NJC. Volume 38 Number 6 June 2014 Pages

Transcription

1 Volume 38 Number 6 June 2014 Pages New Journal of Chemistry A journal for new directions in chemistry ISSN PERSPECTIVE Marta Sowinska and Zofia Urbanczyk-Lipkowska Advances in the chemistry of dendrimers

2 PERSPECTIVE Advances in the chemistry of dendrimers Cite this: New J. Chem., 2014, 38, 2168 Received (in Montpellier, France) 9th October 2013, Accepted 17th February 2014 DOI: /c3nj01239e Marta Sowinska and Zofia Urbanczyk-Lipkowska* Dendrimers, which are highly branched, multivalent and monodisperse polymers, have received continuous interest in recent years because of the global demand for new nanomolecules that are useful in advanced technology and medicine. While dendrimers are advantageous for many highly specialised applications, the high polyvalency of higher-generation dendrimers requires, however, well-controlled, target-tailored regioselective chemical engineering. The structural evolution of dendrimers from simple, monofunctional molecules to the more complex, multifunctional compounds is inextricably associated with a continuous improvement in traditional synthetic strategies, as well as the development of new synthetic tools. The purpose of this review is to provide insight into currently available synthetic methods that yield dendrimers with various morphologies, placing emphasis on the evaluation of their advantages and limitations. In particular, we will review current efforts that focus on simplifying and optimising the existing methods and the development of new strategies that permit control over the targeted introduction of various functionalities in one or more selected areas of the molecule. Introduction Dendrimers emerged from the sea of polymer sciences in the early 1980 s as beautiful, star-like branched molecules with perfect structure and low polydispersity. However, the idea of preparing branched molecules was first envisioned by Flory as early as As in the case of other discoveries that were ahead of their time, the synthetic and analytical methods at Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka Street 44/52, Warsaw, Poland. zofia.lipkowska@icho.edu.pl Electronic supplementary information (ESI) available. See DOI: / c3nj01239e that time were not sufficiently advanced to experimentally support such an idea. The first paper that describes the synthesis of so-called cascade molecules, i.e., low molecular weight dendritic polyamines, was published in 1978 by Vögtle. 2 Three years later, Denkewalter patented the synthesis of poly(lysine) dendrimers up to the 10th generation by a divergent method. 3 In 1985, Tomalia published the synthesis of poly(amidoamine) (PAMAM) dendrimers up to the 10th generation and named this novel class of polymers dendrimers, which directly translated from the Greek language means part of a tree (dendros = tree, meros = part). 4 The same year also ushered in the publication by Newkome, who described the synthesis of poly(ether) dendrimers up to the 3rd generation. 5 These results Marta Sowinska Marta Sowinska earned her MSc degree from the Chemistry Department, Warsaw University, Poland. Then she entered the PhD program at the Institute of Organic Chemistry of the Polish Academy of Sciences in Warsaw. She obtained her PhD in 2013 under the supervision of Prof. Zofia Urbanczyk-Lipkowska, working on the synthesis and properties of biologically active peptide dendrimers. Zofia Urbanczyk-Lipkowska Zofia Urbanczyk-Lipkowska is Professor of Chemistry at the Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw. Her scientific activity is documented by over 220 scientific papers, monographs and patents in the fields of molecular recognition, solid state chemistry, crystallography, and more recently design and applications of dendrimeric mimics of natural antimicrobial peptides. She has received many awards related to research and innovation in the area of dendrimeric antimicrobials New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

3 Perspective were followed by a paper written by Hawker and Frechet who in 1990 presented a new synthetic methodology called a convergent method for obtaining poly(aryl ether) dendrimers, which later became complementary to the formerly applied divergent method. 6 As evidenced by the remarkably high number of patent applications and original papers published in scientific journals, significant breakthroughs in the design, synthesis and applications of dendrimers in various areas of science were made in the 1990 s. More specifically, efforts that were focused on the development of new effective methods for the synthesis of these macromolecules yielded dendritic products that were obtained in a more efficient way, i.e., in fewer reaction steps and in a shorter amount of time. Following more sophisticated prospective applications, new types of dendrimers were designed, i.e., types that possessed different functional groups and building blocks in contrast to the traditional dendrimers, which were constructed from one type of monomer (AB n, where n Z 2) and had a monofunctional surface. The synthesis of new macromolecules that were composed of different building blocks (e.g., monomers of the AB n and CD n structures, where n Z 2), called block dendrimers or codendrimers, was initiated by Frechet s group. 7 Many dendrimers of this type were synthesised and can be classified according to their structural characteristics as layer-block, segment-block and surface-block dendrimers (Fig. 1). 8 The last two groups are often named Janus dendrimers and possess at least two different types of terminal functionalities that are positioned in separate parts of the dendrimer surface. The fact that dendrimers with multifunctional surfaces can find multiple applications due to having a combination of several properties in one molecule made them especially interesting to a broadly based scientific community. As a result, more complex, multifunctional dendrimers with different spatial geometries of terminal functionalities started to be synthesised. Thus, in thelate1990 s,thefirstbifunctional dendrimers with alternating 9 or random 10 distributions of terminal groups were prepared. Nearly a decade later, dendrimers with at least three types of terminal functionalities that might undergo orthogonal postmodification were reported; for example, dendrimers that resembled a fruit salad tree were constructed by Steffensen and Simanek. 11 A departure from the classical notion of having a core or interior of the dendrimers as inactive structural elements advanced into further evolution of these macromolecular compounds. This advancement constituted the incorporation of functionalities into the mentioned structural components of the dendrimer and had already started in the 1990 s. 12 The first decade of the 21st century yielded tremendous progress, both in the field of dendrimers with an active core (e.g., photoactive, electrochemically active or catalytically active core molecules) and dendrimers with functionalised interiors, where functional groups can be localised in every generation 13 or only in certain generations. 14 Dendrimers with their unique branched structure and multivalency were quite early recognised as perfect scaffolds that had tremendous potential to be useful for a variety of different applications. A literature survey of the applications of these compounds clearly indicates the multidisciplinary nature of dendrimer chemistry, which involves medicine, biology, Fig. 1 Structural evolutions of dendrimers. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

4 physics and materials engineering. 15 The rapid growth of applications demands the emergence of new types of dendrimers, i.e., with more advanced, precisely controlled structures. The structural evolution of dendrimers, from simple, monofunctional to the more complex, multifunctional compounds outlined in Fig. 1, is inextricably associated with a continuous improvement in the initial synthetic strategies as well as the development of new approaches. Advantageous for many highly specialised applications, the monodispersity and high polyvalency of higher generation dendrimers often justify the high cost of their production. For most practical applications and further development in the field, however, it is highly desired that the available synthetic methods give reproducible, high purity and well-defined products both in the laboratory and on an industrial scale. In this review, we will provide an update on currently available methods for the synthesis of dendritic nanomolecules with various morphologies. The characterisation of general problems that are encountered in dendrimer synthesis will be followed by a description of the primary methods (divergent, convergent synthesis) and more recent approaches to their preparation. In particular, the accelerated methods and the selected examples of these methods leading to peripherally multifunctionalised or internally functionalised dendrimers will be shown. In this study, we do not intend to present an exhaustive overview on all of the synthetic strategies that have been developed to date, but instead, we provide examples that illustrate the recent progress that has been accomplished in this area and that has resulted in access to a rich diversity of structures of dendrimers. Emphasis is placed on the exposition of advantages and the limitations of specific synthetic strategies. For a more detailed overview of the subject, the reader is directed towards reviews by Newkome and Shreiner. 16 Synthesis of dendrimers general remarks The methods of preparation and the properties of dendrimers are drastically different from those of classical polymers. In contrast to polymers, the synthesis of dendrimers proceeds under wellcontrolled conditions that lead to compounds that are monodisperse with an exact molecular mass and very regular, tree-like branched structure. At present, the creative application of new building blocks in association with the modern methods of organic synthesis has made the chemistry of dendrimers more precise and reliable, which is suitable for designing molecules that have sophisticated three-dimensional structure. Skilful variation of the core molecule and the monomers of various structures enables the construction of dendrimers with programmed structural parameters, such as size, shape, flexibility or the characteristics of internal voids that allow the encapsulation of smaller molecules. Theoretically, one can also program the physical and chemical properties of such molecules to a degree, which is now unattainable in standard polymerisation processes. The synthesis of dendrimers most often relies on multiple sequences of two simple chemical reactions, which involve Perspective operations on two or three different functional groups. Considering a conventional growth approach based on an AB 2 monomer, every upcoming reaction sequence yields a dendrimer of a higher generation and with a doubled number of terminal groups and an approximately two-fold increase in the molecular mass. 17 Theoretically, dendrimer synthesis is an easy process. In real cases, however, it is a laborious and time-consuming adventure. 18 Moreover, despite the application of highly selective reactions, the final yield in the production of dendrimers is often low. The other disadvantages, such as the high cost of the synthesis and the problems with obtaining high-generation dendrimers without structural defects, are relevant to the generation number. These considerations result in the commercial availability of only several classes of dendrimers, namely poly(amidoamine) (PAMAM s, Sigma-Aldrich), poly(propyleneimine) (DAB s, Sigma-Aldrich), phosphorous (PMMH, Sigma-Aldrich) and 2,2-bis(hydroxymethyl)propionic acid-based dendrimers (bis-mpa, Polymer Factory Sweden). Nevertheless, with the emergence of new and robust chemistries, especially click chemistry, 19 the synthesis of dendrimers has matured and their accessibility is foreseen to increase. The dendrimer growth process is mainly determined by the employed chemistry and, at higher generation typically above G4 or G5, by steric factors, i.e., those factors that have an impact on the steric accessibility of the surface functionalities. One of those factors is the backfolding of the terminal groups into the interior of the macromolecule. The degree of this adverse effect on the dendrimer growth depends on both the surrounding chemical environment (the ph, solvent polarity, and ionic strength) and the dendrimer structure (the flexibility of the dendrons and the ability of the structural elements to interact with one another). 20 Another factor that restricts the number of generations is steric hindrance on the surface of the dendrimer. As was predicted in the pioneering work of Maciejewski 21 and later by de Gennes, 22 the number of terminal groups grows faster than the radius of the dendrimer, and therefore, at a certain generation, the surface becomes densely packed. This effect is called the de Gennes dense packing or starburst limit effect, which makes the dendrimer surface not likely to complete, in terms of its stoichiometric chemical modification. The densely-packed generation is reached at different dendrimer generations depending on, e.g., the size and valency of the core and the monomers, the ability of the surface functionalities to create a network with one another via, e.g., hydrogen bonding, thereby consolidating a dense outer shell. The synthesis of dendrimers is at present accomplished with the application of a wide variety of both well-known and novel reactions of organic chemistry, where efficient protection/ deprotection and coupling reactions are of primary importance. This approach enables us to obtain dendritic macromolecules that have variable structures, i.e., that contain exclusively aliphatic, aromatic or mixed structures. Enzymatic methods are used in their synthesis as well. An example is the synthesis of a poly(e-caprolactone) (PCL) monosubstituted G1 dendrimer by lipase catalysis. 23 Dendrimers can also be obtained in non-covalent 2170 New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

5 Perspective Scheme 1 Synthesis of dendrimers according to the divergent method. processes such as the self-assembly of dendrons that possess chelating moieties around a metal ion, which in this case serve as a core moiety. 24 With regard to the methodology of dendrimer synthesis, both in-solution and on-solid-support methods are utilised. The latter method is mainly used for the preparation of peptide or glycopeptide dendrimers, 25 including popular tetramericand octameric poly(lysine) dendrons applied as nanocarriers in so-called Multiple Antigenic Peptide systems (MAP). 26 These poly- (lysine) dendrons are commercially available from Novabiochem, Sigma-Aldrich and Bachem. Several other dendrimers with non-peptide structure were also synthesised using solid-phase methodology. 27 For example, Bradley et al. obtained PAMAM dendrimers with a dipropylenetriamine core up to the fourth generation by this method. 28 The synthesis of dendrimers can be accomplished by various strategies; among the basic strategies are divergent (inside out) and convergent (outside in) syntheses. The most visible difference between these two complementary methods is the direction of the dendrimer growth from the core toward the periphery for the former and from the periphery toward the core for the latter. Over the past 20 years, several groups have significantly contributed to the development of novel, more reliable and simpler synthetic procedures, which yield final dendrimers more time- and costeffectively and with higher yields. Classic methods for the synthesis of dendrimers The synthesis of a dendrimer according to the divergent method proceeds stepwise starting from a multifunctional core molecule, B n (n Z 2), to whose next dendrimer generations are built up via the sequential attachment of building blocks called monomers (Scheme 1). The monomers used are of the type AB n (n Z 2), where A and B denote two types of functional groups. To permit controlled growth of the dendrimer, the A-functional group of the AB n monomer is a reactive group, while the B-functional groups are deactivated/protected. Attaching the monomers to a substrate molecule (a core or growing dendrimer) proceeds by chemical bond formation between the A-functional group of the monomer and one of the activated B-functional groups of the substrate. The activation of the B-functionalities can be conducted by their coupling with a second molecule or the removal of protecting groups. The most commonly used monomers are trifunctional monomers of an AB 2 structure. After coupling of these monomers onto the core (typically di-, tri- or tetra-functional), a first generation dendrimer is obtained. The next two steps, which rely on the activation of the B-functionalities on the first generation dendrimer and their coupling with a new set of monomers, lead to a second generation dendrimer. By the repetition of these two steps, the desired higher generation dendrimer is reached. A well-known example of dendrimers that were synthesised using the divergent growth approach are poly(amidoamine) (PAMAM) dendrimers. PAMAM dendrimers with an ethylenediamine (EDA) core were synthesised by Tomalia et al. up to the tenth generation via the iterative sequence of two basic reactions: a Michael-type addition of amino groups to methyl acrylate followed by aminolysis of the resulting methyl ester with excess ethylenediamine (Scheme 2). 4 In the divergent method with increasing generation, the purity of the intended product decreases due to the increased number of reactions performed on the same molecule that are on-going from one generation to the next. To obtain the desired dendrimer without partly derivatised products, i.e., defect dendrimers, reactions must proceed quantitatively at each coupling and activation step for all of the reactive end-groups This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

6 Perspective Scheme 2 Synthesis of PAMAM dendrimers with a tetrafunctional ethylenediamine core (EDA) by the divergent method. (Note: the numbers of the generations shown in this scheme is coherent with what is generally admitted for dendrimers. However, for PAMAM dendrimers the numbers are different, i.e., G1 in the Scheme corresponds to G0, G2 to G1, etc.) of the dendrimer. However, because the number of terminal groups increases exponentially with each generation, there is dense packing, and associated with the dense packing is steric hindrance; hence, the possibility of conducting a reaction with 100% conversion decreases with the dendrimer growth. The presence of a small number of statistical defects cannot be avoided, even upon the addition of a large excess of reagents coupled with long reaction times. Moreover, purification and separation of structurally perfect dendrimers from defective dendrimers by standard purification techniques are very difficult to achieve because the compounds are very similar, both chemically and in size. This aspect is one of the reasons why the synthesis of dendrimers is tedious and time-consuming and the overall yield is low in spite of using selective reactions. For example, an average selectivity of 99.5% per reaction will, in the case of the synthesis of the fifth generation poly(propylene imine) dendrimer (PPI G5), only result in 29% defect-free dendrimer. 29 An advantage of the divergent method is the ease of fast creation of a library of various generation dendrimers due to the possibility of stopping the reaction at any step as well as the possibility of automating the repetitive steps. For this reason, the synthesis of all commercially available dendrimers, such as PAMAM or PPI, 30 is still performed using this strategy. Moreover, the divergent method enables the preparation of high molecular mass dendrimers, although it is impossible to obtain structurally perfect dendrimers of high generations. It should also be noted that in the divergent process, modification of the full surface of dendrimers in a single step is very easy. Since the properties of dendrimers are mainly driven by the type of terminal groups, dendrimers with the same internal structure can be used in different fields (e.g., as catalysts, drug delivery systems, etc.) by only changing their surface in the last step. Such flexibility, diversity in the properties, is not attainable in the below described convergent synthesis. The convergent method, which was first reported by Hawker and Frechet in , is an alternative route to constructing dendrimers. 6 In this strategy the individual dendrimer wedges, called dendrons, are synthesised first and then coupled to a multifunctional core molecule (Scheme 3). The synthesis of dendrons is conducted by using the conventional AB 2 monomer, which has reactive B-functionalities and the deactivated/protected A-functionality. In the first step, the monomer is subjected to a reaction with a compound that will eventually constitute the periphery (the exterior) of the dendrimer. Most commonly, the key step is the introduction of protecting groups on monomer B-functionalities. In this way, a first generation dendron (G1) is created. The next two steps, which are activation of a dendron focal point and its coupling with the AB 2 monomers, lead to a G2 dendron. Each repetition of these two steps results in an increase in the dendron generation. The dendrimer is formed in the last 2172 New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

7 Perspective Scheme 3 Synthesis of dendrimers according to the convergent method. step of the synthesis by coupling of the activated dendrons that reached the desired generation to the multifunctional core molecule. An example of dendrimers that were prepared by the convergent method are the poly(aryl ether) dendrimers. 6 Their synthesis was started with the construction of peripheral elements, namely, the Williamson etherification reaction between benzyl bromide and 3,5-dihydroxybenzyl alcohol (Scheme 4). The resulting G1 dendron was then activated by substitution of a hydroxyl group by bromide and coupling to the next monomer molecule. Two-fold repetition of this two-step sequence produced a G4 dendron, which in the final step was coupled to a trifunctional core to yield a G4 dendrimer. In the convergent method, because of the limited number of reactions performed on the same molecule ongoing from one generation to the next, it is possible to obtain dendrimers without structural defects. At each propagation step in the growing dendron, there is only one reactive functional group that occurs. Therefore, each coupling step constitutes a reaction of two dendrons with one monomer, whereas the activation step comprises only one reaction per molecule regardless of its generation. The consequences of this arrangement are having greater control over the process, greater purity of the synthesised dendrimer and reduced consumption of the reagents compared to the divergent method. In the convergent strategy, the use of reagents in equimolar quantities or in slight excess is sufficient for the reaction to produce good yields. Additionally, product purification, for example, by chromatographic techniques, is easier than in the case of the divergent strategy because of the large differences in the molar mass and polarity between the fully substituted dendron/dendrimer and the by-products. After each coupling step, the obtained product is over two-fold heavier than the starting dendron in terms of the molecular mass. Larger possibilities of separation of the desired product from unreacted starting materials or defect dendrimers, allow the use of reactions that proceed in somewhat lower than ideal yields (Z90%). This scenario is clearly not the case with the divergent synthesis for which high yield reactions (499%) are required to minimise the introduction of defects. Growth of the convergent dendron is subject to larger limitations in comparison to the growth of dendrimers in the divergent approach. The sources of this effect are both chemical and stereochemical factors. With increasing dendron generation, both reactivity as well as availability of the focal point, at which proceeds the coupling reaction of the dendron to another monomer or the core, decreases. For this reason, the preparation of structurally perfect dendrons at high generations is impossible. Furthermore, bulky dendrons can cause shielding of a multifunctional core, which leads to its incomplete substitution. Consequently, only lower generation dendrimers (typically below the sixth generation) can be formed by the convergent method. The attractive feature of this strategy is that the addition of the same dendrons to different cores immediately gives access to entirely new dendrimers in only one reaction step (Fig. 2A). Hence, a library of dendrimers that differ in the nature of their core and that can be organic or inorganic can be created quickly. As a rule, dendrimers with a functional core (e.g., a photoactive, electrochemically active, redox active or catalytically active core molecule) are synthesised by this method. In contrast to the divergent method, in which the core is used to initiate dendrimer growth and therefore must be stable under the conditions of the subsequent activation and coupling steps, the convergent method enables the introduction of sensitive functional cores because this step takes place in the last synthetic step. Owing to the convergent approach, diverse supramolecular dendrimers are obtained via self-assembly of dendrons that have chelating elements around a suitable core (Fig. 2B and C). Another advantage of the convergent method is that dendrons of different types (different molecular composition and/or generation) can be linked together, introducing regional variations in the final dendrimer. In the case of connecting three or more dendrons, a multifunctional core molecule is used, 7a whereas two dendrons can be coupled directly if their focal points are complementary functions. 31 In this way, dendrimers that have different This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

8 Perspective Scheme 4 Synthesis of dendrimers by the convergent method exampled by the synthesis of G4 poly(aryl ether) dendrimers. morphologies and that possess physical and chemical properties of a few dendrimers can be obtained. Among them, compounds constructed from two dendrons differing in the nature of their terminal functionalities, often called Janus Fig. 2 Convergent synthesis of dendrimers with different core (A) and examples of supramolecular dendrimers obtained by this method: containing metal cation (B) 24 or organic molecule (C) 34 as a core New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

9 Perspective dendrimers, constitute the largest group. However, the nomenclature of these compounds is not fixed yet, and they can be found under various names, such as surface-block dendrimers, segmentblock dendrimers, diblock dendrimers, co-dendrimers, diblock co-dendrimers, asymmetrical dendrimers or bow-tie dendrimers. 8b Dendrons that are obtained by the convergent method are also used for the preparation of dendritic-linear polymer hybrids, 32 such as dendronised polymers. 33 Accelerated methods for the synthesis of dendrimers Double-stage convergent method (also known as the hypercore approach) The double-stage convergent method begins with the synthesis by the convergent approach of low-generation dendrons that have protected terminal groups. Then, these dendrons are coupled to a multifunctional core through their focal point, and the terminal groups of the resulting low-generation dendrimer, called a hypercore, are deprotected. In the last step, dendrons (identical to the previous one or different) are reacted with this hypercore, which leads to the desired higher generation dendrimer (Scheme 5). Compared to conventional convergent synthesis, the doublestage convergent method has significant advantages. This approach relies on the use of the hypercore, which limits the problems of steric hindrance and thereby facilitates access to higher generation monodisperse dendrimers. The surface functionalities of the hypercore are subject to less steric hindrance during the coupling reactions with dendrons than in the case of simple non-dendritic core groups of the conventional convergent method. Moreover, this strategy permits the formation of dendrimers that have chemically differentiated internal and external branching units, i.e., that have two different layers (so-called layer-block dendrimers). This procedure is performed by utilising two different types of monomers in the synthesis of the hypercore and dendrons. The above-mentioned strategy was introduced by Frechet et al. 35 These authors synthesised G7 dendrimers that were characterised by a layered structure that was composed of a flexible inner core surrounded by a more rigid outer layer. The synthesis involved the preparation of a G3 hypercore that possesses 4,4-bis-(4 0 -hydroxyphenyl)pentanol as the building block via the convergent method and G4 poly(aryl ether) dendrons, in this case based on 3,5-dihydroxybenzyl alcohol; then, they are coupled to each other. As a result, the G7 dendrimers were obtained with a 61% yield (Scheme 6). A disadvantage of the double-stage convergent method is the time-consuming synthesis of the hypercore and the dendrons, which is conducted by using the conventional growth approach and therefore requires many synthetic steps. Hypermonomer method (also known as the branched monomer approach) In the hypermonomer method, monomers that have a higher number of functional groups than conventional AB 2 or AB 3 monomers are used. Utilising these hypermonomers (typically of type AB 4 and AB 8 ), dendrimers with the same number or a higher number of functional groups are obtained in fewer steps. For example, while the preparation of a fourth generation dendrimer based on a tetrafunctional core and AB 2 monomers, hence comprising 64 active end-groups, requires eight synthetic steps, the use of AB 4 monomers results in a dendrimer that has the same number of functional groups in only four steps (Scheme 7). The hypermonomer method was applied by the group of Mullen for the synthesis of G2 poly(phenylene) dendrimers via the Diels Alder cycloaddition, using an AB 4 monomer that has four dienophile units and one diene function. 36 Later, this strategy was significantly improved by Blais et al. that took the direct advantage of both the hypermonomer method and the orthogonal strategy (described further in this review). 37 The authors demonstrated a quick access to the third generation phosphorus-containing dendrimers with 750 phosphanyl end groups in only 3 steps as a result of alternate coupling of AB 5 and CD 5 hypermonomers via the condensation reaction (aldehyde with hydrazine) and the Staudinger reaction (Scheme 8). This approach enabled the creation of a new generation at each step and not every two steps as usual. However, the simple multifunctional compounds rarely serve as hypermonomers. For this purpose, the lower generation dendrons are used more often. In such a case, the hypermonomer Scheme 5 Synthesis of dendrimers according to the double-stage convergent method. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

10 Perspective Scheme 6 Synthesis of G7 layer-block dendrimers by the double-stage convergent method. Scheme 7 Synthesis of dendrimers according to the hypermonomer method. strategy can basically be regarded as a multistep growth of the monomer itself, enabling the addition of multiple generations during each coupling step. A good example of such an approach is reported by Frechet et al., who described preparation of a G5 poly(aryl ether) dendron in three steps using a G2 dendron as the AB 4 hypermonomer in the synthesis (Scheme 9). 38 The carboxylic acid terminated hypermonomer allowed facile synthesis of the third generation dendron, which after an activation of its focal point was coupled to the hypermonomer. This one coupling step enabled the growth of a starting dendron by two generations New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

11 Perspective Scheme 8 Synthesis of G3 phosphorus-containing dendrimers by alternate coupling of AB 5 and CD 5 hypermonomers. Scheme 9 Synthesis of G5 poly(aryl ether) dendrons by the hypermonomer method. More recently, a G13 dendrimer containing triazines linked by diamines was synthesised using a monochlorotriazine hypermonomer ensuing in two-generation s worth of growth per synthetic cycle. 39 The synthesis of this giant dendrimer (actually the largest dendrimer reported to date) started with a G1 dendron as a core, which was reacted with the hypermonomer to give the G3 Boc-protected dendron (Fig. 3). Upon deprotection, the two-step process was repeated five more times. Currently, the hypermonomer method is not extensively used for the production of dendrimers because it usually entails the preparation of requisite hypermonomers by a multistep synthesis. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

12 Fig. 3 Structures of a core (G1 dendron) and a monochlorotriazine hypermonomer. Double-exponential method The double-exponential method requires an AB 2 monomer with orthogonally protected A and B functional groups, which have orthogonally protected focal and surface functionalities (Scheme 10). This monomer is activated selectively, either at its focal point or at the periphery, which results in two differently activated monomers. Coupling of the monomers that have reactive B-functionalities with two equivalents of the monomers that have the reactive A-functionality yields a second generation orthogonally protected dendron. Iteration of the synthetic sequence using the G2 dendron, with selective activations and coupling of the resultant activated compounds, leads to the corresponding fourth generation dendron. Growth of the dendron can be continued according to the above scheme or completed by the formation of a dendrimer. For this purpose, the focal points of the dendrons are activated and coupled to a multifunctional core. Perspective In the double-exponential method, the generation of the growing dendron is doubled with each repetition of a three-step sequence that involves two selective activation reactions and one coupling reaction (G1 - G2 - G4 -...). This generation jump leads to a reduction in the number of reaction steps. Compared to the convergent method, the synthesis of a G4 dendron via the double-exponential strategy shortens the procedure by one step, whereas for a G8 dendron, the procedure is shortened by nine steps. The above described strategy was first introduced by Moore et al., who presented it by the synthesis of a fourth generation poly(phenylacetylene) dendron. 40 This approach is of particular interest because it is one of the most rapid methods for the preparation of large dendrons/dendrimers. According to the double-exponential method, a wide variety of dendrimers were obtained including poly(amide), 41 poly(ether urethane) 42 and poly(ester) dendrimers. Among them, the synthesis of dendrimers based on 2,2-bis(hydroxymethyl)propionic acid (bis-mpa) is especially noteworthy. These aliphatic polyester dendrimers belong to a small group of commercially available dendrimers. Their synthesis via the double-exponential method was performed by Gitsov et al. (Scheme 11). 43 They initially conducted a coupling reaction of 2,2-bis(hydroxymethyl)propionic acid derivatives, which results in a second generation dendron bearing two acetonide protective groups at the periphery and a single benzyl ester protective group at the focal point. This dendron was then subjected to a selective deprotection: catalytic hydrogenolysis leading to a dendron with a free carboxylic acid group and hydrolysis in the presence of an acidic polymer resin affording a dendron with free terminal hydroxyl groups. The coupling Scheme 10 Synthesis of dendrimers according to the double-exponential method New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

13 Perspective Scheme 11 Synthesis of bis-mpa dendrimers via the double-exponential method. together of two new dendrons yielded a G4 dendron, which in the next step was activated at the focal point and attached to 1,1,1-tris(hydroxyphenyl)ethane, which was used as the core, with a 85% yield. Deprotection of acetonide protective groups was the last step in the synthesis of a fourth generation bis-mpa dendrimer. The double-exponential method, which is similar to the convergent method, belongs to the versatile procedures that enable the preparation of both simple classical dendrimers and supramolecular or asymmetrical dendrimers. Orthogonal coupling method (also known as the two-step approach, the two monomer approach, and the AB 2 CD 2 approach) The orthogonal coupling method relies on divergent or convergent dendrimer growth using two different monomers, namely AB 2 and CD 2, which have chemoselective functional groups (Scheme 12). The monomers AB 2 and CD 2 are selected such that the focal functionalities of each individual monomer will react only with the periphery of the other monomer; in other words, the functional groups A and C react with the reactive sites D and B, respectively. Such chemoselectivity eliminates the need for activation steps, thereby reducing the number of reaction steps during the dendrimer growth by half. As a result, each synthetic step in this strategy adds a single generation to the dendron/ dendrimer. A consequence of using, in the synthesis, two different types of monomers are dendrimers that are often characterised by a layered structure that contains different covalent linkages between the layers, so-called layer-block dendrimers. If the synthesis of a dendrimer proceeds by the divergent method, then the nature of the monomers also results in alternating terminal functionalities being displayed with each generation. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

14 Perspective Scheme 12 Synthesis of dendrimers according to the orthogonal coupling method via the divergent growth. The orthogonal coupling method was first reported by Spindler and Frechet, who obtained a G3 poly(ether urethane) dendron, using 3,5-diisocyanatobenzyl chloride and 3,5-dihydroxybenzyl alcohol as the AB 2 and CD 2 monomers, in a one-pot synthesis. 44 Unfortunately, the low chemoselectivity of the etherification and urethane formation reactions prohibited the preparation of higher generation dendrons. The first successful synthesis of high generation dendrimers via the orthogonal coupling strategy was demonstrated by Zimmerman et al., who prepared G4 poly(alkyne ester) dendrons with a good yield, employing the Mitsunobu esterification and the Sonogashira coupling reaction of terminal alkyne units with an aryl iodide (Scheme 13). 45 The first orthogonal synthesis of chemically homogeneous dendrons, i.e., dendrons with identical chemical connectivities in their structure, was reported by Yu et al. (Scheme 14). 46 These authors synthesised G4 poly(aryl alkene) dendrons by a double repetition of the sequence that was composed of the Horner Wadsworth Emmons and the Heck coupling reactions. The effectiveness of the orthogonal coupling method is especially high when the synthesis of the dendrimers is performed in a one-pot system. One example of such synthesis is the one-pot synthesis of G4 phosphorus-containing dendrimers based on the AB 2 (A = NH 2, B = PPh 2 )andcd 2 (C = N 3, D = CHO) monomers. The orthogonal reactions utilised were the condensation reaction between an aldehyde and a hydrazine and the Staudinger reaction between an azide and a phosphine. 47 Although the orthogonal coupling method allows the rapid synthesis of dendrimers owing to reduction in the number of Scheme 13 Synthesis of G4 poly(alkyl ester) dendrons by the orthogonal coupling method New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

15 Perspective Scheme 14 Synthesis of G4 poly(aryl alkene) dendrons by the orthogonal coupling method. reaction and purification steps, few syntheses of this type have been reported. This limitation is caused by the complex synthetic requirements of this strategy, i.e., the need to use highly efficient and orthogonal coupling reactions with the participation of two pairs of orthogonal functionalities. Moreover, this orthogonality imposes a limitation on which functionalities can be incorporated into the dendrimer structure without interfering with the synthetic cycle. The click chemistry concept The click chemistry concept introduced by Sharpless et al. in 2001 encompasses a number of versatile reactions that are highly stereo/regioselective, gives readily separable products, usually in excellent yield close to 100%. 48 The click reactions are usually accompanied by the inoffensive byproducts (or none), and utilize readily available starting materials and reagents. Additionally, these reactions are tolerant to the presence of a wide range of functional groups and can be carried out using a broad set of reaction conditions (including water, air, biphasic systems, etc.). Therefore, in recent years the click chemistry methodology had found widespread applications in the synthesis of dendrimers. It has also provided an additional impetus for a greener approach in constructing dendrimers. To date, the click reactions that have been successfully adapted in the field of dendrimer chemistry may be classified as the Cu I -catalysed azide alkyne cycloaddition (CuAAC), 49 the Diels Alder cycloaddition (DA), especially those involving furan and maleimide moieties, 50 and the thiol-based click reactions that can proceed via two routes, namely, the anti-markovnikov radical addition [the thiol ene coupling (TEC) 51 or the thiol yne coupling (TYC) 52 ] or base-catalysed Michael addition (MA) (Scheme 15). The most synthetically exploited click reaction is the CuAAC reaction, which selectively forms a 1,4-disubstituted 1,2,3-triazole moiety. The first synthesis of dendrimers using this reaction was presented by Fokin et al. in 2004 and is recognised today as a starting point of the use of click chemistry in dendrimer synthesis. 53 These authors constructed several third and fourth generation triazole dendrimers via a convergent method starting from AB 2 monomers, containing one chloride and two alkyne functionalities. After each click reaction, the chloride This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

16 Scheme 15 Schematic representation of the click reactions widely utilised for the preparation of dendrimers. functionality was easily converted into the azide group, activating it for the next coupling step. The resulting dendrons containing triazole linkages between each generation were then anchored to a variety of polyacetylene cores in the last step. Subsequently, Wooley et al. demonstrated the divergent growth approach to the G3 Frechet-type dendrimers with triazole linkages. 54 These dendrimers were synthesised through an iterative process involving a click reaction followed by activation via the in situ halogenation and azido nucleophilic substitution. Shortly thereafter, an elegant approach to dendrimer synthesis was introduced by Malkoch et al. that took the Perspective direct advantage of both the click chemistry concept and the orthogonal strategy. 55 The authors demonstrated a quick access to the fourth generation dendrimers of the bis-mpa and Frechet-type as a result of alternate coupling of AB 2 and CD 2 monomers via the CuAAC and traditional esterification/etherification reactions (Scheme 16). The resultant dendrimers had alternating end groups, either azide/acetylene or hydroxyl/phenolic, depending on the generation number. The CuAAC reaction offers numerous advantages, not only by virtue of significantly streamlining dendrimer synthesis, but also during construction of highly sophisticated dendrimers. One of them is the formation of 1,2,3-triazole rings that can serve as ligands to coordinate metals or as hydrogen bond acceptors and donors. 56 For example, Ruiz et al. presented the applicability of dendrimers containing 1,2,3-triazoles for the nanoparticle palladium catalysis. 57 Complexation of the intradendritic triazoles by Pd II followed by the reduction to Pd 0 resulted in dendrimer-stabilized Pd nanoparticles (PdNPs) that were shown to be efficient catalysts for both the olefin hydrogenation and the C C bond coupling reactions. More recently, dendrimers possessing thiadiazole as a core, triazoles as the branching units and chalcones as the terminal groups were synthesised by Maruthamuthu s group. 58 They were utilised as a charge separator component in dye-sensitized solar cells. Furthermore, the CuAAC reaction can be used to easily and effectively achieve dendrimer functionalisation as well as to prepare the unsymmetrical dendrimers that are difficult to obtain otherwise. A general and efficient approach based on CuAAC chemistry for the surface functionalisation of structurally different dendrimers including poly(benzyl ether), PAMAM or bis-mpa dendrimers was described by Hawker et al. in The acetylene-terminated dendrimers of up to G4 were Scheme 16 Synthesis of G4 Frechet-type dendrimers via etherification and CuAAC reactions New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

17 Perspective synthesised first and then utilised as scaffolds for the subsequent reactions with various azido derivatives. This protocol, as well as its reverse version, i.e., the CuAAC reaction between azideterminated dendrimers and acetylene functional molecules, is often used for the decoration of dendrimers with a large number of functional substrates of interest. 60 In the same year, taking advantage of the efficiency and orthogonality of the CuAAC reaction, Hawker et al. reported a robust method for the preparation of unsymmetrical bifunctional dendrimers. 61 In this strategy, two separate dendrons based on the 2,2- bis(hydroxymethyl)propionic acid (bis-mpa) building block were constructed: one that included acetonide groups at the periphery and an alkyne at the focal point, and the other one containing hydroxyl groups at the periphery with an azide at the focal point. Since the CuAAC reaction can be performed selectively in the presence of other functional groups, these G1 to G4 dendrons could be efficiently coupled and as a result the library of amphiphilic dendrimers was created. Additionally, authors conducted further surface functionalisation of one of these dendrimers via two consecutive CuAAC click reactions. The terminal hydroxyl groups were modified by alkynes, which subsequently were coupled with azide-modified coumarin. Removal of acetals gave alcohols and a repetition of alkyne formation and the CuAAC reaction led to the introduction of peripheral mannosides. Using this methodology, the unsymmetrical dendrimer possessing sixteen mannose groups and two fluorescent coumarin dyes located in two specific areas of the surface was prepared (Fig. 4). More recently, Boons et al. synthesised unsymmetrical bifunctional dendrimers with poly(ethylene glycol) chains (PEG) on one side and galactosyl residues on the other side. The analogous dendrimers with RGD peptides were prepared via a similar approach except that the CuAAC reaction was utilised in conjunction with the strain-promoted azide alkyne cycloaddition (SPAAC), which allowed simplification of the overall procedure. 62 This strategy takes advantage of the selective reaction of a strained alkyne with an azide in the presence of terminal alkynes. The SPAAC reaction between two polyester dendrons modified by a focal dibenzo-cyclooctyne or azide and containing also peripheral alkynes or TMS-protected alkynes, respectively, provided dendrimers with free or masked alkyne groups at the periphery (Scheme 17). Then, the terminal Fig. 4 Hawker s bifunctional dendrimer containing coumarin dyes and protein-binding mannose molecules. Scheme 17 General concept of multifunctional dendrimer synthesis by three consecutive click reactions. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

18 alkynes were modified with azide functional molecules using the CuAAC reaction. In a final step, a second type of surface functionality was installed in a controlled manner by removal of the TMS-protecting groups followed by another CuAAC reaction. It should be noted that although the efficiency of the CuAAC reaction is already proven, the use and later removal of the cytotoxic copper catalyst limit its full potential for biomedical applications. Especially in cases where the final structure contains numerous functional groups able to bind copper ions, the removal of the copper catalyst can be problematic. Therefore, copper-free click chemistry, such as the SPAAC reaction, that combines the advantages of the CuAAC reaction without the use of a toxic transition metal is highly valuable for biomedical applications. Weck et al. reported for the first time the use of the SPAAC-based approach as a highly advantageous and efficient functionalisation strategy toward multifunctional dendrimers. 63 To date, the Diels Alder cycloaddition (DA) has been only sporadically explored in dendrimer synthesis. However, this situation has started to change recently, mainly due to recognition of the reversible character of this reaction, known as retro-da that Perspective opens up the possibility to generate the thermoresponsive dendrimers. The power of the DA reaction-based approach to efficiently synthesise dendrimers has already been demonstrated in 1997 by Mullen et al. 64 They successfully obtained polyphenylene dendrimers via the DA reaction of tetraphenyl-cyclopentadienones with different core molecules substituted with multiple alkyne groups. The DA reaction was followed by the release of carbon monoxide leading to the formation of conjugated aromatic polyphenylenes. Later, Mullen s group reported unsymmetrical polyphenylene dendrimers by applying sequential DA reaction with a core molecule containing a protected alkyne (Scheme 18). 65 The groups of McElhanon and Sanyal made a seminal contribution to the subject of thermoresponsive dendrimers. Both teams reported the first examples of thermoreversible dendrimers using the furan maleimide cycloaddition-based approach. McElhanon et al. constructed dendrimers starting from the DA reaction of dendrons containing the furan group at their focal point with the p-hydroxyphenylmaleimide. 66 Thus obtained dendrons bearing a thermally responsive bicyclic unit were condensed with the 1,3,5-benzenetricarbonyl trichloride to give symmetrical dendrimers. The synthesis of the unsymmetrical thermoresponsive dendrimers, described by Sanyal et al., was carried out by a direct Scheme 18 Synthesis of unsymmetrical polyphenylene dendrimers New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

19 Perspective Fig. 5 Sanyal s unsymmetrical dendrimer. conjugation of the furan-functionalised poly(aryl ether) dendrons to the maleimide-functionalised polyester dendrons (Fig. 5). 67 Recent years have brought an increasing number of contributions reporting the thiol-based click reactions. Such reactions attract more and more attention in dendrimer science because they represent the most promising approach considering their green aspects, as well as the presence of a wide range of commercially available thiols and corresponding reagents that can readily react with thiols. The first report of thiol-based click reaction described an effective two-step divergent method for the synthesis of G4 aliphatic poly(thioether) dendrimers. 68 Using commercially available compounds, namely thioglycerol (AB 2 monomer) and 4-pentenoic anhydride (CD monomer), Hawker et al. succeeded in the preparation of these dendrimers in eight steps via alternating thiol ene (TEC) and esterification reactions. Later, this strategy was significantly improved by the utilisation of a library of interchangeable modular AB 2 and CD 2 monomers. 69 Aseries of bis-mpa based dendrimers with the same functional group density was obtained in just half of the number of steps. A combination of the TEC click reaction and a Grignard substitution was used by Rissing and Son 70 to prepare carbosilane-thioether dendrimers of up to G5 with 972 vinyl end-groups. Dendrimers were constructed via a divergent growth strategy applying these two reactions sequentially (Scheme 19). In 2011, Malkoch et al. reported the synthesis of dendronised thiols (macrothiols) from cleavable, disulfide cored dendrimers and the use of them for the preparation of a structurally comprehensive dendritic library including traditional and unsymmetricalbifunctionaldendrimersaswellasdendronised polymers and dendritic linear dendritic (DLD) hybrids. 71 The high molecular weight macrothiols, displaying a single reactive thiol group in the core, were efficiently coupled to various allylic precursors via TEC click chemistry. A very efficient alternating approach employing both the photochemically induced addition of a thiol to an alkyne (TYC) and esterification was described by Stenzel et al. 72 This approach allowed for the formation of dendrimers with a high terminal group density and requiring fewer reaction steps. Photochemically-aided addition of 1-thioglycerol to a trivalent core, namely tris-3-propynyl-1,3,5-benzenetricarboxylate, followed by the esterification with an alkyne-terminated anhydride led to Scheme 19 One iterative sequence during the carbosilane-thioether dendritic growth from G2 to G3 and further functionalisation of G3 with 2-mercaptoethanol through TEC. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

20 Perspective Scheme 20 Dendrimer synthesis from the kinetically selective monomer pair MAEA and cysteamine. G1 dendrimers having the original number of peripheral groups increased by a factor of four. Repetition of that two-step sequence twice gave the third generation dendrimers with 192 hydroxyl groups at the periphery. In the same year, Radosz et al. demonstrated a highly efficient strategy for the synthesis of G4 polyester dendrimers by combining kinetic or mechanistic chemoselectivity and click reactions between the monomers. 73 To obtain alternating dendrimers having terminal alkyne or amine groups, the kinetically asymmetric monomer bearing two different alkene functionalities, namely 2-[(methacryloyl)oxy]ethyl acrylate (MAEA), was subjected to a Michael addition with cysteamine as a CD 2 monomer (Scheme 20). The key to the success of this approach is the orthogonality of methacrylate and acrylate residues. Whereas the acrylate group reacts selectively with amines, the methacrylate group is disposed to undergo reaction with thiols. Using these reactions in a sequential manner through a divergent methodology the authors obtained desired dendrimers. Subsequently, to obtain alternating dendrimers with terminal alkyne or hydroxyl groups, the authors designed a pair of mechanistically asymmetric monomers, namely 2-isocyanatoethyl methacrylate (IEMA) and 1-thioglycerol. In this case, the fact that isocyanates readily react with alcohols in a mechanism different from the Michael addition was employed. Another click-type approach based on the base-mediated thio-etherification of thioglycerol with a-bromoesters (thio-bromo click reaction), followed by an esterification with 2-bromopropionyl bromide was reported by Percec et al. 74 The power of this divergent method was illustrated by the rapid synthesis of G4 bromideterminated poly(thioglycerol-2-propionate) (PTP) dendrimers. One of the most recent synthetic accomplishments in dendrimer chemistry is double click reaction strategy, which is a combination of two chemically and mechanistically different click reactions. 75 This methodology starts to be recognised as an extremely powerful synthetic tool suitable not only to accelerate the synthesis of dendrimers but also to prepare them with diverse topology and composition. To date, only a few click reaction combinations have been successfully adapted to the double click reaction strategy for dendrimer synthesis, namely the CuAAC-DA, the CuAAC-TEC and the TYC-MA reactions. A combination of the CuAAC and furan maleimide Diels Alder reactions was used by Kakkar et al. to obtain G1 to G3 thermoreversible dendrimers that cleaved upon heating via a retro-da reaction. 76 Dendrimers were synthesised via adivergent growth approach, using two click reactions sequentially and two monomers containing furan and maleimide fragments (Scheme 21). Xiong reported the synthesis of G2 and G3 dendrimers with 8 and 16 hydroxyl groups on the periphery, respectively, using the CuAAC and Diels Alder click reactions in a one-pot technique. 77 The groups of Hawker and Malkoch jointly demonstrated for the first time the orthogonality of sequential CuAAC and TEC click reactions for dendrimer synthesis. 78 By successively combining the above-mentioned reactions, they prepared a G6 dendrimer in a single day. The synthesis was accomplished by using tris(allyloxy)triazine (TAT) as a core and two types of monomers prepared from 2,2-bis(hydroxymethyl)propionic acid, nominally an AB 2 monomer, having one thiol and two azide functional groups, and a CD 2 monomer, having one alkyne and two alkene functionalities (Scheme 22). More recently, Li et al. reported a facile methodology to prepare amine- or alkyne-terminated dendrimers by taking advantage of the orthogonality of aza-michael addition (MA) and thiol yne reactions (TYC). 79 The fifth generation dendrimers based on the ethylenediamine core were obtained divergently using but-3-ynyl acrylate and cysteamine as two unsymmetrical monomers in five steps. The authors also showed that the periphery of these dendrimers can be easily decorated with various functional groups through selective coupling with amine or alkyne groups. MCR-based approach to dendrimers Multicomponent reactions (MCRs) combine three or more starting reactants in a one-pot and one-step process to generate single, structurally and functionally complex products under mild conditions. 80 This approach improves the overall efficiency of synthetic strategies and also meets the principles of sustainable chemistry, because MCRs tend to produce benign (if any) byproducts and reduce the number of synthetic or purification steps. Within the most conventional multicomponent processes, those based on the peculiar reactivity of isocyanides, such as the Passerini three-component reaction (Passerini-3CR) and the Ugi four-component reaction (Ugi-4CR) are the most studied (Scheme 23). 81 Currently, these isocyanide-based multicomponent reactions (IMCRs) attract increased interest for the synthesis of highly diverse macromolecular structures including dendrimers New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

21 Perspective Scheme 21 Synthesis of thermoreversible dendrimers via sequential CuAAC and DA click reactions. The first excellent example was reported by Rivera et al. in They demonstrated that the Passerini three-component and the Ugi four-component reactions can be successfully used for the divergent synthesis of structurally diverse peptide-peptoidic dendrimers up to generation four (including also Janus dendrimers). The proposed strategy is based on the repetitive MCR/activation protocol and illustrated in a simplified form in Scheme 24, where by way of example a tetra-carboxylic acid core molecule undergoes a fourfold Ugi-4CR with monoprotected bifunctional building blocks (i.e., isonitriles, primary amines and aldehydes) to produce a G1 dendrimer. Subsequent activation of protected functionalities finally affords up to threefold number of Ugi-reactive functional groups, which in turn are capable of forming a highly branched G2 dendrimer in Ugi-4CRs. The above strategy can utilise either known multifunctional core molecules with Ugi-reactive groups (not only carboxylic acid groups) or new ones synthesised via Ugi-4CRs, as well as bifunctional building blocks with a nonbranching unit whereby the 1-2 branching or linear prolongation is freely accessible in every generation. Moreover, it is possible to apply different Ugi-4CRs in subsequent synthetic cycles and incorporate almost every type of chemical functionalisation. A remarkable diversity of peripheral groups and architectural diversity were demonstrated in the library of G1 to G4 dendrimers, which highlight an important advantage of this MCR-based strategy. An example of the G3 dendrimer, which was additionally functionalised with sugar functions via CuAAC click reactions, is shown in Scheme 25. In 2012, Rudick and co-workers demonstrated the use of the Passerini three-component reaction as a very efficient approach to connect dendrons in a convergent manner. 84 Three component dendrons were first synthesised following the convergent strategy developed for 1,3-propanediol dendrons and then assembled via the Passerini reaction to yield G2 dendrimers containing both benzyl and linear alkyl peripheral groups (Scheme 26). The advantage of this Passerini-3CR-based strategy is that the unprecedented surfacetriblock dendrimers, having three different types of surface groups, can be prepared in a single synthetic transformation. Recently, Meier et al. described a very useful approach for the divergent synthesis of dendrimers using renewable building blocks by a combination of the Passerini three-component reaction and the olefin cross-metathesis. 85 The proposed strategy included the Passerini reaction of castor oil-derived platform chemicals, such as 10-undecenoic acid and 10-undecenal, with an unsaturated This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

22 Perspective Scheme 22 Synthesis of G6 dendrimers by the orthogonal coupling method utilising click chemistry (reactions CuAAC and TEC). Scheme 23 Representation of the Passerini-3CR and the Ugi-4CR. isocyanide to obtain a core molecule having three terminal double bonds (Scheme 27). Subsequent olefin cross-metathesis with tert-butyl acrylate, followed by hydrogenation of the double bonds and hydrolysis of the tert-butyl ester, led to an active core molecule bearing three carboxylic acid groups as reactive sites. Iterative steps of the Passerini reaction with 10-undecenal and 10-isocyanodec-1-ene for branching, and olefin cross-metathesis Scheme 24 Synthesis of dendrimers according to the MCR-based strategy reported by Rivera et al New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

23 Perspective Scheme 25 Synthesis of G3 acetylene-terminated dendrimers and their further functionalisation with mannose functions via CuAAC click reactions. with tert-butyl acrylate, followed by hydrogenation and hydrolysis allowed the synthesis of G3 dendrimers. Approaches to the differentiation of the dendrimer surface synthesis of peripherally multifunctionalised dendrimers Control over the peripheral multifunctionalisation of dendrimers constitutes a considerable challenge. Thus far, there has been no general strategy for the simple and efficient synthesis of dendrimers with chemically differentiated surfaces, i.e., containing more than one type of terminal functionalities, which could allow for the introduction of a wide variety of functional groups at defined locations. The use of the majority of known methods is limited to certain types of dendrimers because of special requirements that must be fulfilled, e.g., the presence of specific surface groups, such as primary amine or P(S)Cl 2 groups. However, making an effort to construct these sophisticated structures is profitable because they are excellent multipurpose scaffolds, offering a larger number of potential applications compared with traditional dendrimers. 86 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

24 Scheme 26 Convergent synthesis of surface-diblock dendrimers via the Passerini reaction between dendron components. From an application viewpoint, especially those related to biology, specific interest is attached to dendrimers that have at least three types of terminal functionalities. Their controlled synthesis is more difficult to conduct than the synthesis of bifunctional dendrimers, Perspective which is certainly the reason for the relatively limited number of examples of such compounds. Basically, peripherally multifunctionalised dendrimers can be prepared via either pre- or post-modification methods, i.e., by the introduction of different desired end-groups during the course of the dendrimer growth or the modification of the existing peripheral groups after completion of the synthesis. In the first strategy, only those peripheral groups that possess the appropriate solubility and that neither cause side reactions nor undergo degradation during the iterative activation and coupling steps can be used. However, functionalities that could be too sensitive for the convergent-type synthesis can be introduced by the postmodification approach. Nevertheless, this strategy has the same limitations as the divergent synthesis; hence, it requires highly efficient synthetic and purification methods, especially in the case of higher generation dendrimers. Given the location of the different terminal functionalities with respect to one another, multifunctional dendrimers can be divided into three subgroups, namely dendrimers with blocks and alternating or random distributions of these groups. Dendrimers with different peripheral groups in separate segments of their surface (surface-block dendrimers) can be obtained, as mentioned in the section devoted to the convergent Scheme 27 Divergent synthesis of G3 dendrimers via the Passerini three-component reaction and olefin cross-metathesis New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

25 Perspective Scheme 28 Synthesis of bifunctionalised dendrimers with perfectly alternating periphery by using one type of unsymmetrical dendrons (the use of at least two different unsymmetrical dendrons leads to dendrimers containing more than two types of terminal functionalities). method, by coupling together two types of dendrons that differ in their terminal groups or by coupling such dendrons (two or more) to a common core molecule. Another possibility constitutes using the focal point of a dendron for the growing of new branches in a divergent way. 87 This approach is less straightforward than the first two approaches, but it offers access to dendrimers that have a variety of functional end groups. 88 One of the possible strategies for the preparation of multifunctional dendrimers with an alternating distribution of peripheral groups relies on the convergent growth of the dendrimer using unsymmetrical dendrons, i.e., having two different terminal groups (Scheme 28). Dendrons of this type are typically constructed by the stepwise incorporation of two different functionalities into an AB 2 monomer. This process proceeds efficiently if the monomer is used in excess or if it is possible to distinguish between the two B-functionalities either because one of them is protected or because they show different reactivities under the required synthesis conditions. Using the above strategy, Grayson and Frechet accomplished the synthesis of aliphatic polyether dendrons with alternating pairs of benzyl- and ketal-protected hydroxyl groups on the periphery. 89 Owing to the fact that benzyl and ketal protecting groups are orthogonal to one another, they can be selectively removed, providing control over further postfunctionalisation of the dendrimer surface. The synthesis utilised methallyl dichloride (MDC) as the monomer (Scheme 29). In a first step, a single chloride group of the MDC monomer (used in 2-fold excess) was functionalised with the alkoxide of the G1 benzylprotected alcohol. Next, the remaining allylic chloride of the obtained MDC derivative was reacted with the alkoxide of the G1 ketal-protected alcohol to yield an unsymmetrical G2 dendron. Repetition of the activation and coupling steps led to the construction of a desired G4 dendron. Possibilities of the unsymmetrical dendron method for the preparation of dendrons containing more than two types of terminal functionalities were demonstrated by Thayumanavan et al. 90 They successfully Scheme 29 Synthesis of aliphatic polyether dendrons with differentiated surface functionalities using one type of unsymmetrical dendron. synthesised third-generation poly(benzyl ether) dendrons in which all eight terminal groups are different (Fig. 6). The synthesis relied on the use of 3-allyloxy-5-hydroxybenzyl alcohol as the AB 2 monomer, in which one of the B-functionalities was protected, and four different unsymmetrical dendrons. Another strategy that permits the preparation of multifunctional dendrimers with alternating peripheral functionalities belongs to postmodification methods and relies on stepwise targeted coupling of two different functional groups to each individual terminal group of a dendrimer (Scheme 30: variant I). This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

26 Perspective Fig. 6 G3 poly(benzyl ether) dendrons with eight types of terminal functionalities. This approach was first reported by Shinkai et al., who prepared selectively bifunctionalised G2 PAMAM dendrimers through reductive amination with 9-anthraldehyde and subsequent reaction with the boronic acid reagent, i.e., 2-[2-(bromomethyl)- phenyl]-5,5-dimethyl-1,3,2-dioxaborinane. 91 Other commercially available dendrimers bearing amine terminal groups, i.e., PPI, were functionalised by sulphonylation followed by substitution of the sulphonamide proton with alkyl, benzyl or dendritic halides (Scheme 31). 92 Later, Frechet et al. presented the multifunctionalisation of polyester dendrimers based on 2,2-bis(hydroxymethyl)propanoic acid (bis-mpa) and having a cyclic carbonate periphery. 93 This goal was accomplished by a ring-opening reaction with amines and subsequent reaction of the activated hydroxyl groups with propargylamine (Scheme 32). A dendrimer that contained both protected aldehydes and alkynes was obtained. More recently, Simanek et al. prepared dendrimers terminated with either four or eight dichlorotriazines by reacting amineterminated dendrimers with cyanuric chloride. 94 Sequential nucleophilic aromatic substitution with two different amine nucleophiles yielded compounds that displayed the desired compositional diversity on the periphery. A more versatile variant of the above-described postmodification method relies on the functionalisation of each peripheral Scheme 31 Synthesis of bifunctionalised G2 PPI dendrimers according to Schalley et al. 92a group of the dendrimer with a trifunctional amino acid, which provides two different or the same functional groups in orthogonally protected form (Scheme 30: variant II). The resulting bifunctional dendrimer with the orthogonal and chemoselective nature of the peripheral groups can be orthogonally deprotected, and according to the need, subjected to further selective postfunctionalisation. A wide variety of trifunctional amino acids, both natural and unnatural, can be used in this one-step strategy to obtain the desired bifunctional dendrimers. Dendrimers with differentiated peripheral functionalities with the application of orthogonally protected trifunctional amino acids, such as lysine and arginine, were first reported by Urbanczyk-Lipkowska et al. 95 They belong to a family of G1 peptide dendrimers and were constructed either based on known Lys(Lys) 2 scaffolds or novel multibranched scaffolds of amino acid origin. 96 Some examples of such dendrimers are shown in Fig. 7. The postfunctionalisation ability of these dendrimers was also confirmed. 97 Frechet et al. further developed this approach for the multifunctionalisation of both amine- and hydroxyl-terminated Scheme 30 Synthesis of bifunctionalised dendrimers with alternating molecular periphery by the postmodification method: variant I two-step approach, variant II one-step approach New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

27 Perspective Scheme 32 Synthesis of bifunctionalised bis-mpa dendrimers by a two-step procedure. Fig. 7 Examples of G1 peptide dendrimers based on different scaffolds and end-capping with orthogonally protected lysine. dendrimers using various trifunctional amino acids natural (aspartic acid, glutamic acid, tyrosine, and serine) as well as unnatural (propargylglycine and 4-acetyl-phenylalanine). 98 For example, they introduced alternating Boc-protected amine and alkyne functionalities onto the surface of a G1 polyester dendrimer by reacting each of its eight peripheral hydroxyl groups with Boc-protected propargylglycine (Scheme 33). Kannan et al. demonstrated that this simple one-step method to convert a peripherally monofunctionalised dendrimer into its bifunctionalised counterpart can also be successfully applied to higher generation amine-, hydroxyl- or carboxylic acid-terminated dendrimers. 99 They prepared a library of amino acid surface-modified G3.5 and G4 PAMAM dendrimers. Appropriate choice of the type of PAMAM dendrimer (G4-PAMAM-NH 2, G4-PAMAM-OH or G3.5-PAMAM-COOH) and amino acids resulted in bifunctional dendrimers that bear diverse peripheral groups (OH + NHBoc, SH + NHBoc, OH + COOMe, NHBoc + COOH, and NHBoc + S-TP). Additionally, the possibility of further postfunctionalisation of these dendrimers was proved by the conjugation of two drugs (dexamethasone and indomethacin) as well as drug and imaging agents (dexamethasone and FITC), on the aspartic acid surfacemodified PAMAM dendrimer. Wide possibilities of multifunctionalisation entail the functions of dendrimers that end with P(X)Cl 2 (X = S,O). 100 Majoral et al. were the first to describe dendrimers that have three or even four different types of peripheral groups. 9b These compounds were obtained by functionalisation of poly(phosphorhydrazone) dendrimers (PPH) 101 that were terminated with P(X)Cl 2 (X = S,O) groups. This type of functionalisation, called by the authors multiplurifunctionalisation, 102 relies on selective monosubstitution of Cl with a variety of primary or secondary difunctionalised amines followed by the second substitution performed with another amine or with phenols (Scheme 34). The aldehyde end groups allow further modification, e.g., through the Schiff and Wittig reactions. A commonly used approach to produce multifunctional dendrimers, which permits the circumvention of often multistep multifunctionalisation, is the stochastic approach, i.e., the statistical grafting of two (or more) different functions onto the surface of the dendrimers. This option is cost-effective and easy This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,

28 Scheme 33 Synthesis of bifunctionalised G1 polyester dendrimers by a one-step procedure. to perform. However, in this case, the resulting dendrimers are not well defined; they have an unpredictable (random) distribution of the various functional groups in their periphery. Strictly speaking, the stochastic approach resulted in polydispersity, i.e., heterogeneous dendrimer populations that have differing numbers, ratios and spacings of the grafted functions (Scheme 35). Such a situation is a consequence of the fact that under conditions of the statistical function attachment, there is an excess of reactive groups on the dendrimer relative to the amount of function added. The random functionalisation of the dendrimer surface proceeds without any degree of control over individual functional group placement. A complex mixture of products following stochastic synthesis is typically reported using simple cartoon models that present the mean number of functions attached. Holl et al. determined that the use of the mean value as a physically meaningful representation for the obtained material underestimates its Perspective heterogeneity. 103 For example, when a G5 PAMAM dendrimer is coupled with an alkyne function to achieve a mean value of 12.9 functions per dendrimer, the actual material contains 27 different populations that range from dendrimers with no functions to dendrimers with 26 functions (not counting dendrimers with the same number of functions but deployed differently relative to one another). Additionally, in most cases, the dendrimer population with the same number of functions as the mean number constitutes only a small percentage in the obtained material. Holl et al. also noted that attaching multiple different functions onto the same dendrimer following sequential reaction steps further increases the material heterogeneity. In light of the above facts, it is not surprising that the stochastic approach applied to obtain multifunctional dendrimers, especially for biomedical application purposes, raises an acute issue related to the consistency of the results and the statistical responses. Recently, Baker et al. showed that the synthesis of dendrimers with defined and uniform ratios of grafted functions is crucial in obtaining consistent and biologically reproducible activity. 104 They synthesised two types of heterofunctional, targeted cancer therapeutics based on G5 PAMAM dendrimers and containing the targeting molecule folic acid (FA) and the chemotherapeutic drug methotrexate (MTX). The first type was prepared through a stochastic approach, which includes a multistep, sequential dendrimer surface modification and was characterised by heterogeneous populations having differing numbers and ratios of the functionally antagonistic FA and MTX (the mean number: 5 FA and 7 MTX per dendrimer). 105 The second type was achieved by an improved stochastic approach that includes a one-step click reaction (a triazine scaffold approach) that resulted in more homogeneously distributed populations with a fixed 1 : 1 ratio of FA and MTX (3.1 molecules per dendrimer). Biological studies showed that a dendrimer therapeutic with the same FA/MTX ratio in all populations induces more reproducible and higher cytotoxicity compared to the sequentially synthesised dendrimer therapeutic, in which populations with variability in the ratios of the two molecules lead to inconsistent and sometimes biologically inactive samples of material. As was suggested by Baker et al., this increased cytotoxicity of the dendrimer therapeutic with a defined FA/MTX ratio can likely be attributed to the Scheme 34 Multiplurifunctionalisation of the surface of PPH dendrimers (only one terminal group P(X)Cl 2 (X = S, O) is shown, representative of all the terminal groups) New J. Chem., 2014, 38, This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

29 Perspective Scheme 35 Heterogeneous dendrimer populations as a result of heterobifunctionalisation via the stochastic approach. The variation of the number and spacing of the grafted functions. presence of more functionally active dendrimers, i.e., dendrimers that have two different functions that are in close proximity to one another. This explanation notes the importance of having the proper distribution of different functions on the dendrimer be convincing. It is well known that the properties of a dendrimer are determined not only by the specific properties of the terminal functional groups, their number and structural variety but also by cooperative effects between different functionalities. 106 Similarly, the mechanism of action of enzymes depends on several functional groups being in spatial proximity. 107 Methods for the synthesis of internally functionalised dendrimers In all of the strategies presented above, functionalisation is introduced into the dendrimer framework at the core, the periphery, or both. The interior of the dendrimer (consisting of the dendritic branches) is usually inactive independently of the synthetic approach. It serves as an inert scaffold, linking the periphery and the core and arranging the surface groups in space without having an inherent function. However, the incorporation of functional groups (chemically functional or physically active groups) in the inner layers, i.e., generations, is desirable for certain applications, e.g., catalysis. The traditionally challenging functionalisation of the dendrimer interior can take place either prior to dendrimer construction (premodification approach) or on the preformed dendrimer (postmodification approach). 108 The syntheses of internally functionalised dendrimers usually rely on the use of new monomers of an AB 2 C structure, where the A functional group can react only with the B functional group and the C group is the functional group that does not take part in a synthetic cycle, being represented in the interior of the final dendrimer. The C-functionality must be Scheme 36 Synthesis of dendrimers by using the AB 2 C-type monomer by the divergent method. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38,