371-1378. 35 Rouxhet, P. G.; Misselyn-Bauduin, A. M.; Ahimou, F.; Genet, M. J.; Adriaensen, Y.; Desille, T.; Bodson, P.; Deroanne, C. XPS analysis of food products: toward chemical functions and molecular compounds. Surface and Interface Analysis 2008, 40, 718-724. 36 Strobel, M.; Lyons, C. S. An Essay on Contact Angle Measurements. Plasma Processes Polym. 2011, 8, 8-13. 37 Ichimura, K.; Oh, S. K.; Nakagawa, M. Light-driven motion of liquids on a photoresponsive surface. Science 2000, 288, 1624-1626. 38 Chen, Y. L.; Helm, C. A.; Israelachvili, J. N. Molecular mechanisms associated with adhesion and contact angle hysteresis of monolayer surfaces. The Journal of Physical Chemistry B. 1991, 95, 10736-10747. 39 Siewierski, L. M.; Brittain, W. J.; Petrash, S.; Foster, M. D. Photoresponsive Monolayers Containing In-Chain Azobenzene. Langmuir 1996, 12, 5838-5844. 40 Delorme, N.; Bardeau, J. F.; Bulou, A.; Poncin-Epaillard, F. Azobenzenecontaining monolayer with photoswitchable wettability. Langmuir 2005, 21, 1227812282. Conclusiones        Characterisation Techniques 287    Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR spectra were obtained on a Nicolet Avatar 360-FT-IR spectrometer (Chapter 2) and Bruker FT-IR spectrometer using KBr pellets. Nuclear Magnetic Resonance Spectroscopy (NMR) 1H-NMR and 13C-NMR spectra were measured on a Bruker AV-400 spectrometer at 400 MHz and on a Bruker AM250 spectrometer at 250 MHz (Chapter 5 and 6) Mass spectrometry (MS) MALDI-TOF MS was performed on an Autoflex mass spectrometer (Bruker Daltonics). Number-average molecular weight (Mn) and polydispersity of the BCs were calculated from the mass spectra using PolyTools 1.0 (Bruker). ESI-MS spectra (Chapter 7) were recorded on an Autoflex mass spectrometer (Bruker Daltonics) and a LXQ mass spectrometer (ThermoFisher Scientific) equipped with an atmospheric pressure ionization source operating in the nebuliser-assisted electrospray mode. The instrument was calibrated in the m/z range 195-1822 using a standard comprising caffeine, Met-Arg-Phe-Ala acetate (MRFA), and a mixture of fluorinated phosphazenes (Ultramark 1621, all from Aldrich). Elemental Analysis (EA) EA was performed using a Perkin–Elmer 2400 microanalyzer. Size Exclusion Chromatography (SEC) SEC was carried out on a Waters e2695 Alliance liquid chromatography system (Chapter 2, 3 and 4) equipped with a Waters 2424 evaporation light scattering 288 Appendix   detector and a Waters 2998 PDA detector using two Ultrastyragel® columns, HR4 and HR2 from Waters, of 500 and 104Å pore size and on a Polymer Laboratories PL-GPC 50 Plus Integrated System (Chapter 5 and 6), comprising an autosampler, a PLgel 5 mm bead-size guard column (50 7.5 mm) followed by three PLgel 5 mm MixedC columns (300 7.5 mm) and a differential refractive index detector. Measurements were performed in THF with a flow of 1 mL/min using narrow molecular weight PS and PMMA standards. Preparative SEC (Chapter 2) was carried out on a Waters 600 pump and a Waters 2998 PDA detector using two UltrastyragelTM columns, 19300 mm, of 500 and 104Å pore size. Separations were carried in THF using a rate of 6 mL/min. UV-Vis Spectroscopy UV-Vis spectra were recoreded on an ATI-Unicam UV4-200 spectrophotometer. Fluorescence Spectroscopy Fluorescence measurements were recorded using a Perkin Elmer LS 50B fluorescence spectrophotometer. Thermogravimetry (TGA) TGA were performed using a Q5000IR from TA Instruments under nitrogen atmosphere using 2-5 mg of the sample. Differential Scanning Calorimetry (DSC) Thermal transitions were determined by DSC using a DSC Q-2000 from TA Instruments with powdered samples (2-5 mg) sealed in aluminium pans. Glass transition temperatures were determined at the midpoint of the baseline jump Characterisation Techniques 289    and the isotropic temperatures were determined at the maximum of the corresponding peaks. Polarised optical microscopy (POM) Mesomorphic behaviour was evaluated by POM using an Olympus BH-2 polarizing microscope fitted with a Linkam THMS600 hot stage. Transmission Electronic Microscopy (TEM) Morphologic study of the polymers was studied by TEM in a JEOL-2000 FXIII and (Chapter 2, 3, 5 and 6) in a Tecnai T20 electron microscope (Chapter 4) electron microscope operating at 200kV. Cryogenic Transmission Electronic Microscopy (Cryo-TEM) Cryo-TEM observations were carried out in a JEM-2011 electronic microscope on samples rapidly frozen in liquid ethane. Dynamic light scattering (DLS) DLS measurements were carried out in a Malvern Instrument Nano ZS using a He-Ne laser with a 633 nm wavelength, a detector angle of 173º at 25ºC using a He-Ne laser with a 633. The self-assembiles concentration was 0.05 mg/mL (Chapter 3, 4 and 5) and 0.10 mg/mL (Chapter 6) and size measurements were performed at least three times on each sample to ensure consistency. Confocal Microscopy Fluorescence vesicles were observed with a Olympus FV10i confocal scanning microscope. Images were collected using a 60x oil immersion lens (lens 290 Appendix   specification, Plan S-APO 60xO, NA 1.35), a line average of 8 and a format of 1024x1024 pixels. The confocal pinhole was 1 Airy unit. Contact Angle Measurements Advancing and receeding contact angle measurements were performed with an OCA5 instrument (Dataphysics, Filderstadt, Germany). HPLC grade water was used and all measurements were performed on several spots of the substrate and averaged (n>3). Characterisation Techniques 291    Surface Analysis Effective and powerful methods for surface analysis are necessary for basic research on solid surfaces as well as for technical applications. These methods must be capable of giving detailed information, particularly about the chemical composition of the surface. In this section, fundamentals of two powerful techniques for surface analysis, i.e X-ray Phototelectron Spectroscopy (XPS) and time-of-flight Secondary Ions Mass Spectrometry (ToF-SIMS), will be briefly described. X-Ray Phototelectron Spectroscopy (XPS) XPS1, also known as Electron Spectroscopy for Chemical Analysis (ESCA) is a widely used technique to investigate the elemental composition, chemical state and electronic state of a surface element. This technique is based on the photoelectric effect outlined by Einstein in 1905. In the photoelectric effect, electrons are emitted from solids, liquids or gases when they are exposed to sufficiently energetic electromagnetic radiation (Figure AP.1a). The kinetic energy, Ek, of these photoelectrons is determined by the energy of the X-ray radiation, h, and the electron binding energy, Eb, as given by: Ek = h - Eb The experimentally measured energies of the photoelectrons are given by: Ek = h - Eb - Ew where Ew is the work function of the spectrometer. Since the energy of the emitted photoelectrons is exactly the energy of the incident photon minus the material's work function or binding energy, the work function of a sample can be determined by bombarding it with a monochromatic X-ray source or UV source, and measuring the kinetic energy distribution of the electrons emitted. The peak areas obtained can be used (with appropriate sensitivity factors) to determine the composition of the materials surface. The shape of each peak and the binding energy can be slightly altered by the 292 Appendix   chemical state of the emitting atom. Therefore, XPS can also provide chemical bonding information. XPS is not sensitive to hydrogen or helium, but can detect all other elements. Figure AP.1 a) Scheme of the photoelectric effect. b) Diagram of an X-ray photoelectron spectrometer. XPS instruments consist of an X-ray source, an energy analyzer for the photoelectrons, and an electron detector (Figure AP.1b) For the analysis and detection of photoelectrons, the sample must be placed in a high-vacuum chamber. Since the photoelectron energy depends on X-ray energy, the excitation source must be monochromatic, Al Kα (1486.6eV) or Mg Kα (1253.6eV) are often the photon energies chosen. The energy of the photoelectrons is analyzed by an electrostatic analyzer, and the photoelectrons are detected by an electron multiplier tube or a multichannel. XPS measurements were performed using a K-Alpha XPS spectrometer (ThermoFisher Scientific, East Grinstead, UK). All samples were analyzed using a microfocused, monochromated Al Kα X-ray source (400 μm spot size). The kinetic energy of the electrons was measured by a 180° hemispherical energy analyzer operated in the constant analyzer energy mode (CAE) at 50 eV pass energy for elemental spectra. Data acquisition and processing using the Thermo Avantage software is described elsewhere.2 The spectra were fitted with one or more Voigt profiles (binding energy uncertainty: + 0.2 eV). The analyzer transmission function, Scofield sensitivity factors,3 and effective a) b) Characterisation Techniques 293    attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism.4 All spectra were referenced to the C1s peak of hydrocarbon at 285.0 eV binding energy controlled by means of the well known photoelectron peaks of metallic Cu, Ag, and Au, respectively. Time-of-Flight Secondary Ions Mass Spectrometry (ToF-SIMS) ToF-SIMS is a very effective and universally applicable method for the chemical analysis of surfaces.5-7 When a surface is bombarded by energetic ions, they penetrated into the solid surface and transfer their kinetic energy to the atoms of the solid in a succession of individual collisions. The majority of species emitted are neutral but it is secondary ions which are detected and analysed by a mass spectrometer (Figure AP.2). Figure AP.2 Scheme of the SIMS process The ToF ion mass spectrometer consists of three main components: the ion gun, the accelerating and flight path system and the detector (Figure AP.3). The primary ion source (tipically Cs or Ga) produces mass separated pulses. The emited secondary ions travel through the time-of-flight analyzer at different velocities, depending on their mass to charge ratio (k=½mv2). For each primary ion pulse, a full mass spectrum is obtained by measuring the arrival times of the secondary ions at the detector and performing a simple time to mass conversion. 294 Appendix   Figure AP.2 Diagram of the time-of-flight ion mass spectrometer By reducing the diameter of the primary ion beam and scanning it over the surface it is possible to measure the lateral distribution of the secondary ion emission and therefore that of the surface constituents responsible for the emission. The primary ions beam is positioned in particular positions of the sample and the spectrum is recorded and stored with its corresponding coordinates. From these data, it is possible to construct an image (chemical map) for each secondary ion species or group of species showing the distribution of the surface. Characterisation Techniques 295    Figure AP.3 ToF-SIMS images of a metal structure showing the Ti and Cu distribution (top) and an overlay (bottom) ToF-SIMS5,7 (Time-of-Flight Secondary Ion Mass Spectrometry) was performed on a TOF.SIMS5 instrument (ION-TOF GmbH, Münster, Germany), equipped with a Bi cluster liquid metal primary ion source and a non-linear time of flight analyzer. UHV base pressure was < 5x10-9 mbar. The Bi source was operated in the bunched mode providing 1.1 ns Bi1+ ion pulses at 25 keV energy and a lateral resolution of approx. 4 μm. The short pulse length allowed for high mass resolution to analyze the complex mass spectra of the immobilised organic layers. Images larger than the maximum deflection range of the primary ion gun of 500×500 μm2 were obtained using the manipulator stage scan mode. Spectra were calibrated on the C-, C2-, C3-, or on the C+, CH+, CH2+, and CH3+ peaks. Primary ion doses were kept below 1011 ions/cm2 (static SIMS limit). Advancing and receding contact angle measurements were performed with an OCA5 instrument (dataphysics, Filderstadt, Germany). HPLC grade water was used and all measurements were performed on several spots of the substrate and averaged (n>3). 296 Appendix   References 1 Elipe, A. R. G.; Munuera, G.; Contreras, G. M.; Universidad de Sevilla. Secretariado de, P. Fundamentos y aplicaciones de la espectroscopia de fotoelectrones: (XPS/ESCA). (Universidad de Sevilla, Secretariado de Publicaciones, 1986). 2 Parry1, K. L.; Shard, A.G.; Short, R.D.; White, R.G.; Whittle, J.D.; Wright, A. ARXPS characterisation of plasma polymerised surface chemical gradients. Surf. Interface Anal. 2006, 28 ,1497-1504. 3 Scofield, J. H. Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129-137. 4 Tanuma, S.; Powell, C. J.; Penn, D. R. Calculations of electron inelastic mean free paths. V. Data for 14 organic compounds over the 50–2000 eV range. Surf. Interface Anal. 1994, 21, 165-176. 5 Leggett, G. J. & Vickerman, J. C. Static secondary ion mass spectrometry (SSIMS) - an emerging surface mass spectrometry. Annu. Rep. Prog. Chem., Sect. C 1993, 88, 77-133. 6 Benninghoven, A. Chemical Analysis of Inorganic and Organic Surfaces and Thin Films by Static Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS). Angewandte Chemie International Edition in English 1994, 33, 1023-1043. 7 Arlinghaus, H. F. Possibilities and limitations of high-resolution mass spectrometry in life sciences. Appl. Surf. Sci. 2008, 1058-1063. 