Intramolecular Cross-Linking Methodologies for the Synthesis of

Review pubs.acs.org/CR

Intramolecular Cross-Linking Methodologies for the Synthesis of Polymer Nanoparticles Sudheendran Mavila, Or Eivgi, Inbal Berkovich, and N. Gabriel Lemcoff* Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva-84105, Israel 3.2. Hydrazone Chemistry 3.3. Enamine Chemistry 3.4. Reversible Cycloaddition 4. SCNPs via Noncovalent Interactions 4.1. Hydrogen Bonding Interactions 4.1.1. Benzamide Dimerization 4.1.2. Benzene Tricarboxamide Stacking and Ureidopyridinone Dimerization 4.1.3. Ureidoguanosine−Diaminonaphthiridine Dimerization 4.1.4. Hamilton’s Wedge−Cyanuric Acid and Thymine−Diaminopyridine Interactions 4.2. π-Stacking 4.3. Hydrophobic Interactions 4.4. Host−Guest Interactions 4.4.1. Cucurbit[n]uril 4.4.2. Cyclodextrins 4.5. Metal Complexation 5. Conclusions and Outlook Author Information Corresponding Author Notes Biographies Acknowledgments References Note Added after ASAP Publication

CONTENTS 1. Introduction 2. SCNPs via Covalent Bonds 2.1. Friedel−Crafts Alkylation 2.2. Free Radical Coupling 2.3. Thermal Cycloadditions 2.3.1. Benzocyclobutene Dimerization 2.3.2. Benzosulfone Dimerization 2.4. Photochemical Cycloadditions 2.4.1. Photoinduced [4 + 2] Cycloaddition 2.4.2. Photoinduced Tetrazole−Ene Cycloaddition 2.4.3. Photo-Cross-Linking of Cinnamoyl Groups 2.4.4. Photodimerization of Anthracene 2.5. Tetrazine−Norbornene Reaction 2.6. Isocyanate−Amine Coupling 2.7. Bergman Cyclization 2.8. CuI-Mediated Click Chemistry 2.8.1. Azide−Alkyne Click Chemistry 2.8.2. Glaser−Hay Coupling 2.9. Thiol−Yne Click Chemistry 2.10. Amine Quaternization 2.11. Amide Formation 2.12. Michael Addition Reaction 2.13. Nitrene Insertion/Coupling 2.14. Olefin Metathesis 2.15. Oxidative Polymerization of Thiophene 2.16. Ring-Opening Polymerization 2.17. Hydrolysis and Polycondensation of Alkoxysilane 2.18. Orthogonal Covalent Cross-Linking 2.19. Intramolecular Cross-Linking of Grafted Copolymers 3. SCNPs via Dynamic Covalent Bonds 3.1. Disulfide Chemistry © 2015 American Chemical Society

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1. INTRODUCTION The advancement of polymer science embraces the invention of molecular constructs that provide functional applications. As a result, the development of novel methodologies for the production of polymers and complex macromolecular architectures and the design of new macromolecular systems stand out as appealing goals enthusiastically pursued by the chemical community. A recent report asks the provocative question “How far can we push polymer architectures?”1 The growing complexity currently found in macromolecular scaffolds suggests a rather broad answer to this enquiry. From the architectural viewpoint, simple synthetic linear polymers evolved throughout the last century to more compound structures, such as block copolymers, brush polymers, hyperbranched macromolecules, and dendrimers, just to name a few.2−8 Also, in the functionality domain, a development from ordinary polymeric building materials to

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DOI: 10.1021/acs.chemrev.5b00290 Chem. Rev. 2016, 116, 878−961

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Figure 1. Modes for intramolecular cross-linking of a polymer chain.

can also be classified according to the type of chemistry used to generate the nanoparticle (Figure 1): Homofunctional cross-linking, where pendant functional groups react with each other to generate a bond, heterobifunctional chain collapse, where two complementary functionalities are attached to the polymer chain to cross-link it, and the cross-linker-mediated collapse, which uses a cross-linking molecule to bind two other functional groups in the polymer.9 In addition, multiorthogonal procedures may also be followed.27,28 Importantly, the method chosen for polymer collapse will also in turn affect the overall structure and potential properties of the single-chain nanoparticle (SCNP) formed. The history of intramolecularly cross-linked polymers actually dates back to 1955, when a seminal contribution by Kuhn and Majer reported the possibility of avoiding the crosslinking between different molecules under very low concentrations of the polymer solution. In such a case, they predicted that the self-cross-linking or internal cross-linking should be the major event.29 Continuing the study, Kuhn and Balmer experimented with the intramolecular cross-linking of poly(vinyl alcohol), in which the cross-linking units (−OH groups) were distributed throughout the polymer chain. Cross-linking was promoted in a slightly acidic aqueous solution of poly(vinyl alcohol) (Mw = 100 kDa) by the reaction of the hydroxyl groups with terephthalaldehyde under dilute conditions.30 The reaction at the time was monitored by spectrophotometry and showed first-order reaction kinetics with quantitative conversion of the cross-linker added. The authors also observed a reduction in the intrinsic viscosity, when the cross-linker was added at very low concentrations of the polymer, suggesting self-cross-linking. Concordantly, at higher polymer concentration, an increase in the intrinsic viscosity was observed due to the intermolecular aggregation. In 1968, Longi et al. attempted the intramolecular crosslinking of α-olefin/allylsilane copolymers using alkali/alcohol mixtures to form Si−O−Si bridges.31 Although the authors confirmed the formation of siloxane cross-links in the polymer chains under dilute conditions, no noticeable change in the intrinsic viscosity was observed, probably due to the labile nature of the cross-links and the low density of reactive sites. In the following year, Longi et al. demonstrated the intramolecular cross-linking of a styrene/methyl acrylate

smart sensor applications, self-healing systems, and catalytic and electro-optical devices has followed. Within this rapidly expanding field we find the emergence of single-chain collapse technologies to bestow novel properties and functions upon the shrunken polymer, sometimes called organic nanoparticles (ONPs).9−15 The exquisite control found in the folding of the structure of the most ubiquitous of polymers, i.e., proteins, leads to chemical functions that for now we can only aspire to emulate. Perhaps enthused by this model of Mother Nature, the design of single-chain collapse has recently flourished, and herein we dwell on the recent advances in intramolecular cross-linking of single-chain synthetic polymers and the characteristics of the ONPs obtained by this process. Several methodologies are used to make ONPs, although the most prevalent involve emulsion polymerization techniques or intramolecular cross-linking and more recently even microfluidics technologies.16−25 As an introductory remark to this review two frequently used expressions must be clarified. The applications of organic nanoparticles are many, as will be detailed below, but it is not a secret among scientists that the prefix “nano” has been extensively used in recent times to describe molecular compounds that were previously known by more earthly alternative words, especially when referring to polymers, proteins, or colloids. However, the fabrication of nanometric scale objects whose size can be precisely controlled and where the change in dimensions and shape brings about new properties that were not present beforehand merits the use of the nano term. Another semantic clarification is needed for the way the word cross-link is used in this review and in other reports in the field. Whereas technically a cross-link is a bond that binds two different polymer chains,26 here it is mainly used to define an additional bond or linking unit between monomers of the same chain. Thus, an intramolecular cross-link is always a type of ring-closing reaction, while the more classical cross-linking processes produce undefined thermoset polymers. More relevant to this review, the term cross-link is frequently used to characterize disulfide linkages within proteins. Three main approaches stand out as the most common methods to induce chain collapse in polymers: irreversible covalent, reversible covalent (dynamic), or noncovalent crosslinking. The strategies typically used to achieve chain collapse 879


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ethane solution using SnCl4 as catalyst.37,38 Surprisingly, the characterization of these microgels by using gel permeation chromatography (GPC), static and dynamic light scattering (DLS), and viscosity measurements revealed only a small reduction in the radius of gyration (Rg) and hydrodynamic radius (Rh) even at high degrees of cross-linking. Other early examples of intramolecularly cross-linked polymers involved the synthesis of water-soluble microgels. Frank and Burchard reported single-chain microgels containing free amino groups as model of living cells to study the interaction with polysaccharides.39 The authors chose watersoluble poly(allylamine) (PALA) as the precursor polymer. Thus, poly(allylamine) of Mw = 76 kDa was cross-linked in dilute aqueous solution by using 1,4-dimethoxybutane-1,4diimine dihydrochloride in the presence of NaOH. The microgels formed were characterized by static and dynamic light scattering experiments and viscosity measurements. While the radius of gyration was reduced slightly after cross-linking, no significant change in the molecular weight and hydrodynamic radius was observed. The decrease of the ρ parameter (Rg/Rh) with increasing amount of cross-linker suggested the formation of a more compact structure. Another example reported by Burchard and Brasch involved the synthesis of reactive microgels (microgels having reactive functional groups at the periphery) starting from poly(vinyl alcohol) (PVA) as the precursor.40 This time, the authors made water-soluble microgels41 via the intramolecular crosslinking of PVA using terephthaladehyde as the cross-linker under dilute conditions (1 mg/mL). Before cross-linking, the PVA solution was filtered successively through 0.22 μm filters to obtain a solution containing PVA of molecular weight 162 kDa, free of H-bonded aggregates. The microgels formed were characterized by static and dynamic light scattering and viscometry measurements. While the molar mass of the polymer increased slowly with time due to partial intermolecular H-bonding interactions, a significant reduction in the radius of gyration Rg and intrinsic viscosity [η] was observed upon increasing the amount of cross-linker. These early works set the stage for the rapid expansion the field is currently experiencing. There is no doubt of the importance of these pioneering efforts and the way they influenced the studies of polymer chemists today. Actually, the seminal works by Walsh et al.33 and Martin34,35 in the early 1980s both experimentally and theoretically demonstrated the relationship between the cross-link density and the contraction factor. The field laid more or less dormant for the next 20 years, until the turn of the century where new chemistries were used or developed both for polymer synthesis42−45 and for intramolecular cross-linking strategies, such as the benzocyclobutene (BCB) method46 and olefin metathesis,47,48 among others. Maybe more importantly, characterization methods became much more readily available in the form of triple-detection size exclusion chromatography (SEC), DLS, and electron and scanning probe microscopies. Most relevant for this review was the modulation of intramolecular crosslinks at high dilution to control the polymer size and alter its properties. Thus, Zimmerman et al. showed a linear relationship between the cross-link percentage and the reduction of a dendrimer’s radii of gyration when olefin termini were cross-linked using Grubbs’ first-generation catalyst.49 Further studies revealed more intricacies on the cross-linking mechanism, which were at the time somewhat counterintuitive, and several additional approaches were put

copolymer containing randomly distributed acrylate crosslinking units.32 Thus, a styrene/methyl acrylate copolymer containing 10 mol % of acrylate units having an intrinsic viscosity of [η] = 0.93 dL/g in chloroform at 30 °C was chosen as the polymer precursor. This polymer was crosslinked by reacting it with 9,10-disodio-9,10-dihydro-anthracene as a bifunctional cross-linker in anhydrous tetrahydrofuran (THF) under dilute conditions (5 mg/mL). Upon increasing the amount of cross-linker, a linear reduction in the intrinsic viscosity was observed without a significant change in the number-average molecular weight, indicative of the intramolecular collapse. A few years later, Walsh et al. conducted a quantitative study on the effect of the amount of intramolecular cross-links in narrowly disperse polystyrene (PS) samples.33 Thus, a chloromethylated polystyrene with varying amounts of chloromethyl groups was reacted with n-butylamine to obtain the secondary amine groups. Disappearance of the −CH2Cl signal at 1266 cm−1 monitored by infrared spectroscopy confirmed the successful substitution. The intramolecular cross-linking of the substituted polystyrene was then achieved by reacting it with hexamethylenediisocyanate (as a bifunctional cross-linker) under dilute conditions (1 mg/5 mL) in cyclohexane. Analysis of the carbonyl absorption band (1645− 1655 cm−1) provided the approximate number of cross-links in the sample. In one of the classical early contributions to the field, Martin and Eichinger reported a complete theoretical and experimental study on the changes in the unperturbed radius of gyration of the linear polymer chain upon intramolecular cross-linking.34 According to their calculations, the ZimmStockmayer contraction factor (g) for an intramolecular is given by g = 1 − 0.7ρx 0.5

where g is the ratio of the unperturbed radius of gyration of the cross-linked polymer to that of the linear polymer and ρx is the cross-link density (moles of cross-links per mole of Gaussian statistical segments). In order to support the predictions, the authors carried out Friedel−Crafts-mediated cross-linking of polystyrene with pbis(chloromethyl)benzene in the presence of SnCl4 in cyclopentane under θ conditions.35 Although the authors were able to obtain a close agreement between the experimental values and the theoretical predictions, only a marginal reduction in the size of the polystyrene precursor was found after the intramolecular cross-linking. In contrast, Walsh et al. observed a large reduction in precursor polymer size when a polystyrene precursor with randomly distributed cross-linking sites was used.33 Martin and Eichinger claimed that the reason for this minimal size reduction was due to the formation of smaller rings upon cross-linking. Since every monomer is a potential cross-linking site, the cross-linking of a homopolymer under dilute condition may result in the formation of smaller rings and thus a less significant reduction in the polymer’s radius of gyration. A similar trend in the reduction of size was observed by Antonietti et al. in the synthesis of microgels of intramolecularly cross-linked polystyrene in a good polymer solvent (dichloroethane).36 The microgels were prepared by Friedel−Crafts reaction-mediated intramolecular cross-linking of linear polystyrene with p-dichloroxylene in dilute dichloro880


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Table 1. Covalent Cross-Linking Chemistry for Generating SCNPs

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Table 1. continued

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Table 1. continued

forth for the advancement of the field as will be detailed in the following sections.50 The field of macromolecule intramolecular cross-linking, or single-polymer chain collapse, has surged during the past decade. At least five reviews covering this topic have been

published lately,9−13 the most recent being a brief user’s guide by Berda et al. discussing the synthesis, characterization, and potential uses of SCNPs. In this review we extensively covered the current literature, organizing it by the type of chemistry used to generate the SCNP. For convenience, we 883


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Scheme 1. Intramolecular Chain Collapse of PS by Friedel−Crafts Alkylation

polystyrene microgels (Scheme 1a). However, only small changes in the structural and dynamic properties could be observed for the intramolecularly cross-linked polystyrene microgel compared to the uncross-linked polystyrene precursor.36 One possible reason for the reduced impact of self-cross-linking could be the tendency to form small rings, as previously stated. In any case, the cross-linking density achieved was found to be less than 10%. Aiming to improve the degree of cross-linking, Davankov et al. utilized a similar strategy to obtain conformationally rigid cross-linking. The term “nanosponges” coined by the authors for these highly cross-linked materials was attributed to their low density and high porosity. The idea was to find a method to involve every monomer of the precursor polymer chain in a cross-linking event.51,52 The authors used monodisperse atactic polystyrene with Mw = 330 kDa as their starting material. Then the pendant phenyl rings of the polymer chain were chloromethylated by using monochlorodimethyl ether either in approximately 50% conversion or quantitatively (determined by elemental analysis). Cross-linking was then carried out by heating a dilute solution (0.5 g/L) of chloromethylated polymer in ethylene dichloride in the presence of a suitable catalyst to promote the Friedel−Crafts alkylation (Scheme 1b). These intramolecularly cross-linked materials exhibited higher porosity (as measured by argon BET) in the dry state than the regular polystyrene and dramatically lower intrinsic viscosity values.

start with the most prevalent type of cross-link chemistry used, the covalent bonds. This section is followed by the reversible dynamic covalent bond strategy, and we finish with noncovalent bonding methods. Finally, a short conclusions and outlook section summarizes the potential of the field and gives some hints as to where it may lead.

2. SCNPS VIA COVALENT BONDS A vast majority of the cross-linking strategies developed for single-chain collapse so far falls in the category of covalent chemistry. Among them, click chemistry, radical coupling, benzocyclobutane dimerization, Bergmann cyclization, Diels/ Alder ligation, etc., are frequently used. Unlike noncovalent and dynamic cross-linking chemistry, covalent cross-linking delivers irreversible nanoparticles that remain stable and unchanged in response to external stimuli. Thus, the route for attaining a desired compact structure in SCNP synthesis by covalent cross-linking has taken the lion’s share of the efforts in the area. The folding of single-polymer chains follows any of the four general cross-linking strategies: homofunctional, heterobifunctional, cross-linker-mediated collapse, and a few examples of orthogonal collapse of a single block within a dior triblock copolymer. However, SCNPs prepared via covalent cross-linking lose their dynamic nature, thereby making them unsuitable for some biomimetic applications. In the following section various covalent bond forming chemistries used for the collapse of single-polymer chains are described. Table 1 summarizes the cross-linking chemistry used to generate the SCNP and whether the method was by homofunctional crosslinking (HOM) or heterofunctional cross-linking (HET) or if it was assisted by an external cross-linker (XL).

2.2. Free Radical Coupling

Radiation-induced cross-linking of polymer chains is a seldom used strategy, which involves the generation of radicals along the polymer chains by applying a short intense pulse of γ-ray ionizing radiation. The OH radicals and H atoms generated (in water) lead to the formation of radical sites on the polymer chain via hydrogen abstraction. One advantage of using this strategy is that there is no need to use any specialized monomers or cross-linkers. However, the main parameter governing inter- and intramolecular cross-linking is the concentration and the number of radicals per chain, which is hard to control by this method. In 1998, Ulanski and coworkers investigated the intramolecular cross-linking of

2.1. Friedel−Crafts Alkylation

Early studies on intramolecular cross-linking used mostly functionalized polystyrene derivatives of narrow molecular weight distributions. Commonly, the cross-linking was achieved through pendant chloromethylated phenyl units across the single-polymer chains. As mentioned above, Antonietti et al. used Friedel−Crafts alkylation chemistry as a cross-linking method to study the dynamic behavior of 884


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Jiang and Thayumanuvan reported another example of radical-mediated chain collapse in 2005. In this work the authors were able to prepare amine-containing nanoparticles that could be further functionalized.57 The precursor copolymer, with varying feed ratios of the comonomers, was synthesized by the reversible addition−fragmentation chain transfer (RAFT) copolymerization of Boc-protected 4-vinyl aniline with 4-chloromethylstyrene using AIBN as the radical initiator. The pendant chloromethyl groups were then further functionalized to produce cross-linkable moieties by the substitution reaction with 4-[(3-hydroxyphenoxy)methyl]styrene. Using AIBN as the radical initiator, the styrene side groups of the resultant copolymer were cross-linked intramolecularly under high dilution (Scheme 4). The cross-linking process was monitored by SEC, which showed a decrease in the molecular weight from Mn = 21 to 12 kDa relative to polystyrene standards. The hydrodynamic radius (Rh), measured by DLS, also showed a significant reduction from 26.1 to 16.9 nm. 1H NMR spectroscopy analyses indicated that approximately 90% of the carbon−carbon double bonds had reacted. The nature of the nanoparticles and that of the precursor polymer were also imaged by atomic force microscopy (AFM). While the polymer precursor appeared as a worm-like structure probably due to aggregation, welldefined spherical particles could be observed for the SCNP, correlating well with the DLS results in terms of size. Boc deptrotection afforded amine functionalized nanoparticles that were insoluble in common organic solvents such as THF, dichloromethane (DCM), and chloroform. The ability of the SCNP to undergo further functionalization was proven by reacting the free amino groups with pivaloyl chloride in the presence of triethylamine as base. The authors were able to functionalize more than 90% of the amino groups with pivaloyl chloride to afford nanoparticles which regained their high solubility in organic solvents. Thus, a pathway toward the preparation of SCNPs containing desired appendages was paved.

various water-soluble polymers poly(acrylic acid) (PAA), PVA, etc., by using this methodology.53 The researchers suggested the formation of intramolecularly cross-linked nanogels after preliminary studies on the irradiation of an aqueous PVA solution (2 × 10−2 mol/dm3) with 1.1 kGy electron pulse. A few years later, the same research group extended their crosslinking method to polyelectrolyte-poly(acrylic acid).54 Unlike the neutral polymers tested previously, the recombination of PAA was strongly pH dependent and many competing processes such as intra- and intermolecular cross-linking, intermolecular disproponation, and chain scission were expected. However, kinetic studies (pH = 2, 17.5 mM PAA 1.1 kGy per pulse) actually revealed that the cross-linking was predominantly intramolecular. A significant drop in the solution viscosity provided further evidence for this conclusion. One year later a detailed a study on radiationinduced intramolecular cross-linking of PAA and the properties of the nanogels formed was reported.55 The authors discovered that the intramolecular cross-linking was preferred when many carbon-centered radicals were generated simultaneously along each PAA chain in dilute solutions. By changing the initial concentration of the polymer precursor and the dose absorbed by the sample, the average molecular weight and dimensions of these nanogels could be controlled (Scheme 2). Scheme 2. Radiation-Induced Cross-Linking of PAA Chains

Also hinging on a free-radical strategy, Miller and coworkers reported the collapse of various polymers bearing acryloyl or methacryloyl functionalities via radical-mediated intramolecular covalent cross-linking.56 The self-cross-linking of these polyesters was realized by radical cross-linking using azobis(isobutyronitrile) (AIBN) as the radical initiator in an ultradilute solution of the precursor polymer (Scheme 3). The cross-linking reaction was confirmed by 1H NMR spectroscopy, monitoring the disappearance of vinyl peaks corresponding to pendant acrylate or methacrylate groups. 1H NMR revealed 72−92% conversion, which led to the formation of nanoparticles in the range of 3−15 nm. The single-chain collapse was also monitored by SEC, which showed a significant decrease in the hydrodynamic radius of the nanoparticles compared to that of the parent polymer chain. Further evidence for SCNP formation by intramolecular covalent cross-links was obtained by using dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) measurements. The formation of internal covalent cross-links inside the nanoparticle were confirmed by transesterification of the cross-linked ester moieties with methanol, which resulted in the disruption of the ester bonds and the increase of the hydrodynamic radius of the nanoparticles to near that of the original precursor polymer chain. Owing to the tendency of these nanoparticles to disperse uniformly with minimum aggregation, they were utilized as sacrificial porogens to generate nanoporous thin films.

2.3. Thermal Cycloadditions

2.3.1. Benzocyclobutene Dimerization. One of the most influential chemistries developed for intramolecular polymer cross-linking was the benzocylcobutene reaction. In order to overcome competing intermolecular cross-linking Hawker and co-workers followed an ingenious continuous addition strategy to prepare well-defined SCNPs using BCB chemistry.46 Since only the reactive species need to be under ultradilute concentration, a continuous addition strategy is more practical than the traditional high-dilution strategy, provided the cross-linking is sufficiently rapid, efficient, and irreversible. The precursor polymer was prepared by nitroxidemediated copolymerization (NMP) of styrene with varying amounts of 4-vinylbenzocyclobutene using an alkoxyamine as the initiator. Polymer folding was carried out by the continuous addition of a polymer benzyl ether solution (0.1 g/mL) at a rate of 12.8 mL/h to 120 mL of benzyl ether at 250 °C with vigorous stirring under argon (Scheme 5). No trace of intermolecular cross-linking was observed up to a concentration of 0.1 M. A comparative study of the synthesis of nanoparticles by both traditional and continuous addition methods by using the styrene/BCB random copolymer of the same molecular weight showed dramatic improvement in nanoparticle formation with minimum intermolecular crosslinking. However, the main advantage of the continuous 885


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Scheme 3. Synthesis of Acrylate Cross-Linked SCNPs by Free Radical Coupling

Scheme 4. Functional PS Nanoparticles via Free Radical Coupling

strategy by using copolymers with additional functionalities, e.g., copolymers of methacrylate monomers and polyethylene glycol (PEG) blocks (Scheme 6). Hawker et al. conducted a detailed theoretical and experimental investigation on BCB cross-linking chemistry to study the morphology and physical properties of the nanoparticles formed by this method.58−60 Due to the fact that the precursor copolymer and the BCB-cross-linked hybrid particles (nanoparticle−coil copolymers) possess the same

addition strategy is that it allows practical large-scale syntheses of the nanoparticles. The formation of nanoparticles was monitored by SEC and DLS measurements, showing a significant 65−75% reduction in the apparent molecular weight. The cross-linking reaction efficiency was also evidenced by 1H NMR analyses that showed the rapid disappearance of the peak corresponding to benzocyclobutene unit at 3.10 ppm. The authors also demonstrated the versatility of the new single-chain collapse 886


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Scheme 5. BCB-Mediated Intramolecular Collapse of PS

Scheme 6. Intramolecular Collapse of PEG-b-PS/BCB Block Copolymer

molecular weight, they serve as perfect candidates for comparative studies. For example, the influence of architecture on the self-assembly of nanoparticles and precursor copolymer could be readily discerned. 58 While the linear block

copolymers (precursors) formed disk-like surface assemblies, the nanoparticle (cross-linked) block copolymer exhibited long (>10 μm) wormlike aggregates. Hawker and co-workers also prepared polystyrene nanoparticles with varying molec887


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Scheme 7. Synthesis of Hybrid Particle−Coil Copolymer from Dendritic Initiator

ular weights and cross-linking densities via BCB chemistry46 and studied the relaxation dynamics of the polymer as a function of their architecture.59 Linear polystyrene and crosslinked nanoparticles were characterized by measuring their

intrinsic viscosity [η], hydrodynamic radius (Rh), and radius of gyration (Rg). Intrinsic viscosity and DLS measurements showed a decrease in size upon cross-linking. Moreover, the relative intrinsic viscosity measurements in different solvents 888


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Scheme 8. Intramolecular Cross-Linking of PS Copolymer via Benzosulfone Dimerization

Scheme 9. Intramolecular Chain Folding of ABA Triblock Copolymer

studied the conformation of BCB-mediated intramolecularly cross-linked polystyrene nanoparticles on mica surface.60 For this purpose, a careful study of the intrinsic rigidity and substrate−nanoparticle interaction was required. Therefore, nanoparticles of varying degrees of cross-linking (lightly (2.5 mol %), tightly (20 mol %), and extremely (60 mol %)) were prepared by following the method developed by Hawker and co-workers using benzocyclobutene cross-linking chemistry. The nanoparticles were spin coated onto the mica surface modified with Sigmacote (a solution consisting of 2.5% chlorosiloxane ((SiCl2C4H9)2O) and 97.5% heptane that functionalizes the surface with short alkane chains), and the deposition was analyzed by AFM measurements. A pancakelike conformation was observed for the lightly cross-linked

(cyclohexane, THF, and benzene) exhibited smaller perturbations in the dimensions for the tightly cross-linked polymer compared to the lightly cross-linked and linear polymer chains, suggestive of reduced swelling of the macromolecular architecture when significant intramolecular cross-linking transpired. The Burchard’s ρ ratio (Rg/Rh) calculated for these nanoparticles showed values close to the Gaussian range for the lightly cross-linked polymers, while tightly cross-linked polymers showed values close to hard spheres, indicative of the particle-like character. Furthermore, this strategy also allowed studying the rheological behavior of the polymer chains with respect to the cross-linking density and the molecular mass. Aiming to find an application for rigid nanoparticles in data storage technology, Dukette et al. 889


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Scheme 10. BCB-Mediated Cross-Linking at 150 °C Using Substituted BCBs

pendant benzosulfone moieties on polystyrene or polyacrylate.62 The random copolymerization of 90% styrene and 10% vinyl benzosulfone by using α-hydrido alkoxy amine afforded a copolymer with a Mw = 30 kDa and low polydispersity (PDI = 1.20). The intramolecular cross-linking was carried out by the continuous addition of a concentrated polymer solution to dibenzyl ether at 250 °C to obtain the SCNP (the reaction temperature was chosen by observing that the polymer afforded an exothermic maximum on the DSC curve at 250 °C) (Scheme 8). The decrease in the apparent molecular weight from 30 to 19 kDa monitored by SEC and 1H NMR spectroscopy analyses clearly indicated the formation of a single-chain nanoparticle by the benzosulfone dimerization. The authors applied the same strategy for polyacrylate copolymers to show the compatibility of this cross-linking strategy with other polymers. In further work, Harth and coworkers used the benzosulfone cross-linking chemistry to prepare polymeric nanoparticles from an ABA triblock copolymer in which a conducting B block was confined within the core of the nanoparticles (Scheme 9).63 A photoluminescence study of these nanoparticles revealed that the molecular weight of block A was crucial for the effective site isolation of the conducting polymer block. Most significantly, the collapsed SCNP displayed 3 times larger quantum efficiency compared to the uncross-linked copolymer, highlighting the utility of polymer folding and steric isolation of the middle block. Notwithstanding the positive features found in the use of benzocyclobutane and benzosulfone dimerization such as fast, stable, and irreversible cross-linking, these chemistries require very high temperatures, typically around 250 °C, which limit their applicability with linear polymers bearing heat-sensitive functional groups. In order to overcome this obstacle, Harth and co-workers used judiciously substituted benzocyclobutenes.64 It has been reported that the introduction of a substituent on the four-membered ring in BCBs dramatically decreases the ring-opening reaction temperature to afford the

and tightly cross-linked nanoparticles. In contrast, the extremely cross-linked nanoparticles showed a spherical conformation, indicating the requirement of a higher degree of cross-linking to retain the spherical conformation. The authors also determined that the type of surface on which the SCNPs are placed may also affect their conformation. In related work, Hawker employed the benzocyclobutene chemistry to prepare nanoparticle−coil copolymers by selective intramolecular folding of one of the blocks in poly(n-butyl acrylate)-block-poly(styrene-random-(4-vinyl benzocyclobutene) diblock copolymer.61 However, the desired nanoparticles were contaminated by traces of nanoparticle generated by the homopolymer impurity found in the mixture. In order to overcome this difficulty, an alternate convergent dendrimer synthetic strategy was developed taking advantage of both dendrimer synthesis and controlled radical polymerization techniques (Scheme 7). For this purpose the authors synthesized an asymmetric star block copolymer that possessed a linear poly(n-butyl acrylate) attached to a dendron bearing eight peripheral benzocyclobutene units. The hybrid nanoparticle−coil copolymer was then prepared by the intramolecular cross-linking of the poly(styrene-r-BCB) arm via the BCB dimerization chemistry. In addition to the typical analytical methods, further evidence of SCNP formation was obtained from high-resolution AFM images, in which globular structures corresponding to the cross-linked poly(styrene) and wormlike morphologies for the linear poly(n-butyl acrylate) were observed with an average diameter in the range of 10−20 nm. 2.3.2. Benzosulfone Dimerization. Although the use of BCB dimerization offers several advantages, tedious multistep synthesis, purification difficulties, and low yields led the Harth group to think about using the five-membered benzosulfone as an alternative. Upon heating to 250 °C, benzothiophene dioxide dimerizes extruding SO2 to form the o-quinodimethanes. Indeed, this chemistry was used for the collapse of a single-polymer chain through thermal dimerization of 890


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Scheme 11. Synthesis of Monofunctional SCNPs via Diels−Alder Photocycloaddition

reactive intermediates.65,66 On the basis of this concept, a RAFT polymerization-derived polyacrylate was grafted with a terminal amine-functionalized benzocyclobutene via chloroformate activation chemistry. The optimal temperature for isomerization was investigated by 1H NMR spectroscopy using model cross-linkers and found to be around 130 °C, a great improvement over unsubstituted BCBs. Thus, a precursor polymer containing 5% of functionalized benzocyclobutene was cross-linked in N,N′-dimethylformamide (DMF) at 150 °C (Scheme 10) by following the continuous addition technique described earlier. The single-chain folding was monitored by DLS and transmission electron microscopy (TEM) and showed the formation of nanoparticles in the range of 3.5−7.5 nm.

polystyrene modified with 4-hydroxy-2,5-dimethyl benzophenone and N-maleimide.70 The precursor poly(styrene-co-4chloromethylstyrene) with varying amounts of comonomers was prepared by nitroxide-mediated radical polymerization of appropriate mixtures of styrene and 4-chloromethylstyrene at 125 °C. An NMP initiator functionalized with a terminal alkyne was used in order to incorporate the alkyne functionality at the end of the polymer chain. The functional monomers 4-hydroxy-2,5-dimethyl benzophenone and Nmaleimide were introduced to the polymer chain via their sequential addition in a one-pot two-step process (Scheme 11). Irradiation of the polymer solutions at an optimal concentration of about 0.01 mg/mL with UV light at 320 nm resulted in single-chain collapse, characterized by 1H NMR, DLS, and AFM measurements. 2.4.2. Photoinduced Tetrazole−Ene Cycloaddition. The nitrile imine-mediated tetrazole−ene cycloaddition (NITEC) reaction has been known for almost 50 years.71 The reaction usually involves the photoinduced generation of a nitrile imine, a highly reactive 1,3-dipole, capable of reacting with olefins to form pyrazoline cycloadducts that exhibit broad fluorescence properties. Therefore, NITEC has been recognized as a useful biocompatible ligation technique for a variety of applications.72,73 Recently, Barner-Kowollik and co-workers exploited the use of phototriggered NITEC chemistry for the generation of

2.4. Photochemical Cycloadditions

2.4.1. Photoinduced [4 + 2] Cycloaddition. The UV light triggered Diels−Alder cycloaddition of o-quinodimethane derivatives with maleimide is another convenient atomefficient method that could be carried out quantitatively at room temperature without the need of any metal catalyst.67,68 Glassner et al. reported the use of photoinduced Diels−Alder reaction in combination with azide−alkyne click chemistry for the modular ambient-temperature synthesis of ABA- and ABC-type triblock copolymers.69 Recently, Altintas et al. applied this chemistry for the intramolecular cross-linking of 891


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Scheme 12. Tetrazole−Ene Cycloaddition SCNP Synthesis

(pro)fluorescent single-chain nanoparticles (Scheme 12).74 For this purpose, the copolymer precursor was synthesized by nitroxide-mediated polymerization of styrene and 4-(chloromethyl)-styrene using 2,2,6,6-tetramethyl-(1-phenylethoxy)piperidine as radical initiator. Postpolymerization modification of pendant chloromethyl groups with various ratios of the corresponding cross-linking partner species afforded the final copolymer precursor. The reaction was carried out under high dilution (0.017 mg/mL), and the formation of the pyrazoline cross-link was monitored by in situ fluorescence spectroscopy after excitation at 315 nm. 1H NMR and AFM analyses were consistent with intramolecular chain collapse. Moreover, the expected change in the hydrodynamic radius, determined by dynamic light scattering, further supported SCNP formation. Other than the hydrodynamic size, the fluorescence properties of SCNPs could also be controlled by varying the amount of

cross-linking units incorporated, which naturally determine the final degree of pyrazoline content in the SCNP. In addition, the authors took advantage of residual tetrazine moieties in the SCNP for the macrofunctionalization of maleimidecontaining polymeric microspheres (pore size 100 nm), which became fluorescent after the desired ligation. 2.4.3. Photo-Cross-Linking of Cinnamoyl Groups. Guojun Liu and co-workers demonstrated the self-assembly of various block copolymers bearing cinnamoyl groups in one of the blocks.75−77 In block-selective solvents these copolymers form micelles with poly(2-cinnamoylethyl methacrylate) (PCMA) block at the core. Upon irradiation, photo-crosslinking of the cinnamoyl groups resulted in the formation of star polymer nanospheres (spherical polymeric nanoparticles) and their cross-linked aggregates. Further work with this type of polymers led to water-soluble cross-linked micelles 892


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Scheme 13. Intramolecular Folding of Copolymer via Photo-Cross-Linking of Cinnamates

Scheme 14. Synthesis of Janus (amphiphilic) Nanoparticles via Intramolecular Collapse of PEO-b-PCEMA

the volume fraction of cyclohexane in the cyclohexane/ chloroform solvent mixture resulted in the shrinking of the PCEMA block and consequently favored exclusive intramolecular cross-linking. Under the optimal 64% volume fraction of cyclohexane and by using the Pt-BA-b-PCEMA of high CEMA content, tadpoles with approximately 33% reduction in the volume compared to the precursor copolymer were produced. More recently, Zhou et al. extended their Janus nanoparticles research by studying the single-block collapse of a poly(ethylene oxide)-b-poly(2-cinnamoyloxyethyl methacrylate) (PEO-b-PCEMA) diblock copolymer (Scheme 14).80 The cross-linkable cinnamoyl groups were incorporated into the polymer chain via the reaction of poly(ethylene oxide)-bpoly(2-hydroxyethyl methacrylate) (PEO-b-PHEMA) with cinnamoyl chloride. The authors followed a high-dilution strategy for the intrachain cross-linking of the PCEMA block using DMF as a common solvent. Upon UV irradiation at 254 nm, the PCEMA block was collapsed intramolecularly to some degree, depending on the concentration of the polymer solution and the irradiation intensity. The irradiation intensity was controlled by simply adjusting the distance of the light source to the sample. For example, when a copolymer (79 kDa) solution at a concentration of 1.26 mg/mL was irradiated at a distance of 1 m for 3 h, a reduction of 23%

(nanospheres) that could take up organic compounds from aqueous mixtures. Under certain solvent conditions the authors could also use this method to achieve single-chain collapse, which they called unimer cross-linking, or “tadpole” molecules. The assumed single-block collapse was monitored by size exclusion chromatography/static light scattering (SEC/ SLS) and 1H NMR measurements. The authors indeed observed the formation of both micelles and intramolecularly cross-linked polymers (SCNPs) depending on the irradiation time. However, the separation of the so-called tadpoles from the nanospheres required tedious GPC fractionation.78 The same research group later improved the method for the single-chain tadpole nanoparticles by applying Hawker’s continuous addition method.79 Thus, poly(tert-butyl acrylateblock-poly(2-cinnamoylethyl methacrylate)-ran-poly(2-hydrocinnamoyloxyethyl methacrylate) (Pt-BA-b-P(CEMA-rhCEMA)) containing 0%, 18%, and 34% hCEMA were used as the polymer precursors. The hCEMA units were incorporated in order to minimize interchain cross-linking. Intramolecular photoinduced cross-linking of cinnamoyl groups was achieved via the dropwise addition of the precursor polymer solution (0.22 mL/min) to a chloroform/cyclohexane mixture under constant UV irradiation and stirring (Scheme 13). Because cyclohexane is a good solvent for the Pt-BA block and a bad solvent for PCEMA, increasing 893


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Figure 2. TEM image of the tadpole-like SCJNPs prepared at a concentration of 1.26 mg/mL (a), and the image at a larger magnification (b). TEM samples were stained by RuO4 for 15 min.

Scheme 15. Synthesis of SCNP with an Available (reactive) Monofunctionality

in the hydrodynamic radius (from 6.1 to 4.7 nm) was observed, while the absolute molecular weight, determined by multiangle laser light scattering (MALLS), only slightly increased to 87.7 kDa, a clear confirmation of intramolecular cross-linking. The tadpole-like morphology was also visualized

by the TEM images, showing clearly the collapsed PCEMA as the head and a PEO chain as the tail (Figure 2). Moreover, under the appropriate conditions the tadpoles could selfassemble. Thus, in a selective solvent for the tail (DMF/ ethanol: 1/4, v/v), the rigid PCEMA head of the SCNPs 894


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Scheme 16. Synthesis of Organic Nanotubes via the Intramolecular Cross-Linking of a Helically Folded Amphiphilic Poly(mphenyleneethynylene)

poly(m-phenyleneethynylene) of Mw = 24.7 kDa and PDI = 1.35 by Pd(0)-catalyzed polycondensation of diiodocinnamate with trimethylsilylacetylene (TMSA) in the presence of traces of water.84 The helically folded conformation of the polymer was monitored by a solvent denaturation experiment using UV−vis and fluorescence studies in chloroform and acetonitrile. Photoirradiation of a dilute solution of poly(mphenyleneethynylene) in a folding-promoting solvent at 316 nm resulted in the intramolecular 2 + 2 cycloaddition of cinnamate units. The cross-linking process was monitored by following the decrease in the UV absorbance and further confirmed by 1H NMR spectroscopy. As indicated by GPC measurements, a short irradiation time preferably led to intramolecular cross-linking, while longer irradiation produced aggregates. 2.4.4. Photodimerization of Anthracene. Berda et al. recently studied the potential of anthracene 4π + 4π lightinduced cycloaddition as a suitable chemistry to prepare organic nanoparticles.85 Thus, copolymers of methyl methacrylate bearing varying amounts of 9-anthracenylmethyl methacrylate collapsed into SCNPs upon irradiation at 350 nm under dilute conditions (Scheme 17).

aggregated to form superparticles, which upon ultrasonic treatment dissociated back to the individual SCNPs. Synthesis of polymeric monofunctional nanoparticles where the functional groups are fully exposed and prone to further modification is difficult to achieve because of the concealment of the functional group by the cross-linked polymer. Xie et al. recently used cinnamoyl cross-linking chemistry to demonstrate the synthesis of covalent monofunctional nanoparticles by the intramolecular cross-linking of PEO-b-PCEMA block copolymer sparsely grafted on sacrificial silica spheres (Scheme 15).81 For this purpose, the silica surface was first modified with a Y-shaped functional molecule (N,Ndipropargyl-3-aminopropyl) triethoxysilane. Grafting of PEOb-PCEMA on the silica was achieved through Cu(I)-catalyzed azide−alkyne click reaction of azide end-functionalized PEOb-PCEMA with alkynes on the silica surface. The inherent steric repulsion from the grafted chains inhibited subsequent introduction of new polymer chains nearby, leading to a sparsely grafted surface. The PEO-b-PCEMA-grafted silica spheres were characterized by FT-IR, TGA, and TEM measurements. Thermogravimetric analysis studies showed a grafting density of approximately 76 polymer chains per silica sphere. By this strategy the sparse grafting eliminated the possibility of intermolecular cross-linking upon photoirradiation. Thus, the polymer chains could then be intramolecularly connected via photoinduced cross-linking of cinnamoyl groups. Intramolecularly cross-linked nanoparticles were visualized by TEM, showing well-defined nanoparticles sporadically grafted on the silica spheres. HF etching followed by dialysis produced SCNPs with a fully exposed azide functional group. The DLS showed a hydrodynamic radius of 4.1 nm, which is less than that of the precursor PEO-bPCEMA, indicative of the successful intramolecular chain collapse. Further confirmation was obtained from TEM measurements showing the nanoparticles with a size of about 6.0 nm. The reactivity of the monoalkynyl functional single-chain nanoparticles was examined by the reaction of the alkynyl group with azide end-functionalized poly(dimethylaminoethyl methacrylate) (PDMAEMA-N3) to give a PEO-collapsedPCEMA−PDMAEMA hybrid particle. An increase in the hydrodynamic radius from 4.1 to 4.6 nm and a decrease in the retention time confirmed the reactivity of the monofunctional nanoparticle. An interesting example by Hecht et al. demonstrated the construction of organic nanotubes via the intramolecular cross-linking of a helically folded amphiphilic poly(m-phenyleneethynylene).82 Folding of poly(m-phenyleneethynylene) through the π−π stacking of the neighboring unit83 positions the cross-linking moieties (cinnamate groups) close to each other (Scheme 16). For this purpose, the authors prepared

Scheme 17. Intrachain Folding of an Acrylate Polymer via Anthracene Dimerization

The chain folding and consequent nanoparticle formation by dimerization was monitored by UV−vis spectroscopy, triple-detector SEC, and TEM. Slight aggregation due to the interchain cross-links could be observed by the MALLS measurements which are more sensitive to the larger particles. Partial reversibility of the cycloaddition reaction could also be promoted by irradiation at 254 nm, as indicated by the 895


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2.6. Isocyanate−Amine Coupling

reappearance of the characteristic anthracene peak in UV spectra (about 20% of the original value), although curiously no change in the SEC retention time could be observed.

Even though the use of electron-rich benzocyclobutenes broadened the scope of SCNP formation considerably, crosslinking with BCB chemistry was still limited to polymers not bearing heat-sensitive functional groups. Beck et al. introduced a related strategy in which linear polymers bearing pendant isocyanate groups could be cross-linked without the aid of any catalyst at ambient temperature by using diamines as crosslinkers.89 As in the case of BCB chemistry, intramolecular cross-linking occurred without any side products. The precursor polymer backbone of varying isocyanate (5−20%) incorporation was prepared by RAFT-mediated radical copolymerization of methyl methacrylate (MMA) with various vinyl monomers containing the isocyanate group, e.g., isocyanatoethyl methacrylate (ICEMA). The chain collapse was achieved by the continuous dropwise addition of the precursor polymer solution to a dilute solution of 2,2′(ethylenedioxy)diethylamine (Scheme 20). In order to avoid nanoparticle aggregation, the unreacted isocyanate groups at the surface of the nanoparticles were capped with 2-methoxy ethylamine. The degree of cross-linking determined by 1H NMR spectroscopy showed that approximately 75% of the isocyanate groups participated in cross-linking. The formation of nanoparticles by single-chain collapse was monitored, as usual, by size exclusion chromatography, showing a systematic increase in the retention time compared to the parent polymer chain, highly correlated to the relative amount of isocyanate groups in the precursor. However, in contrast to BCB-based nanoparticles, a slight increase in the polydispersity for the nanoparticles compared to the precursor polymer was observed, maybe due to the two-component cross-linking system.

2.5. Tetrazine−Norbornene Reaction

The catalyst-free inverse electron demand pericyclic cycloaddition reaction between a tetrazine and a strained olefin fulfills all the requirements of a “click” reaction (Scheme 18). It is atom efficient and can be performed under mild reaction conditions, offering quantitative conversion and no byproducts.86,87 Scheme 18. Tetrazine Norbornene Inverse Electron Demand DA Ligation

This appealing methodolgy was recently studied by Hansell et al. for the single-chain collapse of RAFT-derived linear polystyrene-containing pendant norbornene groups (Scheme 19).88 The reaction under slow addition of the norbornenecontaining copolymer with a bis-tetrazine cross-linker was examined by 1H NMR spectroscopy and SEC, among other typical characterization methods. Notably, the polymers with the higher norbornene ratios (20%) showed greater decrease of apparent molecular weights, while copolymers with just 5% norbornene loading showed even higher molecular weights after cross-linking. The fact that the control polymer, without norbornene, also showed increased molecular weights determined that the cause for this deleterious consequence was prolonged heating (24 h), which induced degradation and thiol−norbornene coupling (the thiol originates from the polymer end group). The drawback of having to use high temperatures may be solved by having a more reactive tetrazine cross-linker; however, it is important to be aware, as in many other SNCP procedures, that aggregation or side reactions do not occur in order to achieve well-defined particles.

2.7. Bergman Cyclization

The Bergman cyclization is an intramolecular cyclization of enediyne derivatives, which occurs under thermal or photochemical conditions.90 The cyclization occurs through the formation of a highly reactive aromatic 1,4-diradical intermediate (Scheme 21). Although this chemistry is not very common in polymer synthesis,91 recently Hu and co-workers exploited the use Bergman cyclization for the intramolecular folding of singlechain polymers (Scheme 22).92 Like in the case BCB chemistry, the thermal triggering nature of Bergman

Scheme 19. Tetrazine−Norbornene-Mediated Cross-Linking of a PS Precursor

896


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Scheme 20. Intrachain Cross-Linking by Urea Formation

h. The resulting collapsed polymer chain showed a decrease of up to 26% in the apparent molecular weight. Control experiments with polymers that did not include the enediyne monomer showed no change after UV irradiation, supporting the assumption that the chain collapse was caused by photoBergman cyclization. More evidence for the formation of the nanoparticles was obtained from SEC, 1H NMR, UV−vis, and Raman spectroscopies. AFM analysis of these nanoparticles showed very distinguishable particles in the range of 10 nm that were in agreement with the data obtained by SEC measurements. The method was generalized for other similar acrylate-based monomers such as methyl acrylate, ethyl acrylate, and tert-butyl acrylate. These linear polymers exhibited different Tg’s at relatively low temperatures (depending on the polymer from −15 to 60 °C) and a characteristic exothermic peak above 300 °C due to the onset of the thermal Bergman cyclization reaction. On the other hand, the corresponding nanoparticles displayed no characteristic glass transition, suggesting decreased chain mobility for the cross-linked nanoparticles, and no exothermic peak, concomitant with the absence of enediyne moieties. A salient feature of this chemistry is the fact that the crosslink is not just a link between two monomers as in most other examples discussed here but a “zipper”-like moiety, where several monomers may be bound simultaneously, maybe creating an even more compact structure. Although the exact morphology of the cross-link within the SCNP was not detailed in this paper, the authors mentioned that irradiation of the enediyne monomer itself afforded dimers, trimers, and mainly tetramers. The same group reported on the synthesis of hydrophilic nanoparticles via the Bergman cyclization and their use as a size-tunable nanoreactor to fabricate and encapsulate ZnS quantum dots (QDs) (Scheme 24).95 The QDs were generated by encapsulation of the Zn2+ ions followed by the addition of sodium sulfide. The crystalline QDs formed were characterized by high-resolution TEM. This one-pot synthetic strategy allows for the synthesis and assembling of a precise number of QDs in the reactor just by adjusting the size of the polymer precursor. Unfortunately,

Scheme 21. Bergman Cyclization Polymerization

cyclization (nearly at 150 °C) and the rapid coupling of the radical generated meets the critical factors required for the continuous addition strategy developed by Hawker et al.46 The versatility of the Bergman cyclization was demonstrated by incorporating the enediyne in two ways, i.e., by attaching the enediyne units to the monomer (Scheme 22a) or by attaching it to the preformed polymer via a postpolymerization functionalization technique (Scheme 22b). In order to avoid any unwanted cyclization during the polymerization, silyl-protected alkynes were used. Either way, following a continuous addition strategy, the folding of single-chain polymers was successfully accomplished. GPC measurements evidenced the intramolecular cross-linking showing a significant drop in the apparent molecular weight up to 47% depending on the percentage of enediyne units incorporated into the polymer chain. The 1H NMR, FT-IR, and AFM measurements provided further evidence for the single-chain collapse. The use of thermal cyclization may sometimes be detrimental for thermally sensitive polymers; thus, it was appealing to probe an alternative method for the Bergmann cyclization by irradiation with UV light at room temperature.93 Therefore, Hu and co-workers explored the photoinduced Bergman cyclization for folding acrylate based polymers.94 A precursor copolymer was prepared by single electron transfer living radical polymerization (SET-LRP) of n-butyl acrylate and an enediyne-containing acrylate (Scheme 23). The incorporation of enediyne moieties in the polymer chain was evidenced by the appearance of the characteristic aromatic protons between 6.8 and 8.5 ppm. The linear polymer precursor was irradiated using a medium-pressure Hg lamp at a dilute concentration (0.06 mg/mL) in toluene for 6 897


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Scheme 22. Use of Bergman Cyclization for the Intramolecular Cross-Linking of Linear Polymer Chains

Scheme 23. Cross-Linking of Acrylate Polymer via Photoinduced Bergman Cyclization

were produced.96 The authors followed a three-step strategy to prepare the C dot in which a polymethacrylate prepared via the single-electron transfer radical polymerization was postfuctionalized with varying amounts of enediyne units via transesterification with 4-(2-(2-(trimethylsilyl)ethynyl)-

the comparison of QD properties before and after single-chain collapse by Bergman cyclization was not reported. Continuing the Bergman cyclization strategy, size-tunable photoluminescent carbon dots (C dots) via a “bijective” approach, where each C dot is generated from one SCNP, 898


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Scheme 24. Synthesis of ZnS Quantum Dots within SCNP Prepared via Bergman Cyclization

materials. In 2002, the Sharpless109 and Meldal110 groups independently discovered that copper catalysis accelerates the rate of triazole formation by approximately 7 orders of magnitude compared to the uncatalyzed reaction. This variant of the Huisgen cycloaddition reactions was termed the Cucatalyzed azide−alkyne reaction (CuAAC). At the same time Sharpless et al. coined the term click chemistry to describe a set of chemical reactions that efficiently link two components in high yield with high regiospecifity and minimal byproduct. The CuAAC reaction is known as the archetype version of click reactions.111 In the quest for near-perfect chemical reactions, the click reaction has emerged as an ideal bioorthogonal protocol for the preparation of rich chemical diversity.112−115 1,2,3-Triazole moieties are attractive connecting units because they have advantageous properties such as high chemical stability, generally inert to severe hydrolytic, oxidizing, and reducing conditions, and they exhibit large dipole moment (4.8−5.6 D) and aromatic character.116,117 In addition, they are highly stable to metabolic degradation and capable of hydrogen bonding, which can be favorable in the binding of molecular targets. 118 For example, as a bioconjugation tool, the use of click chemistry has been extended to contemporary biomedical studies,119 especially in chemical biology120−125 where DNA or proteins are functionalized for imaging purposes and detection.126−129 Since its lucid formulation, the versatility of the click reaction involving Huisgen azide−alkyne couplings has gained considerable momentum in a wide range of synthetic domains. Moreover, within the realm of polymer science, it has become so ubiquitous that a very large amount of novel polymers are now being made or modified by click reactions130 In particular, these new methodologies open new applications for functional macromolecular materials, especially for biocompatible polymerization protocols. The convenience of the CuAAC as described above has led many researchers to use it for polymer collapse. Its high orthogonality to other reactions, typical high conversions, and the synthetic simplicity for the introduction of the coupling partners have made it the method of choice to manipulate macromolecular structure in many research groups.

phenyl)but-3-yn-1-ol. Single-chain folding followed by carbonization produced the photoluminescent C dots of defined size as indicated by TEM measurements. A comparison with the uncross-linked polymer to discern how the single-chain collapse affects the process was also not reported in this study. 2.8. CuI-Mediated Click Chemistry

The assembly of 1,2,3-triazoles by 1,3-dipolar cycloaddition of azides and alkynes was discovered more than a century ago in 189397 and thoroughly studied in the 1960s by Huisgen.98 Since then these reactions have been referred to as Huisgen cycloaddition reactions (Scheme 25). Although the Huisgen Scheme 25. 1,3-Dipolar Cycloaddition of Azides and Alkynes

cycloaddition reaction is thermodynamically favored with a relatively large driving force (ΔG° ≈ 61 kcal/mol), the high activation energy barrier99 renders it with poor regioselectivity and relatively low chemical yield. Thereafter, Wittig and Krebs presented a new efficient synthetic procedure to obtain a single product of 1,2,3-triazole using cycloalkyne with azidobenzene.100 The enhanced reactivity compared to linear unstrained alkynes was explained by the high ring-strain energy of cyclooctyne (∼18 kcal/mol), which is partially released after the reaction.101 Houk et al. reported that the stress of the cycloalkyne leads to a lower transition state distortion of the azide and the alkyne,102 significantly lowering the energy barrier of the cycloaddition reaction.103 Experimental as well as computational studies revealed that fluorine substitution also had a significant effect on reactivity at ambient temperature,104,105 lowering the electronic and free energy activation barrier106 and validating the potential of this strategy in biomedical studies.107,108 However, this method was limited to bicyclic triazole products and required specialized tailormade functional cyclooctyne 899


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Scheme 26. Synthetic Route to Bioconjugable Single-Chain Nanoparticles via Click Chemistry

Scheme 27. General Click Chemistry Route to SCNPs Using Dialkyne Cross-Linkers

CuI as a catalyst at room temperature by the continuous addition protocol produced well-defined nanoparticles quantitatively. As expected, a significant reduction in the hydrodynamic volume and a reduction in the apparent molecular weight of about 40% were observed by size exclusion chromatography. Further evidence for the formation of welldefined nanoparticles was obtained from TEM images that showed well-defined spherical nanoparticles in the range of 5−20 nm. The utility of click chemistry for the preparation of potentially useful functionalized nanoparticles was exemplified by the reaction of leftover excess azide groups on the nanoparticles with propargyl glycine.

2.8.1. Azide−Alkyne Click Chemistry. In 2008, Luzuriaga et al. utilized for the first time the copper(I)catalyzed click chemistry for an efficient and convenient synthesis of bioconjugable single-chain nanoparticles under mild reaction conditions (Scheme 26).131 Advantages of using metal-mediated click chemistry include the easy incorporation of coupling partners in the polymer chain and a highly efficient and selective cross-linking that can be conducted at room temperature. The authors prepared RAFT-derived terpolymers of methyl methacrylate, 3-azidopropyl methacrylate, and 3-(trimethylsilyl)propyn-1-yl methacryalate. Deprotection of the propargyl units followed by cross-linking using 900


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Scheme 28. Synthesis of a Versatile SCNP with a Predefined Amount of Functional Monomers

Scheme 29. Synthetic Route to GdIII-Loaded SCNP

the easy synthesis of SCNPs with different chemical structure and functionality, which naturally affects its physicochemical characteristics and hence potential applications.132 Pomposo et al. reported another interesting application of azide−alkyne click chemistry.133 In their work, a combination of controlled RAFT polymerization and azide−alkyne click chemistry was used to produce ultrasmall polystyrene functional nanoparticles (Scheme 28). A versatile copolymer nanoparticle precursor with a predefined amount of functional monomers was synthesized by controlled statistical random

In an expansion of the scope of this method, various copolymers bearing pendant chloro groups were prepared by RAFT polymerization of alkyl chloride-containing monomers with styrene, alkyl methacrylates, 4-styrenesulfonate, Nisopropylacrylamide, etc. Replacement of the chloro functionality by azide groups and subsequent cross-linking via dialkyne cross-linkers furnished nanoparticles in the range of 3−20 nm with various types of polymers (Scheme 27). The nanoparticles formed were characterized by FT-IR, SEC, DLS ,and AFM measurements. The general method presented allows 901


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Scheme 30. Formation of PS SCNPs via CuAAC Using Diethynyl Benzene Cross-Linker

Scheme 31. Formation of Shaped SCNPs by Folding of a PS Precursor with Precisely Positioned Functional Groups

approximately 4.2 ± 0.9 nm by DLS analysis in THF. However, AFM measurements showed the dry nanoparticles height to be around 2.5 nm. According to the authors this difference may be due to the partial swelling of nanoparticles in solution. Actually the height measured for soft organic nanoparticles by AFM has been shown to be very dependent on the rigidity of the polymer analyzed; thus, an alternative explanation may be a slight flattening of the SCNP upon

copolymerization of 4-chloromethylstyrene and 2-methylacrylic acid 3-trimethylsilanyl-prop-2-ynyl ester. Partial azidation of the chloromethylstyrene monomers with sodium azide in DMF resulted in well-defined terpolymers having chloride, azide, and alkyne functionalities. Upon cross-linking under appropriate conditions, ultrasmall polystyrene nanoparticles bearing chloromethyl groups were obtained. The hydrodynamic diameters of these nanoaprticles were found to be 902


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Scheme 32. Synthetic Route for SCNPs Derived from NIPAM-Based Terpolymer Using CuAAC

deposition on the surface.49 The usefulness of these multifunctional SCNPs was demonstrated by preparing nanocomposites with elastomeric polymers that showed improved rheology behavior and by exhibiting useful fluorescence emission by employing the right chemistry. Another advantage of this method is that the excess chloromethyl functionality can be further functionalized and used for various purposes. In 2010, Perez-Baena et al. prepared nanoparticles bearing GdIII complexes as potential MRI image contrast agents.134 For this purpose a polyacryate bearing 10% chloroethyl groups along the polymer chain was prepared as discussed earlier by the controlled RAFT-mediated copolymerization of tert-butyl methacrylate and 2-chloroethyl methacrylate. Subsequent azidation of the chloroethyl group by using sodium azide in DMF produced an azide-bearing polyacrylate of Mw = 50 kDa and polydispersity 1.08. The authors designed and synthesized a cross-linker containing two terminal alkyne groups and a diethylenetriaminepentaacetic acid (DTPA) unit to complex the GdIII ions. Thus, the azide polyacrylate was reacted with the cross-linker using CuI-catalyzed cycloaddition under dilute conditions. Quantitative intrachain folding was achieved, monitored by the disappearance of the azide asymmetric stretching band at 2103 cm−1 (FT-IR). Deprotection of tert-butyl groups by using trifluoroacetic acid (TFA) at 40 °C yielded water-soluble nanoparticles (a requirement for biological imaging applications). The GdIII ions were then loaded to the nanoparticles by reaction with GdCl3·6H2O. Inductively coupled plasma (ICP) analysis showed 5.6 -wt % GdIII content, which corresponds to almost complete occupancy of the DTPA chelating sites in the nanoparticles (Scheme 29). These Gd-loaded rigid nanoparticles showed a relaxivity of 6.78 mM−1 s−1 on a per Gd basis, two times more efficient compared to the commercially available GdIIIbased image contrast agent, Magnevist. Another example of a bifunctional cross-linker-mediated approach using click chemistry was reported by Cengiz et al. in 2011(Scheme 30).135 In their work, diethynylbenzene and

1,10-dipropargyloxy decane were used as bifunctional crosslinkers for the single-chain folding of an azide-functionalized polystyrene copolymer. The precursor polymer was prepared by free radical polymerization of styrene and 4-chloromethylstyrene followed by nucleophilic substitution with azide. Intramolecular cycloaddition was confirmed by following the disappearance of the N3 band at 2095 cm−1 in FT-IR as well as the shift of the 1H NMR peak at 4.2 ppm that corresponds to the methylene adjacent to the azido group. Further evidence for the chain collapse was obtained from the DSC measurements, where a shift in the Tg value from 95 to 132 °C was observed. In addition, the expected decrease in the apparent molecular weight was observed by SEC. Most significantly, the shear-rate-dependent viscosity measured at 25 °C for these nanoparticles showed an order of magnitude increase compared to the precursor polymer, an important change in properties due to the particle-like nature of the collapsed polymer chain. Lutz and co-workers introduced an ingenious and a very convenient strategy for the synthesis of wide variety of foldable linear polymer chains of well-defined primary structure.136 In contrast to most works in the SCNP area, in these systems the cross-linking functionalities can be precisely positioned; thus, P-shaped, Q-shaped, 8-shaped, and α-shaped cyclic structures could be made by a coppercatalyzed azide−alkyne coupling reaction (or Glaser coupling for the α-shape) (Scheme 31). The compaction parameter (SEC maximum peak value for the linear polymer divided by the value for the folded polymer) was compared between all shapes, showing the greatest compression for the 8-shape polymer, although this is not so surprising taking into account that two cross-links occurred in these molecules compared to just one in all others. Nonetheless, the development of this important proof of concept is essential if the emulation of the unique properties derived from folding in biopolymers is to be achieved. Recently, Ormategui et al. used the azide−alkyne click chemistry for the synthesis of thermoresponsive polymer 903


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Scheme 33. SCNPs Formation via Glaser−Hay Coupling Chemistry

Scheme 34. SCNPs Formation Using Photoactivated Thiol−Yne Cross-Linking Chemistry

nanoparticles via chain collapse of an N-isopropylacrylamide (NIPAM)-based terpolymer (Scheme 32).137 RAFT-derived poly(N-isopropylacrylamide-co-3-chloroethyl methacrylate-co3-(trimethylsilyl)propyn-1yl methacrylate) terpolymers containing equimolar amounts of protected alkyne and halogenated monomers were synthesized using S-1-dodecylS-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate (DDMAT) as the chain transfer agent and AIBN as radical initiator. The halogenated monomer units were then converted to azides by the reaction with sodium azide in DMF. Deprotection of alkynyl functional groups followed by the intramolecular click reaction produced well-defined nanoparticles. Successful crosslinking through azide−alkyne click reaction was conveniently monitored by FT-IR that showed the disappearance of azide and alkyne bands, respectively, at 2103 and 2187 cm−1. Further evidence for the conformational change in the polymer chains as a consequence of chain collapse was obtained from matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), AFM, and diffusion-ordered spectroscopy (DOSY) experiments. Size determination by DLS experiments revealed the effect of chain collapse directly correlated to the amount of crosslinker. The thermal phase transition behavior of the nanoparticles and the precursor polymers was also studied by using temperature-dependent turbidity measurements that showed a gradual decrease in the transmittance for nanoparticles compared to that of the linear precursor polymer. 2.8.2. Glaser−Hay Coupling. Another click-type crosslinking chemistry used in single-chain folding is the coppercatalyzed alkyne homocoupling Glaser-Hay coupling. Sanchez-

Sanchez et al. used this C−C click chemistry recently for the preparation of nanoparticles from poly(methyl methacrylateco-propargyl acrylate) (poly(MMA-co-PgA) (Scheme 33).138 The use of alkyne groups to effect cross-linking is very attractive, due to the orthogonality of this reaction and the tolerance to many other functional groups throughout the coupling process. However, polymers bearing free propargyl pendant groups are difficult to synthesize by usual RAFT polymerization due to unwanted secondary reactions. The authors succeeded to circumvent this difficulty by using a redox-initiated RAFT copolymerization of MMA and propargyl acrylate (PgA) at room temperature, which significantly decreased side reactions. Accurate control over the composition, molecular weight, and polydispersity of poly(MMA-co-PgA) was observed with a maximum PgA content of 35%. The naked propargyl units were then coupled by the Glaser−Hay method under dilute conditions. The SCNPs were characterized by FT-IR, NMR, and SEC measurements. To note in this work is the use of relatively simple building blocks and high atom economy, which should foment the relatively rapid development of these types of nanoparticles. 2.9. Thiol−Yne Click Chemistry

Photoactivated thiol−ene and thiol−yne chemistry is yet another type of click chemistry that has recently received much attention as a powerful method for providing access to various advanced macromolecular architectures. High functional group tolerance of the alkyne group and rapid kinetics under mild reaction conditions without the need for a metal 904


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Scheme 35. Route for SCNPs Formation by Amine Quaternization Cross-Linking Using Diiodobutane

Figure 3. TEM images of (a) SCNPs of PDMAEMA-b-PS, (b) SCNPs of PDMAEMA74-b-PS297, and (d) SCNPs of PDMAEMA15-b-PS151 prepared by casting from THF solutions. (c) Dynamic light scattering curves of SCNPs of PDMAEMA74-b-PS297 and SCNPs of PDMAEMA74-bPS297.

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Scheme 36. Amphiphilic Monotethered SCNPs Preparation by Amine Quaternization

2.10. Amine Quaternization

catalyst make the thiol−ene/yne chemistry an effective tool for cross-linking chemistries. Accordingly, the group of Pomposo recently studied the use of photoactivated thiol− ene/yne chemistry for the intramolecular chain folding of acrylate copolymers bearing unprotected alkene/alkyne functional groups (Scheme 34). To avoid side reactions the authors followed the redox-initiated RAFT polymerization technique discussed above to prepare a precursor copolymer of molar mass around 125 kDa bearing nearly 20 mol % of naked alkene/alkyne groups.139 Intramolecular thiol−yne reaction using 3,6-dioxa-1,8-octane-dithiol as homobifunctional cross-linker and 2,2-dimethoxy-2-phenyl acetophenone as photoinitiator produced single-chain nanoparticles with nearly globular morphology. The single-chain collapse process was followed by SEC/MALLS measurements. Characterization of SCNPs by SEC, small-angle X-ray scattering (SAXS), and DLS revealed that the cross-linking by thiol− yne chemistry leads to a higher compaction degree than the thiol−ene chemistry. This again was probably to be expected, taking into account that the thiol−yne chemistry brings about twice as many cross-links. Molecular dynamic (MD) simulations accompanied the experimental work. Notably, the SAXS results of these single-chain nanoparticles revealed a scaling exponent of v = 0.37 which is very close to the value assigned for globular objects. The authors claimed that the formation of nearly globular nanoparticles can be attributed to the formation of long-range intrachain loops formed by the use of relatively long cross-linkers.

The quaternization of dimethyl amine-bearing polymers using a dihaloalkane can be used as a cross-linking method, particularly in the synthesis of amphiphilic SCNPs.140 Wen et al. exploited the use of quaternization chemistry to prepare tadpole-like single-chain nanoparticles that are capable of selfassembling to form micelles.141 These tadpole-like single-chain nanoparticles are usually prepared by the collapse of one block of a diblock polymer (vide supra). In their work, Wen et al. used RAFT polymerization-derived poly(2(dimethylamino)ethyl methacrylate)-block-polystyrene (PDMAEMA-b-PS) as the precursor polymer. Cross-linking was achieved via quartenization of pendant dimethylamino groups by 1,4-diiodobutane (Scheme 35). Under dilute conditions, cross-linking occurred intramolecularly to afford the charged SCNP. The degree of cross-linking in the singlechain collapse was controlled by the amount of cross-linker added. The increased retention time relative to the decrease in the hydrodynamic volume and the apparent molecular weight monitored by size exclusion chromatography confirmed the contraction of the PDMAEMA block. The polar collapsed block together with the polystyrene random coil block formed macromolecular amphiphiles that led to the formation of micelles and vesicles depending on the size of the SCNP. In aqueous solution, self-assembly resulted in micelles with the hydrophilic nanoparticle in the corona and the tethered polystyrene chain in the core, while in cyclohexane the opposite was observed. As a curious note, 906


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Scheme 37. SCNPs Formation via Pyridine Quaternization with Cross-Linker

Scheme 38. SCNPs Preparation by Cross-Linking of Pendant Propargyl Groups

increasing the quaternization degree to 60%, the CMC value was raised to 0.033 mg/mL. The authors also demonstrated the use of these SCNPs as stabilizers for the suspension polymerization of styrene. TEM measurements showed that the average size of the polystyrene colloidal particles decreases with increasing the amount of stabilizers. Finally, the cleavage of the disulfide linkage of the monotethered SCNPs using tri-n-butyl phosphine afforded reactive thiol functional groups on the surface of the colloidal particles. The thiol groups on the surface were further modified by a disulfide exchange reaction with 2,2′-dipyridyl disulfide and used as a platform for further functionalization. The quaternization of pyridine with suitable alkyl halides is another widely used strategy for cross-linking of pyridinecontaining polymers.144−146 For example, Zhu and co-workers reported the use of pyridine quaternization chemistry for the large-scale synthesis of single-chain Janus nanoparticles from a commercially available ABC triblock copolymer, with a long A hydrophobic polystyrene block, a short B poly-2-vinylpyridine (P2VP) block, and a long C hydrophilic polyethylene glycol block (Scheme 37) .147 In their work, the middle P2VP block was selectively cross-linked via quaternization using 1,4dibromobutane (DBB) as the cross-linker in DMF. An efficient intramolecular cross-linking of the P2VP block was observed at relatively high polymer concentrations (20 mg/ mL). The lack of intermolecular binding at such a high concentration of the polymer precursor was attributed to steric shielding of the PS and PEO end blocks. Moreover, the high effective molarity of the grafted quaternization product was essential to enhance the intramolecular cross-linking through the second quaternization. Upon cross-linking, the absolute molecular weight (304 kDa and a PDI of 1.15) of the copolymer was slightly increased to 352 kDa, probably due to the added weight of the DBB cross-linker. DLS measurements showed two distinct peaks that corresponded to unimolecular Janus nanoparticles of Rh = 8.7 nm and large aggregates in the range of 50−115 nm. Upon dilution, the intensity and size of the larger assemblies were reduced, indicating that these are actually self-assembled moieties that can be dispersed below a critical concentration. The morphology of the Janus nanoparticle was visualized by

the authors referred to the micelles formed by the collapsed polymers as “bunchy” micelles or strawberry-like micelles, probably due to some of the images obtained by TEM which resembled the texture of the strawberry exterior (Figure 3). The self-assembly of these amphiphilic SCNPs (or shape amphiphiles) was found to be greatly influenced by their surface charge density. Wen et al. showed that by varying the amount of 1,4-diiodobutane, the charge density on the nanoparticles can be altered.142 Significantly, upon increasing the degree of quaternization the average size of the micelles increased. This was attributed to a repulsion of positively charged nanoparticles upon increasing the charge density. Therefore, in order to minimize the free energy of the micelles, the equilibrium micellar morphologies will favor larger aggregates. In contrast, the morphology of the micelles prepared from shape amphiphiles with smaller single-chain nanoparticles afforded a mixture of wormlike cylinders and vesicles upon increasing the surface charge density. Quite recently, Zhang and Zhao explored the utility of similar amphiphilic monotethered SCNPs for the synthesis and stabilization of surface-tunable polystyrene colloidal particles.143 The monotethered SCNPs were prepared from a diblock copolymer consisting of poly(ε-caprolactone) (PCL) and PDMAEMA linked by a redox-responsive disulfide linkage. As in previous work, the intramolecular chain collapse of PDMAEMA block via amine quaternization using 1,4diiodobutane under dilute conditions (0.2 mg/mL) afforded the SCNPs (Scheme 36). Their formation was monitored as usual by SEC, DLS, and 1 H NMR measurements. Furthermore, TEM measurements revealed the formation of SCNPs in the range of 5−10 nm, which was in good agreement with the average hydrodynamic diameter (6 nm) observed by DLS. As expected, the amphiphilic SCNPs showed surfactant-like properties in water. A comparative study of the surfactant behavior of the linear diblock copolymer (the precursor) and the monotethered SCNPs (15% degree of cross-linking) exhibited a relatively higher CMC value (0.022 mg/mL) for the SCNPs compared to the linear block copolymer (0.014 mg/mL). The diminished tendency of the nanoparticles to form micelles was attributed to the repulsion of positively charged SCNPs. Upon 907


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Scheme 39. Single-Chain Collapse of PGA by Amide Bond Formation

viscosity studies showed that intramolecular cross-linking was preferred at lower concentration of cross-linkers leading to contraction of the PGA chain. In contrast, a higher amount of cross-linker resulted in the formation of aggregates via intermolecular cross-linking. In an alternative strategy, Pomposo and co-workers reported intrachain collapse based on amide formation using hexamethylene diisocyanate (HMDI) as a bifunctional crosslinker (Scheme 40).151 The precursor polymers bearing β-

TEM imaging. Regarding the cross-linking chemistry used, it must be noted that an excess of 8:1 of the dibromo crosslinker to vinylpyridine was needed in order to efficiently collapse the middle block; this is a consequence of the relatively dilute conditions that need greater amounts of crosslinker for the reaction to happen at an appreciable rate (the reactions at 30 °C took 2 weeks, while heating to 100 °C still required one full day for reaction completion at the higher concentrations). Chen and co-workers reported another example that involved the collapse of the poly-4-vinylpyridine (P4VP) block of a PMMA-b-P4VP diblock copolymer using propargyl bromide as cross-linker (Scheme 38).148 In this case the crosslinking occurred through a “spontaneous” polymerization of the propargyl groups grafted to the main polymer chain.149 Unlike the above case, a low polymer concentration was necessary to achieve intramolecular cross-linking. An attempt to cross-link at higher concentrations (5 mg/mL) favored intermolecular cross-linking. SEC, NMR, and TEM analyses were used to further characterize the SCNPs obtained. The amphiphilic tadpole-like single-chain nanoparticles (TSCPNs) produced by this method exhibited excellent emulsifying performance in an oil/water system even with content as low as 0.0075% vs the total weight of the oil and water. These emulsions presented long-term stability, e.g., the water layer was not separated even after a period of 4 months. The application of these extremely stable emulsions having large interfacial area was demonstrated by performing a reduction of p-nitroaniline with sodium sulfide at the interface of the oil/water emulsion giving >91% conversion in 3 h reaction time. The drawback for this collapse method is that the crosslinking chemistry is not well understood, and it is not clear what the mechanism of the alkyne self-polymerization entails. Having said this, appropriate characterization of the purported polyacetylene cross-linking unit could bring about interesting properties due to its extended conjugated system.

Scheme 40. SCNPs Formation via HMDI Cross-Linker

ketoester units were prepared by the RAFT copolymerization of MMA and 2-acetoacetoxy-ethyl methacrylate (AEM) using 2-cyanoprop-2-yl-dithiobenzoate (CPDB) as chain transfer agent and AIBN as radical initiator. Polymers with two different molecular weights, each containing 30 mol % of βacetoacetoxy units, were used for this purpose. The folding was achieved under high dilution (1 mg/mL) via reaction of the isocyanate group of the cross-linker with the β-ketoester units of the polymer chain in the presence of 1,5diazabicyclo[4,3,0]non-5-ene (DBN) as catalyst. The compaction was verified by SEC/SLS and DLS experiments. More evidence for the chain-folding process was obtained from FTIR and 1H NMR spectroscopies. The appearance of the IR band corresponding to the NH vibration between 3100 and 3700 cm−1 confirmed the amide bond formation. Moreover, the collapsed polymer was also characterized by elemental analysis, which indicated an approximate degree of crosslinking of 97%. Thus, these two different strategies display important models for the use of the biologically relevant amide bond to effect the single-chain collapse of synthetic polymers.

2.11. Amide Formation

In 2008, Borbély and co-workers reported the formation of water-soluble nanoparticles via the intramolecular amidation of biosynthetic poly-γ-glutamic acid (PGA) (Scheme 39).150 The precursor PGA was produced from Bacillus licheniformis under appropriate cultivation conditions. The cross-linking of PGA (100 mg dissolved in 10 mL of distilled water at pH 4.5) was carried out with different amounts of 2,2′(ethylenedioxy)bis(ethylamine) cross-linker (10%, 25%, 50%, and 100% relative to the free carboxyl groups) in the presence of a water-soluble carbodiimide (CDI). The structure of the cross-linked PGA was studied by 1H NMR spectroscopy. The degree of cross-linking was calculated from the ratio of integral values of the α-CH (PGA) at δ = 4.25 ppm and 1CH2 (diamine cross-linker) at δ = 3.75 ppm. SEC measurements showed a decrease in the hydrodynamic volume upon increasing the degree of cross-linking. The TEM micrographs showed the nanoparticles of the size in the range of 20−90 nm. Further investigation using DLS and

2.12. Michael Addition Reaction

Pomposo and co-workers also reported on the construction of transient-binding protein-mimetic nano-objects via Michael 908


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demonstrated a possible application in the combined delivery of dermal protective and anticancer bioactive cargos.153

addition cross-linking of RAFT-derived acrylate copolymers.152 The polymer precursor bearing randomly distributed β-ketoester functionalities (Michael donors) was prepared by RAFT polymerization of MMA and AEM (vide supra). Intramolecular cross-linking of these copolymers was then achieved under dilute conditions (1 mg/mL) using multifunctional acrylates (Michael acceptor) as cross-linkers and potassium hydroxide as catalyst (Scheme 41). Most

2.13. Nitrene Insertion/Coupling

Wang and co-workers used sulfonyl azide copolymers for the intramolecular folding of single-polymer chains.154 The precursor polymer was prepared by RAFT-mediated copolymerization of 4-styrenesulfonyl azide with various vinyl monomers such as styrene, methyl methacrylate, etc. The chain collapse mechanism involved the formation of a nitrene intermediate via the thermal decomposition of pendant sulfonyl azide moieties (Scheme 42). The nitrene intermediate thus generated readily inserts into C−H bonds, generating the intramolecular bond.155,156 The authors followed the continuous addition strategy46 at a concentration of 0.1−1.0 M in benzyl ether at 190 °C. A decrease in the apparent molecular weight of the cross-linked polymer compared to the precursor polymer chain was found by SEC analysis, indicating SCNP formation. As typical for these processes, the reduction of the hydrodynamic volume was found to be dependent on the amount of sulfonyl azide groups in the precursor polymer chain. While the polymer precursor bearing 5% sulfonyl azide groups showed a reduction in the hydrodynamic radius by 17%, the one with 20% sulfonyl azide groups was reduced by 56%. The morphology of the nanoparticles was also studied by AFM and TEM, which showed nanoparticles in the range of 14−15 nm. Highly reactive nitrene intermediates may also be obtained by azide photolysis at ambient temperature. Upon irradiation with UV light, the azide groups decompose to produce nitrenes that may be used for C−H insertion chemistry. Accordingly, this method has been used for cross-linking of polymers in various applications.157−160 Relevant to this review, Li et al. recently reported on the single-chain collapse of polystyrenes bearing pendant azide functionalities.161 The precursor polymers were synthesized by RAFT-mediated copolymerization of 4-vinyl benzyl azide with styrene. Intramolecular cross-linking was carried out by irradiating a precursor chloroform solution (1 mg/mL) with UV light at 365 nm (Scheme 43). Upon increasing the UV irradiation time, a decrease in the characteristic azide band at 2094 cm−1 band, monitored by FT-IR spectroscopy, indicated the successful azide decomposition. Further evidence for the cross-linking was obtained from 1H NMR spectroscopy, showing the disappearance of the methylene protons at 4.26 ppm adjacent to the azide group. The reduction in the apparent molecular weight proportional to the original amount of azide groups confirmed the chain collapse through intramolecular cross-linking. Due to the reduced segmental chain mobility, the Tg of the nanoparticles also rose

Scheme 41. SCNPs via Michael Addition of Multifunctional Acrylate Cross-Linker

interestingly, the study determined that the triacrylate was found to be the optimum cross-linker, while tetra-, penta-, and hexa-acrylate cross-linkers failed to give well-defined nanostructures, giving gels instead. Thus, using the triacrylate, the reduction in the hydrodynamic radius determined by SEC together with the unchanged value of absolute molecular mass determined by SLS measurements confirmed the exclusive intramolecular cross-linking. The actual structure in solution of the nanocarrier experimentally examined by small angle neutron scattering (SANS) in combination with MD simulations indicated the formation partially folded proteinslike structures. Use of these nano-objects as vitamin B9 nanocarriers in water at neutral pH exhibited excellent drugreleasing activity, with 41% weight loading and total release in 5−6 h. In the same year, the authors employed this crosslinking chemistry for the synthesis of single-chain nanoobjects mimicking intrinsically disordered proteins (IDPs) and

Scheme 42. Single-Chain Collapse of Sufonyl Azide PS via Nitrene Insertion

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Scheme 43. Cross-Linking of PS via Photolysis of Azide Groups

Scheme 44. Preparation of SCNP by Metathesis of Terminal Olefin Pendant Groups

Scheme 45. Preparation of SCNP by Tandem ROMP-RCM Protocol

consistently with the amount of azide in the precursor

2.14. Olefin Metathesis

polymer. TEM showed an average nanoparticle diameter in

Recent developments in olefin metathesis polymerizations have made this reaction the method of choice for the construction of novel macromolecular structures.162−164 Besides the robustness and stability of the carbon−carbon double bond, one of the advantages of using olefin metathesis is that the olefin functionality retained in the metathesis product may be used for further functionalization. More

the range of 5 nm. The authors also expanded their work by making fluorescent nanoparticles through a common click chemistry approach taking advantage of the unreacted azide groups in the SCNP. 910


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Scheme 46. SCNP Formation via Electrochemical Oxidative Coupling of proDOT Side Chain

nanoparticles. The authors demonstrated the versatility of this strategy by preparing copolymers of various functional groups by ROMP and subsequent cross-linking via RCM; thus, both organic and water-soluble tunable SCNPs could be easily made even at multigram scale. Moreover, the living nature of the ROMP allowed for the synthesis of useful monofunctional nanoparticles. The authors finally showed the ability of these SCNPs to carry multiple fluorescent probes with increased photostability and their use in bioimaging applications.

specifically for cross-linking purposes, the only byproduct formed during the cross-metathesis of terminal alkenes is ethylene gas, which may be easily removed. The first example of organic nanoparticle formation using olefin metathesis cross-links was carried out by tying the ends of an olefinterminated dendrimer. The nanoparticle study was inspired by Zimmerman’s monomolecular imprinted dendrimers,47,165 which led to the realization that this strategy could be used for dendrimer collapse and rigidification. In this work, the relationship between size and cross-link percentage was clearly determined for a series of dendrimers.49 Even though the generation of the organic nanoparticles was quite effective, the synthesis of dendrimers is a tedious and time-consuming process. In 2007, Coates and co-workers utilized the intramolecular cross-metathesis strategy for the preparation of single-chain nanoparticles.48 Thus, a 38 mol % vinylcontaining polycarbonate with Mn = 54.1 kDa and PDI = 1.20 was prepared by the terpolymerization of cyclohexene oxide, vinyl cyclohexene oxide, and CO2 with a zinc catalyst (Scheme 44). The formation of nanoparticles under dilute conditions (1 mg/mL) using Grubbs second-generation catalyst was confirmed by 1H NMR spectroscopy. AFM was extensively used in this study to monitor the covalent crosslinking, and a linear relationship was found between the square of the extension ratio and the degree of cross-linking. Zimmerman, Lemcoff, and co-workers recently reported the synthesis of mono- or polyfunctional organic nanoparticles via a sequential ring-opening metathesis polymerization (ROMP) and ring-closing metathesis (RCM) protocol.166 The method involved the ring-opening metathesis polymerization of a norbornene dicarboxyimide monomer carrying an activated ester group (N-hydroxy succinimide ester) using Grubbs third-generation catalyst. The activated ester groups of the ROMP-derived polymer were then grafted with tris(allyloxymethyl)aminomethane to produce a large number of terminal olefin groups across the polymer chain. Ringclosing metathesis reactions using Grubbs first-generation catalyst under dilute conditions resulted in intramolecular chain folding to produce well-defined nanoparticles (Scheme 45). NMR and GPC/MALLS provided evidence for the single-chain collapse. The large numbers of olefin groups generated on the cross-linked polymer nanoparticles were further modified by dihydroxylation to produce water-soluble

2.15. Oxidative Polymerization of Thiophene

The synthesis of single-chain nanoparticles via tandem atom transfer radical polymerization (ATRP) and oxidative polymerization was recently reported.167 The electroactive monomer, a styrene-functionalized propylene dioxythiophene (proDOT-Sty) unit, afforded the corresponding homopolymer by a typical ATRP methodology. Subsequent electrochemical oxidative coupling of the ProDOT side chain resulted in an intramolecular collapse, which was monitored by the change in the apparent molecular mass using SEC and by the disappearance of aromatic protons of thiophene in the 1H NMR spectrum (Scheme 46). However, prolonged reaction times resulted in intermolecular coupling, as indicated by the appearance of a higher molecular mass shoulder in SEC plot. Reductive cleavage of the ester linkage of the polystyrene backbone by LiBH4 provided poly(vinyl benzyl alcohol) and oligo(ProDOT−OH) as further evidence for the structure and formation of the electrochemically polymerized ProDOT. Modification of the precursor polymer chain by introducing a tert-butyl acrylate block followed by oxidative polymerization of ProDOT block resulted in the formation of hybrid linear− coil nanoparticle without any intermolecular cross-linking. It may be worth mentioning at this point that Advincula and coworkers used a similar electrochemical strategy to prepare conjugated nanoparticles via the intramolecular cross-linking of a third-generation carbazole-terminated Fréchet-type polybenzylether dendrimer. As mentioned before, the rigidification of the organic structure caused the height of the cross-linked dendrimer to be higher than its uncross-linked form.168 2.16. Ring-Opening Polymerization

Ring-opening polymerization (ROP) of benzoxazine is another thermal cross-linking strategy reported by Wang et 911


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Scheme 47. Synthetic Route for SCNP Folded by Ring-Opening Polymerization of Pendant Benzoxazine Groups

Scheme 48. Intramolecular Chain Collapse Mediated by Ring-Opening Polymerization of Glycidyl Groups

al. in 2011.169 Like in the case of BCB chemistry a continuous addition protocol was followed by the authors to demonstrate the use of this strategy to fold a linear polystyrene bearing pendant benzoxazine moieties. The precursor polymer used in this case was a RAFT-derived poly(styrene-co-chloromethylstyrene), which was postfunctionalized with benzoxazine groups via copper-catalyzed alkyne azide click chemistry. Upon heating to 250 °C, ring-opening polymerization of benzoxazine groups ensued, resulting in cross-linking (Scheme 47). Intramolecular chain folding was evidenced by size exclusion chromatography analyses, where a reduction in the hydrodynamic volume upon increasing the amount of benzoxazine groups on the precursor polymer chain was

found. The morphology of the nanoparticles formed was further evaluated by AFM, TEM, and DLS measurements. Wang et al. demonstrated yet another use for benzoxazine chemistry by reporting the synthesis of hydrophilic fluorescent nanoparticles with a diameter in the range of 5−20 nm from poly(styrene-co-methyl methacrylate-co-1-(azidomethyl)-4-vinylbenzene) (PS-N3) copolymer postmodified with fluorescent anthracene and cross-linkable benzoxazine moieties.170 Intermolecular cross-linking was carried out by dropwise addition of the precursor polymer solution to dibenzyl ether at 250 °C. Disappearance of the 1H signals at 5.27 and 4.55 ppm (corresponding to the benzoxazine moiety) indicated successful cross-linking, while the decrease in the apparent 912


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Scheme 49. Preparation of Polyacrylate SCNP by Intramolecular Ring Opening of Pendant Caprolactone Groups

experiments, showing a decrease in the hydrodynamic diameter of one precursor polymer from 7.2 to 4.8 nm and the other from 3.6 to 2.8 nm. These nanoparticles, synthesized without any metal contamination, were tested for the biocompatibility toward human embryonic kidney cells (HEK293T) and found to be nontoxic up to concentrations of 120 μg/mL. All ROP-based cross-linking reactions in this report were performed at a polymer concentration of about 100 mg·mL−1, much higher than the typical concentration used for typical intramolecular chain folding. It is interesting to note that no sign of intermolecular cross-linking was observed even at such a higher concentration. In order to investigate the formation of SCNPs at higher polymer concentration, a detailed study on the factors influencing the formation of SCNPs by the ROP technique was carried out.173 The influence of side-chain functionality was studied by preparing a copolymer bearing three side chains of varying polarity, i.e., poly(4-(acryloyloxy)ε-caprolactone)-r-(oligo(ethylene glycol)acrylate), poly(4(acryloyloxy)-ε-caprolactone)-r-styrene), and poly(4-(acryloyloxy)-ε-caprolactone)-r-methyl acrylate). The folding studies, monitored by GPC, DLS, and 1H NMR, showed that the copolymer consisting of PEO side chains collapsed intramolecularly forming SCNPs, whereas those having styrene and methacrylate functionalities resulted in multichain aggregates. This experiment clearly emphasized the role of PEO in single-chain collapse at higher concentrations. Varying the initiator structure from benzyl alcohol (monohydroxy) to triethylene glycol (dihydroxy) and pentaerythritol ethoxylate (tetrahydroxy) failed to show any noticeable influence in the single-chain collapse of the copolymer bearing PEO side chains under the same experimental conditions. Finally, the influence of initiator concentration was also studied. In this case the SCNPs with maximum compaction were produced when the initiator and polymer precursors were used in equimolar concentrations.

molecular weight (SEC) determined that it was intramolecular. The average size of the nanoparticles, studied by DLS, TEM, and AFM, was found to be in the range of 5−20 nm. The nanoparticles thus prepared were hydrolyzed under acidic conditions to make them hydrophilic. Furthermore, these nanoparticles displayed fluorescent behavior similar to that of the monomers and linear precursor, with an emission at 412 nm when excited with 367 nm UV light. Ring opening of cyclic lactones and cyclic ethers has also been used for covalent intramolecular cross-linking of linear polymer chains. Pomposo and co-workers reported one such example that involved the ring-opening polymerization of precursor polymers bearing glycidyl moieties.171 Precursor methacrylate copolymers of molecular weight below 100 kDa with PDI < 1.1 were prepared by controlled RAFT polymerization, whereas those above 1000 kDa with PDI < 1.6 were obtained by free radical polymerization. The glycidyl methacrylate content in the nanoparticles was set around 30% to minimize intermolecular coupling but to allow extensive chain collapse. B(C6F5)3-assisted ring-opening polymerization of glycidyl groups under high dilution led to the formation of the SCNP (Scheme 48). Notably, the nanoparticles retained the B(C6F5)3 units due to strong B···O interactions with the ether or carbonyl oxygen atoms, as confirmed by Tg analysis and 19F NMR spectroscopy. As a proof of concept, the SCNP were tested as organocatalysts in the reduction of α-diketones to silyl-protected 1,2-diols in the presence of a reducing silane and also as polymerization initiators in cationic ring opening of tetrahydrofuran. In both cases reactions could be observed, although the results were not outstanding. In any case, these represent pioneering examples toward the achievement of enzyme-mimetic activities with SCNPs. Qiao and co-workers recently reported another example of intramolecular chain collapse through ring-opening polymerization.172 Polymer precursors of low polydispersity (PDI < 1.4) were prepared via controlled RAFT random copolymerization of oligo(ethylene glycol) methyl ether acrylate, di(ethylene glycol) ethyl ether acrylate, and 4-(acryloyloxy)-εcaprolactone. Incorporation of lactone moieties was confirmed by 1H NMR spectroscopy and found to be approximately 20 mol %. Intramolecular ring opening of the pendant caprolactone moieties was achieved by using benzyl alcohol as a nucleophilic initiator and methanesulfonic acid to achieve the SCNPs (Scheme 49). The chain folding was evidenced (among other methods) by size exclusion chromatography and dynamic light scattering

2.17. Hydrolysis and Polycondensation of Alkoxysilane

The polycondensation of alkoxysilanes is a popular method for the preparation of silica-like porous materials. Jie He and co-workers recently employed this robust chemistry for the synthesis of amphiphilic tadpole-ike nanoparticles, consisting of silica-like polymethacrylate heads decorated with hydrophilic poly(ethylene oxide) (PEO) tethers.174 A precursor polymer containing an inert PEG block and a copoly(methyl methacrylate)-poly(3-trimethoxysilyl)propyl methacrylate (PEO-b-P(MMA-co-TMSPMA) was synthesized by using a 913


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Scheme 50. Formation of SCNP via Hydrolysis and Polycondensation of Trimethoxysilyl Groups

Figure 4. (a, b) Representative TEM images for SCNPs prepared from PEO-b-P(MMA-co-TMSPMA) in THF at different magnifications. Scale bars are 50 nm in a and 200 nm in b. (c) Size distribution of SCNPs measured from TEM images by averaging more than 200 particles. (d) Hydrodynamic diameter and exponential decay of the correlation function of PEO-b-P(MMA-co-TMSPMA) before (dashed, black) and after (red) intramolecular cross-linking, measured by dynamic light scattering.

macroinitiator (MeO-PEO-Br) of Mw = 5 kDa via atom transfer radical polymerization. The chain collapse of the active polymethacrylate block was triggered under high dilution (0.25 mg/mL) by using ammonium hydroxide in the presence of trace amounts of water, whereupon condensation of the trimethoxysilyl groups ensued (Scheme 50). The SEC traces showed an increase in the retention time and the corresponding decrease in the apparent molecular weight of the polymer from 56.8 to 32.1 kDa in 12 h, suggesting intramolecular cross-linking. A plot of apparent molecular weight against the reaction time disclosed two distinct slopes, indicative of the two-step process (hydrolysis followed by slower polycondensation) involved in the chain

collapse. The SEC analysis also showed that by varying the degree of polymerization of the two blocks, the final size of the nanoparticle could be easily controlled. Furthermore, TEM micrographs showed high-contrast images of the silicalike nanoparticles in the range of 10−20 nm, consistent with the observed DLS results (Figure 4). In THF/water mixed solvent, the amphiphilic silica-like nanoparticles self-assembled to spherical micelles or vesicles depending on the size of the silica heads and the initial concentration. Moreover, these nanoparticles presented distinctive surfactant-like behavior and a rigid hydrophobic head. With respect to the unique chemistry used for the SCNP, the formation of a rigid cross-linked inorganic polymer as the basis for intramolecular 914


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Scheme 51. Sequential Intramolecular Cross-Linking of Poly(oxo-norbornene-co-cyclooctadiene) Using an Orthogonal Covalent Cross-Linking Strategy

leads to the formation of sparse rather than spherical globular objects.27,28,175 The reason for this spread out morphology was attributed to the self-avoiding character of the precursor polymer in good polymer solvent, where bonding at long contour distances is a rare event. Moreover, SEC/MALLS and SAXS techniques, supported by molecular dynamic (MD) simulations, revealed that more compact nanoparticles may be formed when chain folding was carried out using heterofunctional polymers containing two different cross-linkers or by a sequential orthogonal folding of the polymer chain.28 In this context, Berda and co-workers reported the sequential folding of ROMP-derived poly(oxo-norbornene-co-

collapse is certainly novel, and further study would be needed to understand the full potential of this methodology in polymer nanoparticle formation. As an interesting note, even though only one block of the polymer was actually collapsed, the percentage of shrinking was similar to fully collapsed polymers by other chemistries. 2.18. Orthogonal Covalent Cross-Linking

Pomposo and co-workers significantly advanced the field of macromolecular intrachain cross-linking by thoroughly analyzing the architectures obtained after the collapse, both theoretically and experimentally. Thus, they found that homofunctional collapse in single-polymer chains mainly 915


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Scheme 52. Sequential Folding of PS Polymer Using Orthogonal Covalent Chemistries

with two different cross-linkable zones separated by a polystyrene spacer. The compartmentalization of this polymer was achieved in three steps. Initially, the more labile pentafluorophenyl activated ester-containing zone was folded under dilute conditions using ethylene diamine as bifunctional cross-linker. TIPS deprotection followed this first step, and finally, Eglinton coupling of the resulting terminal akynes resulted in the collapse of the other cross-linkable zone (Scheme 52). The reaction progress at each stage was confirmed by 1H NMR spectroscopy and SEC measurements.

cyclooctadiene) to create folded electroactive polymer chains.176 The electroactive aniline tetramer was first introduced by reacting it with about one-half of the anhydride groups. The folding process was started by the reaction of the remaining anhydride groups with p-phenylenediamine as the cross-linker (Scheme 51). The partial chain folding was monitored by following the decrease of the hydrodynamic volume using SEC equipped with a MALS detector. The second folding step was achieved via thiol−ene chemistry between a dithiol cross-linker and the internal double bonds of the ROMP-derived polymer. Both polymer collapse processes were carried out under dilute conditions at a polymer concentration of 1 mg/mL. By using the sequential orthogonal protocol, the authors were able to achieve a tightly folded nanoparticle with a hydrodynamic volume that was 70% smaller than the parent polymer chain. The thus prepared SCNP displayed interesting spectroscopic and electrochromic properties. It will be important in the near future to determine how the tight compression of the polymer affects the electrooptical properties of the nanoparticle, especially compared to unfolded or less compact polymeric analogues. The orthogonal folding strategy is beneficial not only to prepare more compact globular structures but also for the compartmentalization of the folded polymer chains. For example, Meijer and co-workers used a noncovalent orthogonal chain-folding strategy for the compartmentalization of single-chain nanoparticles and for designing enzyme mimics (vide infra).177 Continuing with this model, Lutz and co-workers reported on the use of the orthogonal covalent cross-linking strategy for the compartmentalization of a sequence-controlled polystyrene copolymer via sequential compactation.178 The copolymer precursor was equipped

2.19. Intramolecular Cross-Linking of Grafted Copolymers

Many reports in the recent literature deal with the intramolecular cross-linking grafted copolymers such as star and brush copolymers. Even though technically these macromolecular structures are not pure single-chain polymers, the relevance to the topic merits their inclusion in this review. Miller and co-workers reported on the synthesis of crosslinked polymeric nanoparticles from a functionalized multiarm star copolymer synthesized by anionic polymerization.179 The precursor star polymer was synthesized with a tert-butyl dimethylsilyloxy end-functionalized living polystyrene macroinitiator containing randomly distributed 4-vinyl benzocyclobutene moieties (VBCB). The macroinitiator was then appended to a cross-linked divinylbenzene (DVB) core following an arm-first synthetic approach. Subsequent addition of styrene and VBCB to the living macroinitiator produced half-star and full-star copolymers. The number of arms incorporated was estimated from the molecular weight of macroinitiator and that of the star copolymer by using SEC/ SLS measurements. The incorporation of VBCB moieties was confirmed by monitoring the benzocyclobutene signals at δ = 3.1 ppm. The star polymers thus produced were cross-linked by activating the BCB units under pseudo-high-dilution 916


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Scheme 53. Preparation of Cross-Linked Star Copolymers Using Benzocyclobutene Chemistry

Scheme 54. Cross-Linking of Star Copolymers through Intramolecular Polymerization of Methacrylate Termini

conditions at 250 °C (Scheme 53). Disappearance of the signals assigned to the 4-membered ring protons in the 1H NMR confirmed the successful cross-linking. The intramolecular cross-linking was further confirmed by SEC/SLS, DLS, and SAXS measurements. In contrast to the folded linear single-chain polymers, the cross-linked star polymers exhibited only relatively minor changes in their hydrodynamic radii compared the precursor polymers. The

benzocyclobutene chemistry in the above is significantly stable toward anionic polymerization conditions and allows easy incorporation of thermally latent BCB units throughout the star side arms. Alternative strategies are based on postsynthetic functionalization of the cross-linking agent. For example, Du Prez et al. utilized the secondary bromine end groups of an ATRP-derived star copolymer to introduce acrylates for intramolecular cross-linking.180 The preparation 917


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Scheme 55. Preparation of “Cored” Cross-Linked Star Copolymers via RCM of Terminal Olefins

arms were found per star polymer. Intramolecular crosslinking was carried out by cross-metathesis of pendant olefins using Grubbs second-generation catalyst (Scheme 55). Evidence for the cross-linking was obtained from the disappearance of the peaks between 5 and 6 ppm in the 1H NMR spectrum. Furthermore, an increase in the SEC retention time was also observed as expected for intramolecular cross-linking (and the evolution of ethylene). As indicated by UV−vis spectroscopic measurements, basemediated hydrolysis of cross-linked polymer resulted in the formation of a “cored” nanoparticle of approximately 20 kDa and a polydispersity index of 1.5 via removal of >99% porphyrene core. However, the more flexible star polymer structure could not reproduce the monomolecular imprinting recognition characteristics of its more symmetric dendritic counterparts. In contrast to the star copolymers, the cross-linking of brush copolymers results in the formation of fairly welldefined cross-linked core−shell nano-objects with higher stability than the self-assembled block copolymer micelles. Wooley and co-workers communicated the synthesis of peripherally cross-linked nanoparticles and hollowed frameworks.185 In this report, the authors followed a tandem synthetic strategy combining ROMP and NMP to synthesize brush copolymers by a “grafting-from” approach. A

of cross-linkable precursor star polymer involved two steps. In the first step, a star-shaped poly(isobornyl acrylate) (PiBA) was prepared starting from a previously reported multifunctional initiator with 4, 6, and 12 bromine end groups.181,182 PiBA was selected as the polyacrylate arm for its high Tg value (95 °C). In the second step the secondary bromine end groups of multiarm star polymer were exchanged by methacrylate end groups. 1H NMR experiments revealed almost 95% substitution. Finally, the nanoparticles were prepared by the intramolecular polymerization of the methacrylate end groups by UV initiation under dilute conditions (5 mg/mL) (Scheme 54). The cross-linking was confirmed by SEC measurements and also by monitoring the disappearance of the methacrylate signals in 1H NMR spectra. Zimmerman et al. reported another example of a core-first approach for the synthesis of “cored” intramolecularly crosslinked star polymers.183 In their work, an 8-armed precursor star polymer containing cross-linkable terminal olefins was prepared by the polymerization of 1-but-3-enyl-4-vinylbenzene and styrene under radical conditions starting from a porphyrin-based ATRP macroinitiator bearing 8 initiator sites. In order to achieve the intramolecular cross-linking at higher concentrations,184 the star polymer was further copolymerized with styrene under ATRP conditions. On the basis of MALS SEC measurements an average amount of 7.6 918


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Scheme 56. Preparation of Peripherally Cross-Linked SCNP and Hollowed Frameworks

norbornene monomer bearing an alkoxy amine (for NMP) was first polymerized using Grubbs first-generation catalyst to obtain a well-defined polymer of Mw = 122 kDa and PDI = 1.13. The alkoxyamine functionalities along the polymer chain

were then used for the sequential polymerization of isoprene (Ip) and tert-butyl acrylate (t-BA) to obtain a well-defined core−shell brush polymer of Mw = 1410 kDa and PDI = 1.23. Hydrolysis of pendant tert-butyl ester groups of the grafted 919


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Scheme 57. Preparation of Cylindrical Shape SCNP via Vulcanization Technique

Degradation of the polyisoprene without cross-linking lead to complete dissociation, highlighting how the collapse procedure allows the formation of novel nanosized architectures. A year later the same group reported on the use of core− shell brush polymers as templates for the synthesis of core cross-linked nanoparticles with controlled cylindrical shape.187 The precursor core−shell brush polymer containing a grafted Pi-P-b-Pt-BA diblock was core cross-linked using sulfur monochloride (S2Cl2) (Scheme 57). The reaction was monitored by FTIR spectroscopy. Further evidence for the vulcanization reaction was obtained from 1H NMR spectroscopy, which showed the disappearance of alkenyl protons at 4.17−5.6 ppm, while the peak corresponding to t-Bu groups remained clearly visible. AFM measurements verified the rigidified cylindrical morphology and narrowly dispersed size.

copolymer followed by cross-linking of the resulting acid functionalities with 2,2′-(ethylenedioxy)bis(ethylamine) (in the presence of a carbodiimide as catalyst) in water afforded the peripherally cross-linked copolymer brushes. As determined by DLS, an increase in the hydrodynamic diameter from 11.5 ± 0.3 to 17.2 ± 1.6 nm was observed, probably due to the higher hydrophilicity and the volume occupied by the cross-linker. AFM measurements of these cross-linked brush polymers on mica exhibited an average diameter of 36.1 ± 8.5 nm and an average height of 2.22 ± 0.32 nm, showing that the cross-linking did not rigidify the structures to a great extent. Finally, the selective degradation of the polyisoprene core with ozone followed by reduction with Na2SO3186 afforded hollowed nanostructures having an average diameter of about 40 nm and an average height of 2 nm (Scheme 56). 920


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Table 2. Dynamic Covalent Cross-Linking Chemistry for Generating SCNPs

Scheme 58. Intramolecular Chain Collapse of Polyacrylamide via the Oxidation of Thiol Groups

tures.191−193 Another important property of the dynamic covalent bond strategy is the possibility to undergo monomer exchange. Therefore, the use of a dynamic covalent crosslinking strategy in single-chain folding of linear polymers can result in the formation of structurally adaptive nanoparticles that are able to respond to external stimuli.13,194,195 The reversible nature of the cross-link bonds allows the incorporation and release of components inside the nanoparticles; therefore, these structurally dynamic polymeric materials are used widely for drug delivery studies involving the controlled release of drug molecules. The following section deals with various dynamic covalent strategies that are used in the collapse of linear polymers for the formation of single-chain nanoparticles.

Furthermore, these well-defined nanostructures exhibited high robustness and higher resistance to collapse due to their highly core cross-linked domain. It is likely to assume that changing the initial polymer to brush size ratio may allow forming interesting nanostructures with tunable properties by applying the selective cross-linking strategy detailed above.

3. SCNPS VIA DYNAMIC COVALENT BONDS Over the past few decades dynamic covalent chemistry has emerged as an efficient tool for organic and polymer chemists. Catapulted into the spotlight by the groundbreaking works carried out by the groups of Jean Marie Lehn and Jeremy Sanders, dynamic covalent bonds exhibit all the characteristic properties of a normal covalent bond but have the ability to break and reform in response to an external stimuli such as pH, oxidizing or reducing agents, chemical agents, etc.188−190 In principle, the dynamic covalent methodology allows for the development of constitutionally flexible polymeric architectures and paves a pathway toward the approach to thermodynamically, rather than kinetically, controlled struc-

3.1. Disulfide Chemistry

Disulfide chemistry is undoubtedly one of the hallmark reactions in dynamic covalent chemistry. Mainly popularized by Sanders and Otto, many impressive examples have been reported in the literature.188,190,196−198 In addition, disulfide 921


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Scheme 59. Reversible SCNP Formation Mediated by Disulfide Chemistry

single-chain collapse strategies mediated by an external crosslinker is that the reaction that binds the cross-linker to the polymer must be fast and efficient; otherwise, under the highdilution conditions required, it will just not occur. The formation of SCNPs was conveniently monitored by SEC. Further evidence on the morphology of the nanoparticles was obtained by MALLS and TEM measurements. The reversible nature of the dynamic covalent cross-links was demonstrated by unfolding the nanoparticles via the reduction of disulfide linkages using dithiothreitol (DTT) and monitoring the change in size by SEC. Moreover, the random coil linear polymer chain bearing thiol functional groups was then refolded by FeCl3-catalyzed oxidation under high dilution. Both the original SCNP produced by the addition of the disulfide cross-linker and the refolded SCNP produced by the oxidatively cross-linking of thiols had approximately the same size, as expected for the formation of probably very similar SCNP structures. An additional important experiment in this work illustrates how small amounts of intermolecular binding can lead to larger structures that may go undetected in SEC analysis with refractive index (RI) detectors. The additional use of a MALS detector clearly showed the formation of these larger structures over time. The use of DLS without chromatographic separation in these cases can lead to skewed data that overestimates the true size of the SCNP due to the much stronger light scattering of larger objects. Inspired by the folding pathways of natural proteins, Lutz and co-workers demonstrated a guided folding protocol for the localized intramolecular binding of atactic polystyrene decorated with positionable cysteine-arginine-cysteine (CRC) motifs.201 The sequence-controlled polymer precursor was synthesized via nitroxide-mediated copolymerization of styrene and pentafluorophenyl 4-maleimidobenzoate, in which the latter functional monomer was positioned at preferred locations. 202 The CRC motifs where then introduced to the copolymer via the amidation reaction of a

chemistry is the method of choice in nature for making covalent bridges in proteins. It is thus not surprising that this chemistry has also been used for the preparation of reversible SCNPs. The following examples detail how this was done. An early study on the disulfide-promoted intramolecular cross-linking of a water-soluble polyacrylamide was reported by Ravi and co-workers.199 In their work, the authors followed a “green chemistry” approach to prepare nonbiodegradable polyacrylamide-based polymeric proteo-mimetics. The polyacrylamide precursor bearing pendant thiol groups was prepared in two steps. Initially, a hydrogel was prepared via the radical copolymerization of acrylamide and N,N′bisacryloylcystamine (about 4 mol %). Reduction of the disulfide bonds with dithiothreitol (DTT) produced the desired water-soluble polyacrylamide bearing pendant thiol groups, albeit with relatively high polydispersities due to the methodology used. Intramolecular oxidative cross-linking of the thiol functionalities produced SCNPs when carried out under high dilution (below 0.3% w/w) (Scheme 58). By this manner, a decrease in the intrinsic viscosity and radius of gyration indicated that the chain folding predominantly occurred through intramolecular cross-linking. However, the DLS and AFM measurements still showed the formation of nanogels in the range of 20−200 nm owing to intermolecular disulfide bond formation. It could be that the high polydispersity of the precursor polymer hindered unblemished intramolecular reactions. Berda and co-workers demonstrated an interesting approach to reversible single-chain collapse based on disulfide linkages.200 In their work, the authors followed a cross-linkermediated approach using a difunctional p-aminophenyl disulfide as redox-responsive connector. The collapse of ROMP-derived poly(norbornene-exo-anhydride) (made with Grubbs third-generation catalyst) was achieved through the ring-opening reaction (aminolysis) of the anhydride by the amino groups of the disulfide cross-linker, creating a linkage under high dilution (Scheme 59). A recurrent motif in these 922


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Scheme 60. Localized Intramolecular Cross-Linking of PS Copolymer Bearing Cysteine-Arginine-Cysteine (CRC) Motifs

derived acrylamide by NMRP. Deprotection and oxidation of the thiol functional groups under dilute conditions (10−3 M) resulted in intramolecular cross-linking through disulfide bond formation (Scheme 61). A decrease in the apparent molecular weight after oxidation from 12.8 to 8.7 kDa (68% of the linear precursor) clearly indicated the collapse through intramolecular cross-linking. However, the partial reduction of the disulfide bonds with DTT followed by capping of thiol groups by a Michael addition with N-phenyl maleimide gave a mixture of collapsed nanoparticles and linear polymers. The authors claimed that this may be due to the reduced accessibility of all the disulfide bonds within the collapsed environment of the SCNP. On the other hand, the presence of higher molecular weight polymers (not present in the original macromolecular precursor) attests to the facile disulfide exchange that occurred during the handling of the reduction of the SCNP cross-links. Inspired by their previous work204,205 Thayumanavan and co-workers developed a new approach for the synthesis of redox responsive single-chain nanoparticles that are capable of

primary amine in a CRC tripeptide-protected derivative with the activated pentafluorophenyl ester functions of the sequence-controlled polymer. Subsequent deprotection and oxidation under high dilution resulted in single-chain folding at the predefined locations (Scheme 60). Even though reversibility was not studied in this case, the important takehome message here is the precise positioning of the crosslinking agents within the polymer chain to affect the SCNP architecture. Another recent example reported by Braslau and co-workers described a manipulation of the redox property of thiol/ disulfide functionalities to prepare various cross-linked polymer networks.203 The authors synthesized several styreneand acrylate-based copolymers bearing protected thiol groups by nitroxide-mediated radical polymerization (NMRP). Thiol deprotection followed by oxidation upon exposure to air resulted mainly in cross-linked networks via disulfide formation. In this report, an intramolecular chain collapse was exemplified by synthesizing a copolymer (Mn = 12.8 kDa, PDI = 1.55) of tert-butyl acrylate and tritylated cysteine923


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Scheme 61. Preparation of SCNP by Cysteine-Derived Disulfide Bond Formation

Scheme 62. Approach for Redox-Responsive SCNPs

encapsulating hydrophobic guest molecules and release them in response to a redox stimulus.206 Thus, a random copolymer poly((HEMA)x-co-(PDSEMA)y) (Mn = 76.9 kDa, PDI = 1.24) was synthesized by RAFT polymerization of 2-hydroxyethyl

methacrylate (HEMA) and pyridyl disulfide ethyl methacrylate (PDSEMA). The pyridyl disulfide bonds were partially cleaved by using 50% of DTT to generate reactive pendant thiol units. These thiol groups exchanged the remaining pyridyl disulfide 924


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Scheme 63. Preparation of SCNP by Condensation of PVBA and a Bishydrazide Cross-Linker

In order to promote exclusive intramolecular chain collapse, an optimum polymer concentration of 0.1 mM was used, beyond which intermolecular cross-linking was observed. Moreover, the degree of cross-linking could be controlled easily by varying the amount of cross-linker added. The SCNPs could be isolated after removal of all traces of catalyst by the addition of excess triethylamine (essential to avoid internanoparticle cross-linking during concentration). Incorporation of the bishydrazide linker was confirmed both by 1H NMR spectroscopy and SEC. In the latter, an increase in the retention time was found, proportional to the amount of cross-linker added, indicating single-chain collapse. By taking advantage of acylhydrazone bonds that do not participate in the cross-linking reaction the nanoparticles could be further functionalized with various low molecular weight organic hydrazides and alkoxyamines to obtain unimolecular nanoparticles with a very high density (80%) of functional groups embedded within their structure. Furthermore, the authors were able to demonstrate the adaptive property of these SCNPs via an ingenious dynamic covalent exchange reaction that altered the structure of the organic nanoparticle. For example, the precursor polymer fully (100%) adorned with monoacyl hydrazide groups was reacted with a bishydrazide cross-linker under acid-catalyzed exchange conditions and monitored by SEC. Over time, the hydrodynamic volume of the fully adorned polymer precursor was reduced, indicating an intramolecular collapse via the exchange of conjugated monohydrazide with the cross-linker. Further evidence for the exchange process was obtained from 1H NMR spectroscopy in which the distinctive methylene peak associated with crosslinker was visible. However, longer reaction times resulted in the formation of aggregated polymeric material of high molecular weight (precipitation was observed after 5 days) due to intermolecular exchange. More recently, the hydrazone cross-linking chemistry was used to prepare thermoresponsive dynamic covalent nanoparticles that can reversibly transform into a hydrogel.208 The polymer precursor in this case was a water soluble, oligo(ethylene glycol)methoxy methacrylate and p-(methacryloxyethoxy) benzaldehyde thermoresponsive copolymer.209

groups resulting in the collapse of the polymer chain via intramolecular disulfide bond formation (Scheme 62). In order to investigate the folding process, SCNPs of varying cross-linking densities (20%, 40%, 60%, 80%, 100%) were prepared and characterized by monitoring the decrease of 1H NMR signal intensity of thiopyridine groups at 7.0−8.5 ppm of PDSEMA groups. SEC measurements confirmed the collapse of the polymer chain through intramolecular crosslinking, showing increased retention times with higher degrees of cross-linking. Further evidence for the intrachain collapse was obtained from DLS experiments that revealed a systematic decrease in hydrodynamic diameter from 9.9 to 8.5 nm when cross-linking density was raised. Moreover, upon increasing the degree of cross-linking, a significant increase in the glass transition temperature (from 76 °C for parent polymer to 110 °C for 100% cross-linked SCNPs) was also observed as a result of the reduced segmental mobility. AFM analysis displayed a uniform distribution of SCNPs with an average size of about 10 nm. Polymers with varying molecular weight also formed uniform SCNPs, indicating the possibility of controlled folding over a considerable molecular weight range. The encapsulation and release capability of these SCNPs was probed using Nile red dye as the guest. Reduction of disulfide bonds at 5 mM DTT concentration led to a rapid release of the encapsulated guest. This interesting proof of concept may be envisaged for the release of certain therapeutic molecules within the appropriate desired environment. 3.2. Hydrazone Chemistry

The first reported example of structurally adaptable SCNPs used hydrazone chemistry as the cross-linking method. In their work, Fulton and co-workers used the dynamic covalent acylhydrazone bonds to bring about the collapse of poly(vinylbenzaldehyde) (PVBA) using a bishydrazide as the cross-linker.207 Trifluoroacetic-acid-catalyzed the condensation reaction of a bishydrazide cross-linker with pendant benzaldehyde moieties of the polymer chain that could be kinetically fixed by neutralization (Scheme 63). 925


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Scheme 64. Preparation of Thermoresponsive Dynamic Covalent Nanoparticles Using Acylhydrazone Chemistry

These kinds of polymers show thermoresponsive properties and reversibly precipitate when heated above their lower critical solution temperature (LCST).210 Thus, the precursor polymer was cross-linked with a bishydrazide cross-linker in 0.1 M AcOH/AcONH4 buffer at pH 4.5 to afford SCNPs (Scheme 64). The SCNPs formed exhibited the thermoresponsive property of the parent polymer chain and formed a hydrogel network by simply raising the temperature above the LCST. This was attributed to a reorganization of the acylhydrazone bonds, making intermolecular cross-links and rendering the cross-linked structure insoluble in common organic solvents. Notably, cooling the sample to room temperature allowed the reverse transformation of the hydrogel back to the SCNPs. The transformation of the random coil polymer to its collapsed state also led to a rise in the LCST, although this could be caused by the hydrophilicity of the cross-linker groups.

Scheme 65. Formation of Reversible SCNPs by the Reaction of Pendant β-Ketoester Groups with a Diamine Cross-Linker

3.3. Enamine Chemistry

Enamine chemistry is another class of dynamic covalent chemistry that was recently used for reversible single-chain collapse. Enamine bonds can be formed easily by condensation of ketones with amines and can be hydrolyzed back to their constituents under acidic conditions. Pomposo and co-workers exploited the use of enamine cross-linking chemistry to obtain reversible single-chain PMMA nanoparticles by a metal-free methodology.211 In their work, the precursor polymer was prepared by the copolymerization of MMA and 2-(acetoxyaceto) ethyl methacrylate (AEMA) via RAFT polymerization using 2-cyanoprop-2-yl-dithiobenzoate (CDB) as the chain transfer agent and AIBN as the freeradical initiator. Reaction of pendant β-ketoester groups with an alkyl diamine cross-linker resulted in the formation of dynamic covalent single-chain nanoparticles (Scheme 65). Both 1H NMR and FT-IR spectroscopy confirmed the formation of enamine bonds. The reduction in the hydrodynamic volume during the chain collapse was followed by SEC with MALLS detection and DLS experiments, showing nanoparticles of 320 nm and their subsequent photodegradation at 254 nm (Scheme 69). The SEC and DLS measurements revealed that the size of the SCNPs could be controlled by adjusting the intrachain crosslinking density, determined by the degree of photodimerization of the incorporated coumarin. The authors suggested that the biocompatible nature of the SCNPs and their simple degradability bestow them the potential to be used in biomedical applications, most likely in light-controlled drug delivery.

reversibility in this case was not carried out in situ, the importance of amines and diamines in biological surroundings may bring about important applications for this kind of SCNPs. 3.4. Reversible Cycloaddition

Polymers bearing coumarin and derivatives are suitable for the synthesis of photosensitive polymers.213 Coumarin-containing polymers can be reversibly cross-linked upon irradiation at different UV wavelengths (Scheme 67), ideal for the synthesis of dynamic covalent SCNP formation.214,215 Scheme 67. Photochemical Reaction of Coumarin Derivatives

Zhao and co-workers exploited the use of reversible photoinduced coumarin cycloaddition for the collapse of single-chain copolymers bearing coumarin units.216 In their work, RAFT-derived random copolymers composed of N,Ndimethylamino ethyl methacrylate (DMAEMA) and 4-methyl[7-(methacryloyl)oxy]coumarin (CMA) with 7 and 13 mol % of CMA were used as the single-chain precursors. Irradiation of a dilute copolymer solution (1 mg/mL) at λ > 310 nm effected the cross-link via dimerization of coumarin units (Scheme 68). In addition, upon irradiation at 254 nm UV

4. SCNPS VIA NONCOVALENT INTERACTIONS The formation of SCNPs using noncovalent interactions is a very appealing concept that may also be used to achieve thermodynamically stable collapsed polymers, as opposed to the kinetic products obtained with covalent nonreversible

Scheme 68. Reversible Single-Chain Folding of Poly(DMAEMA-co-CMA) by Coumarin Photodimerization

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thus, highly dilute solutions must be used to prevent intramolecular aggregates. 4.1. Hydrogen Bonding Interactions

Hydrogen bonding is a key factor in the overall conformation of many macromolecules and, naturally, biomacromolecules. A proper understanding of hydrogen bonding allows chemists to predict the formation of secondary and tertiary structures in proteins and even to plan sequential folding in synthetic foldamers.219 In this section we will summarize the uses of hydrogen bonds to achieve the synthesis of SCNPs. 4.1.1. Benzamide Dimerization. In the pioneering work of Hawker and co-workers,220 a series of linear polymers was prepared and functionalized with dendritic self-complementary hydrogen-bonding (SHB) units (Figure 6). Thus, a methacrylate linked to the benzamide dendron containing the SHBs was copolymerized with MMA by a RAFT methodology (Scheme 70). Two types of polymers were synthesized: A1 with 159 kDa with 1.5% incorporation of SHB and A2 with 131 kDa and 6.1% of the SHB unit. A two-solvent system was used to prepare the polymers: apolar toluene and more polar THF. Naturally, the presence of THF weakened the self-complementary hydrogen bonds by competition. Ingeniously, the polymer chain was collapsed by evaporation of the volatile THF from the solvent mixture used to dissolve the polymers, allowing for the hydrogen bonds to be formed under high-dilution conditions. After filtration, DLS and scanning force microscopy (SFM) data indicated the formation of well-dispersed and stable nanoparticles with a measured mean effective diameter of 24 nm, probably indicating some sort of intermolecular aggregation. Magnifying this unwanted process for the low loading of SHB unit (A1) following the same protocol, irregular aggregates with diameters of >100 nm appeared, suggesting extensive intermolecular interactions. Nonetheless, this seminal work described the first tools to engage hydrogen bonding in the preparation of single-chain nanoparticles.

Figure 5. TEM images of coumarin cycloaddition-derived SCNPs in THF (a) and H2O (b).

cross-links. Noncovalent bonds allow reversible binding and may be fine tuned by changing reactions conditions (e.g., pH, solvent polarity, concentration, temperature) or by external triggers. Due to the relatively low energy barriers of these interactions218 it may be possible to exploit the controlled collapse of a single-chain polymer for specific applications, for example, drug delivery, smart adaptable systems, and selfhealing. As an intrinsic drawback to this method it must be mentioned that the continuous addition protocol is usually incompatible with the reversible nature of the cross-links, and

Scheme 69. Reversible Photochemical Formation of SCNPs by Coumarin Chemistry

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Table 3. Noncovalent Cross-Linking Chemistry for Generating SCNPs

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Table 3. continued

4.1.2. Benzene Tricarboxamide Stacking and Ureidopyridinone Dimerization. Mes et al.221 demonstrated the formation of single-chain polymeric nanoparticles that adopt a helical conformation using the H-bonding interactions of a 1,3,5-benzene tricarboxamide (BTA) moiety (Scheme 71). Isobornyl methacrylate and propargyl methacrylate were copolymerized with a ratio of 80:20, respectively, using ATRP with activators regenerated by electron transfer (ARGET). oNitrobenzyl-protected BTA moieties were installed on the propargyl side chain via a CuAAC reaction. The synthesized copolymer molecular weight was 26 kDa, as indicated by SEC

data (polystyrene standards). A feasibility study on a model caged 1,3,5-benzene tricarboxamide molecule revealed that irradiation at 354 nm UV light in methyl cyclohexane (MCH) induced stacking of the BTA molecules. This procedure was applied on the BTA-containing copolymer at a concentration of 3 mg/mL in a solvent mixture of dichloroethane (DCE) and MCH. After irradiation and heating to 80 °C, the CD spectrum showed a pronounced negative Cotton effect with a maximum at 225 nm, indicating the formation of helical conformation. The maximum value for the Cotton effect was found when 930


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Figure 6. Hydrogen-bond cross-linking interactions between benzamide dendrons.

Scheme 70. Synthesis of Polymer with Grafted Hydrogen-Bonding Dendron

Figure 7. Terpolymer containing PEGMA, a chiral BTA methacrylate ester, and SDP-containing ligands.

Characterization of this polymer by UV spectroscopy and ICP analysis estimated 2.5 Ru atoms per polymer chain. SAXS and DLS measurements in water confirmed that the hydrodynamic radius was indeed smaller for the BTAcontaining polymers, yet SEC data suggest that the hydrodynamic radius is strongly dependent on the solvent. All of the BTA-containing polymers formed compact conformations in water and were detected as black spots of 3−4 nm diameter using cryo-TEM. The folding process of these polymers was monitored by temperature-dependent CD, between 80 and 0 °C, at 10 degree intervals. A negative Cotton effect with a minimum at 225 nm was observed at 0 °C and disappeared upon heating the solution. To examine the activity of the catalyst embedded in the hydrophobic cavity created by the folding of the BTA units, a Ru-catalyzed transfer hydrogenation reaction was probed. The hydrogenation of partially water-soluble cyclohexanone was executed with 0.5% Ru loading at 40 °C to afford 98% conversion. Similar results were obtained for the reduction for poorly water-soluble acetophenone (86% conversion). CD

irradiation was carried out in a solvent mixture of 70:30 DCE:MCH; this ratio was crucial for the solubility of the main chain and the establishment of the hydrogen bonding between the BTA units. The addition of 15 equiv of hexafluoroisopropanol (HFIP) disrupted the Cotton effect, while excess HFIP completely prevented H-bond formation. BTA hydrogen bonds were also used to form single-chain organic nanoparticle catalysts in water.177 This was realized making a terpolymer-containing polyethylene glycol methyl ether methacrylate (PEGMA), a chiral BTA methacrylate ester (BTAMA), and using Ru(Ind)Cl-(PPh3)2 as the metal catalyst (Figure 7). Thus, a series of copolymers was synthesized in high yield (>90%) with low PDIs of 1.2−1.3, differing in size, length of the PEG methyl ether chain, and percentage of incorporation of chiral BTA moieties. In order to install the catalytic function, 4-(diphenylphosphino)styrene (SDP) and a Ru living radical polymerization initiator were added in a onepot procedure, thus inducing both polymerization and ligand exchange. 931


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Scheme 71. Light-Triggered SCNPs Formation by 3-Fold Hydrogen Bonding of BTA Units

Figure 8. Organocatalytic SCNP folded by BTA units.

group.222 For this, 5 amphiphilic copolymers were synthesized (P1−P5). All polymers had a DP of about 100, with different combinations of monomers. The formation of a hydrophobic core was confirmed using the solvatochromic dye Nile Red. The dye intensity in the polymers containing BTA (P1, P2, and P5) was much stronger that the polymer without BTA (P3 and P4), suggesting that BTA aggregation provides a more stable hydrophobic environment facilitating the encapsulation. However, in the reduction of cyclohexanone to cyclohexanol in water, all polymers containing SDP ligands

measurements showed that the chiral helical conformation remained intact, and the catalyst did not decompose or hydrolyze after the reactions, as confirmed by 1H NMR. This system showed great efficiency in the reduction of prochiral molecules; however, no enantioselectivity was observed in the products, probably because the internal chiral center is remote from the active site of the catalysis and hence does not induce asymmetric reactions. The question whether this unique helical hydrophobic structure has a key role in Ru-catalyzed transfer hydrogenation reactions was addressed by the Meijer 932


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Figure 9. BTA-dye and (S)-BTA-L-Pro molecules.

the reaction with P1a in chloroform, where the polymer adopts a random coil conformation, also showed no conversion. This finding is consistent with the hypothesis that high effective molarity within the hydrophobic cavities of the formed SCNP is crucial for the system to be active. Recently,224 the Meijer group developed a modular approach for introducing catalytic functionalities to SCNPs by introducing “free” chiral BTA units with covalently attached L-proline units. The system studied was a random copolymer of oEGMA and BTAMA with a 10:1 ratio, respectively (DP = 223, Mn = 111 kDa, PDI = 1.50). To investigate the level of incorporation of the free chiral (S)-BTA units into the core of the nanoparticles, a fluorescent BTA functionalized with 1,8naphthalimide (BTA-dye) was chosen (Figure 9). Upon decreasing the solvent polarity the emission spectrum of BTA-dye was blue shifted and became more intense. Indeed, the BTA-dye emission shifted from 464 nm in water to 433 nm in the polymer mixture. Concomitantly, the fluorescence intensity increased as expected, providing evidence for the incorporation of the free BTAs inside the hydrophobic core of the polymer. Importantly, the fluorescence was independent of the BTA-dye concentration meaning that all the BTA-dye was located in the same environment within the SCNPs core. As a control experiment, the BTA-dye in the presence of just the oEGMA homopolymer had a maximum fluorescence emission at 458 nm, indicating the lack of a hydrophobic surrounding environment. The L-proline containing BTA, (S)-BTA-L-pro, was incorporated into the polymer in the same manner as the BTA-dye. CD measurements confirmed the incorporation of the chiral BTA units into the stacks as a negative Cotton effect developed due to asymmetric induction. The catalytic activity of the SCNPs containing (S)-BTA-L-pro was tested by an aldol condensation between cyclohexanone and p-nitrobenzaldehyde. The results showed good stereoselectivity and a high ee of 95%. The concentration effect was tested by reducing the number of (S)-BTA-L-pro from 4 units per polymer chain to just 2. To the authors’ surprise and in sharp contrast to the previous reports, reducing the (S)-BTA-L-pro concentration increased the catalyst activity dramatically and higher stereoselectivity was observed. The a posteriori rationalization was that reducing the (S)-BTA-pro units per chain increased the hydrophobicity of the cavity bringing about the desired results. The biomimicry of this system is quite extraordinary, only showing catalytic activity when properly folded. Thus, the special architecture formed by the chain collapse is what allows the proper positioning of the catalytic units by noncovalent interactions and the high efficiency observed (in yields, diastereoselectivity, and

(P1−P3) performed similarly, regardless of whether BTA was present or not. The polymers without the SDP ligands (P4 and P5), where the Ru was not attached to the polymer scaffold, performed poorly. These results indicate that although the hydrophobic pocket in the SCNPs is stabilized by the BTA self-assembly, it is not essential for this specific catalytic system. Continuing the quest toward synthetic enzyme mimics,223 it was shown that organocatalysis in water could be achieved by introducing L-proline residues inside BTA-folded organic nanoparticles. The hydrophobic structured cavities within the SCNP bring reaction substrates into close proximity, thus increasing the effective molarity and enhancing reaction rates. The random copolymer precursor (P1a) was made by a RAFT technique from 85% watersoluble oligoethylene glycol methacrylate (oEGMA), 10% (S)BTA carrying methacrylate, and 5% L-proline methacrylate as the catalytic unit (Figure 8). SEC data for P1a revealed Mn of 28.5 kDa with PDI of 1.56, and DLS data showed that the polymer forms SCNPs in water with a hydrodynamic radius of 6.3 nm. Monitoring the polymer-folding process by CD spectroscopy showed similar results with and without the L-proline moiety. Moreover, CD showed that the addition of the cyclohexanone substrate also did not interfere with BTA single-chain collapse. The catalytic activity of P1a was tested on the model aldol reaction in water between p-nitrobenzaldehyde and cyclohexanone. The aldehyde concentration was set to 50 mM with variable amounts of the ketone. Best results were obtained after 24 h (72% conversion, 94% de, and 70% ee) with an aldehyde/ ketone ratio of 1:10 with 1.6 mol % polymer loading. After 120 h the aldol product was obtained in quantitative yield with 91−92% de and 70−74% ee regardless of the aldehyde/ ketone ratio and with polymer loading decreased to 0.8 mol %. To further study the catalytic mechanism, P1a analogues were synthesized. Increasing the polymer molecular weight led to increased conversion yields, albeit with loss of diastereoselectivity and enantioselectivity. Performing the reaction with a polymer containing also nonchiral BTA moieties led to the same conversion after 24 h with a moderate decrease in de and no change in ee, which suggests that the chirality (or shape) of the pocket does not affect the transition state of the L-proline-catalyzed aldol reaction. A direct relationship was found between conversion and L-proline content in the polymer; more amino acid incorporation led to higher conversions, while decreasing L-proline led to a decrease in reaction yields. Remarkably, control polymers, similar to P1a but without BTA units (n-docecyl instead), showed no catalytic activity at all, thus showing the importance of singlechain collapse for catalytic competence. Moreover, performing 933


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Figure 10. Pn[BB*B] soldier−sergeant−soldier triblock copolymers.

P3[BB*B] with Mn of 31.0 (PDI = 1.2), 53.0 (PDI = 1.4), and 120.8 kDa (PDI = 1.5), respectively (Figure 10). The chiral BTA content in the middle sergeant blocks was 37% of the total BTA content in the polymer chain, well over the 5% necessary in free BTA stacking. To probe the sergeant−soldier principle in this case, 3 control polymers Pn[B*B*B*] with only chiral BTA units were additionally synthesized with matching Mn and polydispersities. Temperature-dependent CD measurements in 1,2-DCE as solvent were carried out to study the conformational behavior of these polymers. Above 60 °C all polymers were CD silent, and all polymers developed a negative Cotton effect upon cooling, typical for a left-handed helix column. The CD signal intensities for the Pn[BB*B] polymers were always lower than the Pn[B*B*B*] ones, meaning that chiral amplification within triblock copolymers could not replicate the fully chiral architecture despite the high local chiral BTA concentration in the sergeant fraction. The shape of the CD spectra was also changed for the differing polymer types, which may imply the disruption of chiral BTA stacks by insertion of achiral BTA in a type of interblock communication (which would mean that the folding is not fully controlled by the block separation and probably multiple cores are present within the collapsed polymer). In an additional experiment, P3[BB*B] was synthesized with higher incorporation of the B* unit (45%, 62%, 66%, 72%, and 100%), and the Cotton effect intensity was plotted against the B* incorporation. This analysis revealed that midrange chiral BTA incorporation (37−72%) had almost no effect on the intensity of the CD signal. This thorough and important analysis on the mechanism of singlepolymer chain collapse is a pioneering effort in the quest to better understand and control future functions in collapsed macromolecular architectures. Meijer and co-workers,229 also developed another hydrogen-bonding scaffold and showed the formation of metastable SCNPs using the strong association of 2-ureidopyridinone (UPy) units incorporated as side chains in a ROMP-derived polynorbornene (Scheme 72). Two types of UPy linkers were synthesized. In one polymer the Upy moiety was attached to the polynorbornene chain via urea units (Upy Urea) and in

enantioselectivity). The folding process of water-soluble polymers in water was further probed by the Meijer group.225 The polymers tested were a random copolymer of oEGMA and 9% chiral BTAMA (P10%, Mn(SEC) 34.2 kDa, PDI = 1.26) and an oEGMA polymer (P0%, Mn(SEC)= 36 kDa, PDI = 1.17), both with a DP of about 100 units. Temperature-dependent CD measurements determined that the P0% polymer was CD silent, while P10% showed a negative Cotton effect at 225 nm, which is typical for the 3fold hydrogen bonding between the BTA units, forming a M helix. Upon heating the solution, the Cotton effect decreased gradually up until 90 °C, where the Cotton effect is completely absent, indicating a lack of BTA association. In isopropanol, a less polar solvent, yet competitive for hydrogen bonding, no Cotton effect was observed. By using water:isopropanol mixtures or changing the temperature the BTA self-assembly could be finely tuned. The Cotton effect signal could be seen starting from a 1:1 solvent mixture and increased its intensity as the water content was raised. SANS measurements revealed that at a high molar fraction of isopropanol or at low temperatures in water the polymer adopts more elongated conformations, while raising the temperature in water led to a more compact and globular structure. Continuing the study on this system 4 copolymers (10:90 BTAMA:oEGMA) (P1−P4) were synthesized with DPs from 110 to 461 to understand whether folding enforced by the BTA units is dependent on chain length.226 The overall conclusion from this study was that the polymer initial size does not affect the folding process to a great extent, and all polymers behaved similar to those previously described. In a recent contribution, critical for the understanding of the folding process in the BTA systems, Hosono et al. reported “soldiers−sergeant−soldiers” (S−S−S) experiments using SCNPs with a triblock architecture,227 taking into account that just 5% of “sergeant” (with chiral side chains) free BTA units are required to induce full chiral amplification of achiral BTA stacks.228 ABA block copolymers were designed with the A block carrying achiral BTA (soldiers, B) and the B block carrying chiral BTA (B*, sergeants). Three such ABA block copolymers were synthesized P1[BB*B], P2[BB*B], and 934


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opposed to the single-chain collapse achieved by irradiation with UV light under dilute conditions. This concept was further expanded to PMMA-based polymers.231 The advantages for using polymethacrylates are better control over the molecular weight by using living polymerization techniques such as RAFT, SET-LRP, or ATRP and increased backbone polarity. Two synthetic approaches were probed: one was copolymerization of a protected UPy-bearing monomer with MMA, and the other path was the copolymerization of propargyl and methyl methacrylate using SET-LRP and then “clicking” an azide-UPy protected with an o-NB group moiety via a CuAAC. This latter approach proved to be more successful. The collapse of these polymers was done in the same manner reported previously, i.e., by dissolving the polymers in chloroform (1 mg/mL) and irradiating with 350 nm UV light for 2 h (up to 54% decrease in Mw). AFM data showed that after just 30 min of irradiation well-defined nanoparticles were observed, with a size decrease from 100 to 50 nm after 90 min (Figure 11).

Scheme 72. SCNPs Formation by Light-Triggered Hydrogen-Bonding Interactions of UPy Units

the other through a carbamate linker. To avoid solubility issues, the polymer scaffolds were decorated with alkyl substituents, and to reduce the association constant during polymer formation, the carboxyl of the UPy units was protected with a light-sensitive o-nitrobenzyl group. Upon irradiation with 350 nm UV light photocleavage of the o-NB group ensued, and UPy dimerization at high dilution led to the collapse of the polymer chain. GPC analysis showed that polymers with 10% cross-linker decreased their apparent molecular weight by about 20%. At higher cross-linker concentrations the decrease in Mn was 26% for the Upy urea and 34% for the Upy urethane. The reversibility of the collapse was demonstrated by addition of formic acid, disrupting intramolecular hydrogen bonds and expanding the SCNPs. Collapsed polymers casted from chloroform on a glass surface could be solubilized easily; however, heating for 20 min at 80 °C caused intramolecular bonds and rendered the network insoluble, demonstrating the dynamic behavior of the SCNPs. The direct correlation between polymer chain molecular weight and nanoparticle size was evaluated by AFM. To further investigate this link, 6 polymers from the above-mentioned type were synthesized with molecular weights between 52 and 1200 kDa,230 with 10% incorporation of the cross-linkable monomer. Collapsing these polymers by UV irradiation into nanoparticles and monitoring by GPC and AFM revealed a decrease in the apparent Mw from 23% to 31%, although no correlation could be found between the percentage of size reduction to the initial polymer size. Polymers where the UPy unit was attached by an alkyl linker showed a slight molecular weight decrease upon irradiation but not as sharp as the polymers with the urea or urethane linkers, determining the important role of these secondary attractive interactions (“orthogonal to the UPy stacking”). The aggregation process was also probed by solvent evaporation. Varying the solvent evaporation time led to the formation of several aggregated morphologies, as

Figure 11. Supramolecular cross-linking monitored by AFM. All images were taken from samples of PMMA-based polymer drop cast (2 μL of a 10−8 mg/mL solution) on freshly cleaved mica. (A) Protected polymer. (B) Following 30 min of irradiation (scale bar common for height and phase insets). (C) Following 60 min of irradiation. (D) Following 90 min of irradiation (scale bars are common in C and D for both height and phase panels).

The thermal behavior of the PMMA-based nanoparticles was preserved, forming insoluble supramolecular networks upon heating. The formation of SCNPs using the UPy motif was recently systematically investigated by the Meijer group232 as a function of the polymer backbone, polymer length, nature of the connecting linker, and solvent. Four types of polymers were selected: PMMA, poly n-butyl acrylate, polystyrene, and polynorbornene (Scheme 73). First, the polymers synthesized were tested for the effects of backbone rigidity and solvent on single-chain collapse. The 935


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Scheme 73. Cross-Linking of Different Polymer Scaffolds Using the SHB UPy Units

Figure 12. ABA-type triblock copolymer containing UPy and BTA hydrogen-bonding motifs.

In an extensive work conducted by the Meijer group,234 the orthogonal self-assembly of SCNPs was demonstrated by combining the two previously reported hydrogen-bonding motifs, BTA and UPy, into an ABA-type block copolymer. To simplify the synthetic effort, a postpolymerization approach was used for installation of the functional groups. Block A consisted of the copolymer of isobornyl methacrylate (IBMA) and hydroxyethyl methacrylate (HEMA), while block B was obtained via copolymerization of IBMA with propargyl methacrylate. The BTA unit was attached using CuAAC, while the o-NB-protected Upy was installed through a carbamate linker using an isocyanate− alcohol reaction. The furnished triblock copolymer, denoted as Pn[UBU] (Figure 12), was thus equipped with two orthogonal hydrogen-bonding motifs. UPy dimerization was triggered by photocleavage of the o-NB protecting group at 354 nm UV light, while BTA stacking occurred in a coordinated manner upon heating and cooling of the solution (Scheme 74).

main conclusions were that with the less polar solvent, chloroform, interparticle aggregation was observed and with the most polar solvent, DMF, the hydrogen-bonding dimerization was completely annulled. The “Goldilocks” principle worked in this case, as the hydrogen-bond-accepting THF was just the right solvent to promote folding. Interestingly, the different polymer backbones, UPy linking units, and polymer sizes had a very minor effect on polymer folding. The important take-home message here was that the strength of the cross-linking interactions (attenuated by the THF solvent) is the critical parameter for SCNP formation. In order to better understand the behavior of SCNPs on surfaces De Greef studied the morphologies of dried solutions of PMMA SCNPs prepared by dimerization of UPy units, and rationalized them by employing 2D lattice gas simulations.233 Image processing algorithms allowed quantitative comparison between computer simulations and experimental observations providing evidence that evaporative self-assembly of SCNPs proceeds via a spinodal dewetting instability triggered by thinning of the liquid film. 936


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Scheme 74. Orthogonal Hydrogen-Bonding Interactions of UPy and BTA blocks

gyration of P2[UBU] was slightly reduced after UV irradiation from 11.8 ± 0.3 to 11.1 ± 0.2. In later work,235 the folding of triblock copolymers with a BAB-type sequence was investigated and compared with the ABA triblock system. The polymer scaffold blocks were constructed by the same manner as before. Six polymers were examined in this work, the BAB series denoted as Pn[BUB] with molecular weights of 24, 57, and 167 kDa and weights of 25, 60, and 181 kDa for their Pn[UBU] counterparts. The effect of UPy dimerization on the folding was analyzed by SEC (in chloroform). The samples were prepared by dissolving the polymers in 0.5 mg/mL concentration in 1,2-DCE and subsequently irradiated with 350 nm UV light to remove the o-nitrobenzyl group. GPC and CD studies seemed to indicate that the Pn[UBU] chains were more tightly packed than Pn[BUB]. However, AFM experiments failed to show major differences between the two polymer series. Kaitz et al.236 combined a depolymerizable low-ceiling temperature (Tc) polymer with the strong DDAA quadruple hydrogen-bonding motif of UPy. The combination of the unique properties of the backbone along with the noncovalent cross-linking resulted in depolymerizable SCNPs at low concentrations and supramolecular networks at higher concentrations. The polymer backbone was constructed by anionic polymerization of o-phthalaldehyde (PPA) with 5nitroisiophtaladehyde (NIPA). The NIPA unit was transformed later into a UPy-containing moiety in two steps. The final random copolymer had a degree of polymerization of ca. 110 and about 8−9 UPy units per polymer chain. The formation of the SCNPs was confirmed by DLS, which showed a 17% decrease in the hydrodynamic radius (Rh) after the introduction of the UPy framework. PPA is known to depolymerize when exposed to acid.237 Indeed, when boron trifluoride etherate was added to the SCNP at room temperature immediate polymer-to-monomer unzipping was observed in the 1H NMR. The reconstitution of the polymer chains was conducted in −78 °C at low concentrations, below the Tc of the polymer, by applying acid and then base to

To investigate the influence of the molecular weight on the folding process, three triblock copolymers P1[UBU], P2[UBU], and P3[UBU] were synthesized with DPs varying from 90 to 420, which corresponded to molecular weights of 28, 53, and 124 kDa, respectively. Monitoring the folding process by VT-NMR (variable-temperature NMR) analysis revealed a pronounced peak shift and broadening of the signals upon slow cooling from 80 to 20 °C; notably, the amide protons which were significantly deshielded provided strong evidence for H bonding. Upon irradiation at 350 nm UV light, the benzylic protons from the protected Upy moieties disappeared and new peaks attributed to the Upy dimerization appeared at 10.1, 11.8, and 13.1 ppm. As evidence for reversibility, NMR analysis detected sharpening of the BTA proton signals and disappearance of the Upy dimer telltale signals above 50 °C, indicating that hydrogen bonds were disrupted. Recooling the sample led to BTA signal broadening and reappearance of the Upy dimer protons. The self-assembly of the chiral BTA moieties was probed using temperature-dependent CD spectroscopy. The BTA moiety concentration was set to 50 μM in a solvent mixture of 25/75 MCH/DCE to achieve a fine balance between backbone solubility and self-assembly. To ensure selfassembly under thermodynamic conditions, a slow cooling of 1 °C/min was applied. Above 65 °C, all polymers were CD silent, meaning the polymers are well solvated. Upon cooling, a negative Cotton effect developed, an indication for the lefthanded helical conformation (vide supra). The signal intensity was proportional to the polymer size and number of BTA moieties, implying that the aggregation occurs within a single chain. Subsequent irradiation at 20 °C led to no change in the CD spectrum, meaning that UPy dimerization does not affect the stability of the BTA helical aggregates, with only the largest polymer, showing a small deviation in the CD cooling curve before and after irradiation. Finally, the triblock SCNP formation process was also studied by SEC, AFM, and SAXS. SEC data showed a large decrease in polymer size varying from 34% to 62%, while SAXS indicated that the radius of 937


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Scheme 75. Depolymerization or Reconstitution of SCNPs Based on Low Tc Scaffolds

produce polymers of different molecular weights or even networks depending on the initial polymer concentration (Scheme 75). These PPA−UPy polymers thus exhibited two types of control: one by the dynamic polymerization of the PPA and the other by the self-assembly of the UPy moieties. In this manner, the reconstitution strategy provides an alternative method for the control of SCNPs size without changing the cross-linker unit loading or the original polymer mass. In a study designed for further understanding the forces involved in the noncovalent intramolecular self-assembly of polymer chains into SCNPs the Meijer and Guan groups used an AFM-based technique called single-molecule force spectroscopy (SMFS) to demonstrate the mechano-unfolding of localized SCNPs formed by interactions of BTA and UPy motifs.238 This innovative approach provided insight into the interior structure of SCNPs and afforded kinetic parameters governing the self-assembly of the BTA stacks. The polymer to be analyzed is first fixed in between the gold-coated AFM cantilever and a gold substrate using gold−sulfur chemoadsorption and then stretched mechanically to obtain a force vs extension (F−E) curve displaying the rupture of the hydrogen-bond cross-links. The polymer was produced by the random copolymerization of n-butyl acrylate (BA) and hydroxyethyl acrylate (HEA) using Cu-mediated ATRP with a bifunctional initiator followed by azidation of both terminal end groups and attachment of a propargyl lipoic acid ester using CuAAC. The furnished parent polymer P[−] (22.2 kDa, PDI = 1.26) had a DP ≈ 180, and the number of HEA residues was set to be ∼35. This parent polymer was subsequently equipped with UPy or BTA units to give the P[UPyn] and P[BTAn] series, where n denotes the estimated number of hydrogen-bonding entities per polymer chain (Scheme 76). As previously shown, the P[UPyn] folding is controlled by removing a photolabile o-nitrobenzyl protecting group upon irradiation, and for the P[BTAn] series the folding is induced upon heating and slow cooling. The F−E curve for the SCNPs based on the UPy motif displayed a “sawtooth” pattern, where each “tooth” is attributed to the

rupture of a UPy dimer under the stretching force applied. The UPy dimers are broken sequentially to give multiple rupture events, with the curves being fitted successfully to the wormlike chain (WLC) model. The rupture events occur within extension lengths of 5−40 nm, which is in good agreement with the overall contour length of the polymer chain (∼45 nm). As anticipated, the number of rupture events increased as the total number of UPy units increased from P[UPy2] to P[UPy6] and P[UPy18] with the total number of rupture events corresponding to the formula (n/2 + 1), where the extra rupture event was attributed to the detachment of the polymer chain from the gold surface or the AFM cantilever. As expected, the increase in the contour length is dependent on the number of UPy units, 15 nm in average for P[UPy2], 6.4 nm for P[UPy6], and 2.3 nm for P[UPy18]. The influence of the solvent was also investigated. Thus, in competing solvents such as 1,4 dioxane, the F−E plot displays mostly a single peak which is attributed to the detachment of the polymer chain from the surface or the AFM cantilever. In the same fashion, pulling experiments were performed with a P[BTAn] series using polymers with different BTA incorporation levels, P[BTA5], P[BTA9], and P[BTA12]. Similar to the experiments with the UPy motif, the F−E histogram showed the characteristic sawtooth” pattern, with comparable force required for breaking the BTA stacks. The BTA stacks have two possible trajectories in which they can unfold: the first in which an individual BTA molecule is peeled off from the end of the helical column or the second in which the BTA stack breaks in the middle to two smaller stacks. In both trajectories, the same number of rupture events is anticipated, 5 for P[BTA5], 9 for P[BTA9], and 12 for P[BTA12]. To the authors surprise, the F−E histogram showed significantly fewer rupture events than anticipated, 3 events for P[BTA5] and 4 events for both P[BTA5] and P[BTA12]. These results were explained by assuming that the stacking mechanism of BTAs in SCNPs not necessarily follows the well-studied cooperative process in free BTAs and that it is possible that the complex structure of the SCNPs does not permit all the BTA units to participate in the stacking process. Similar to the 938


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Scheme 76. Synthesis of Dithiolane Telechelic Polymers for Mechano-Unfolding Study

intramolecular self-assembly of a triblock copolymer using complementary hydrogen-bonding motifs, ureidoguanosinediaminonaphthiridine (UG-DAN), as side chain recognition units. The ABC-type triblock had the A and C blocks bearing the complementary hydrogen-bonding units, while block B played the role of a flexible linker. The polymers were synthesized by ROMP of norbornene carboxylic acid esters, the DAN-containing monomer was the first block, and then the norbornene octyl ester (NOE) spacer block and UGcontaining monomers were polymerized as the third block (Scheme 77). The key to the success of this design lies with the fact that the UG−DAN pair has a very strong association constant of 107 M−1 due to its complementary quadruple hydrogen-bond motif (Figure 13). The self-assembly process was studied by DLS and 1H NMR. DLS data showed that large aggregates over 10 nm are formed with concentrations over 30 mM. However, when concentrations were around 10−20 mM, DLS showed a single peak corresponding to a size of about 0.5 nm. 1H NMR of

UPy, a linear trend was observed for the contour length increase with the increase of the BTA units. The average contour length increase measured was 5, 4.5, and 3.5 nm for P[BTA5], P[BTA9], and P[BTA12], respectively. Combining the data on the number of rupture events and the average contour length increase it was concluded by the authors that for polymer chains with a low density of BTA units the BTAs tend to organize into one BTA stack, while for polymer chains with higher BTA density the formation of multiple and separated BTA stacks are formed. Dynamic force spectroscopy analysis experiments conducted on P[UPy6] and P[BTA9] allowed the calculation of the kinetic parameters such as the dissociation rate constants kd (for the first time for BTA stacking) and extrapolation of the ΔG° of the folding. This study represents an important step toward understanding the intricacies of the folding mechanism in SCNPs, especially with noncovalent forces that may show reversible behavior. 4.1.3. Ureidoguanosine−Diaminonaphthiridine Dimerization. A triblock copolymer motif was also used successfully by Romulus and Weck239 in work describing the 939


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Scheme 77. Polymerization of UG and DAN Containing ABC Triblock Copolymer

(CA) as the hydrogen-bonding donor unit at the α terminus and a Hamilton wedge (HW) unit at the other end (Scheme 78). The resulting α,ω-functionalized polymer chain (Mn = 6 kDa, PDI = 1.04) collapsed into a circular form when placed in chloroform at ambient temperature and under high dilution conditions (4 mM). The formation of a single-chain macrocycle was evidenced by the reduction of the hydrodynamic radius at low concentrations (below 1.5 mM) and its increase at higher concentrations due to interchain aggregation. The concept of forming cyclic SCNPs using heterotelechelic polymers was further explored241 using the complementary hydrogen-bonding interactions of thymine (Thy) and diaminopyridine (DAP). Thus, an α,ω Thy-DAPfunctionalized polystyrene was prepared via living radical polymerization using a thymine-functionalized ATRP initiator. The DAP moiety was attached postpolymerization to furnish a bifunctional polymer, Thy-PS-DAP with Mn = 9.3 kDa (Scheme 79). The self-assembly of Thy-PS-DAP into cyclic SCNPs was characterized using 1H NMR and DLS. At concentrations of 0.5 and 0.75 mM, the hydrodynamic diameter (Dh) measured was 1.2 and 1.4 nm, respectively, while the Dh for the polystyrene standards of similar molecular weight was 4.6 nm.

Figure 13. Complementary quadruple hydrogen-bonding interaction between UG−DAN.

samples under high-dilution conditions (100 μM) indicated intramolecular hydrogen-bonding and SCNP formation. Thus, for this system, the limit between intermolecular and intramolecular interactions was set around the 20 mM mark. 4.1.4. Hamilton’s Wedge−Cyanuric Acid and Thymine−Diaminopyridine Interactions. The preparation of PS chains by living radical polymerization equipped with α,ω complementary hydrogen-bonding motifs was reported by Altintas et al.240 The system was designed with cyanuric acid 940


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Scheme 78. Preparation of α,ω CA- and HW-Functionalized Polystyrene

Scheme 79. Preparation of α,ω Thy- and DAP-Functionalized Polystyrene

At concentrations higher than 1 mM the Dh increased significantly, suggesting intermolecular binding. The CA−W and Thy−DAP hydrogen-bonding chemistries were combined in a single polymer to form SCNPs.242 The target system was designed to feature the cyanuric acid in the α position, the thymine and DAP moieties at preselected positions along the polymer backbone, and the HW in the ω position. The orthogonality of the hydrogen-bonding pairs was examined using 1H NMR spectroscopy using small molecules containing the functional groups as models. After verifying the orthogonality of the molecule pairs, the challenge was to synthesize a single-polymer chain containing the 4 hydrogenbonding motifs. The methodology selected to produce the complex CA-PS-Thy-PS-DAP-PS-HW polymer system was ATRP and modular ligation relying on functionalized alkyne

bearing ATRP initiators (Mn = 154 kDa). To study the selfassembly process, a 1 mM CD2Cl2 solution of the polymer was left for 12 h at ambient temperature. The strong peak shifts in the NMR spectrum indicated that hydrogen bonding had occurred for both pairs. Upon standing for 7 days, no change in the NMR spectrum of the self-assembled polymer was observed. However, temperature-dependent 1H NMR revealed that the polymer chain goes through a rapid assembly disassembly processes. These NMR experiments allowed the calculation of the association constants, with the Kass of the CA−HW pair calculated to be 2.4 × 106 M−1 and the Kass for the Thy−DAP pair calculated to be 653 M−1. As expected, the 6-point hydrogen-bonding interaction of the CA−HW pair was much stronger than the triple hydrogenbonding interaction of Thy−DAP pair. SLS measurement confirmed the molecular weight of the polymer at high 941


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Scheme 80. Preparation of BTA-BiPy-Functionalized ROMP-Derived Copolymers

4.2. π-Stacking

dilution, showing that it is indeed a folded single chain. This report constituted one of the first steps toward highly controlled SCNP formation by using orthogonal folding chemistry. Recently, the Barner-Kowollik group implemented this strategy in the single-chain folding of a diblock copolymer.243 The design was to install the orthogonal Hbonding CA−HW and Thy−DAP pairs in designated positions along the polystyrene-b-poly(n-butyl acrylate) polymer backbone and trigger the single-chain folding to form an eight shape under the proper conditions, reminiscent of the work with click chemistry by Lutz et al.90 The precursor diblock copolymer (Mn = 15 kDa, PDI = 1.11) contained the CA in the α position and the DAP in the ω position with the orthogonal HW and the Thy units between both blocks. The self-assembly was induced by incubating the polymer (6.5 mg/mL) in CD2Cl2 for 1 h and confirmed by 1 H NMR. The disassembly of the SCNP could be achieved using a competitive solvent that can disrupt hydrogen bonding, e.g., THF or DMSO. DLS measurements for the polymer sample showed that the Dh in THF was 8.1 nm, while in DCM, the Dh was 5.9 nm, suggesting folding of the polymer chain. To show the significance of these results, a polystyrene standard sample (Mn = 17.4 kDa, PDI = 1.04, C = 6.5 mg/mL) was also measured in DCM and THF, affording a Dh of 5.6 and 5.8 nm, respectively. The threshold concentration below which only SCNPs are obtained (Csingle‑fold) and not multichain aggregates was determined by diffusion coefficient measurements using DOSY NMR techniques. Below a concentration of 10 mg/mL the diffusion coefficient became invariant with respect to the polymer concentration, thus determining the Csingle‑fold to be about 0.6 mM.

In recent work by Gillissen,244 SCNPs were formed via π−π interaction of BTA-BiPy (3,3′-bis(acylamino)-2,2′-bipyridinesubstituted benzene 1,3,5-tricarboxamide) substituents on a poly-5-norbornene exo-2,3 dicarboxylic acid anhydride scaffold (Scheme 80). The compact conformation of the folded polymers could be affected by increasing the BTA-BiPy functionalization degree or by changing the solvent polarity. Formation of the collapsed particles was monitored by UV−vis and fluorescence spectroscopy and by the increasing fluorescence attributed to the loss of rotational freedom of the bipyridine moiety upon cross-linking. Rigidification induced a red shift of the λmax in the UV region from 355 to 365 nm and the appearance of a shoulder at 385 nm. In addition, an increase in the fluorescence emission at 520 nm was observed. Furthermore, DLS and SLS measurements showed significant size reductions for the tested polymers upon changing the solvent polarity. By exploiting the bipyridine moiety affinity toward transition metal ions, these nanoparticles were used as ion sensors as the binding of the metal ions brings about quenching of the 520 nm fluorescence band. The affinity for CuII ions was 3 times higher than for other transition metal ions tested, with no dependence in the polymer molecular weight but a strong dependence on Bipy-BTA incorporation. A slight drawback for this sensing method is that it cannot discriminate between different metals, and the selectivity was not high enough to detect just a single ion. The Weck group245 recently reported on an ABC triblock system for mimicking β-hairpin structures using quadrupole π−π interactions246,247 between an electron-deficient polypentafluorostyrene (PPFS) block to an electron-rich PS block. A terpolymer containing styrene and pentafluorostyrene 942


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Scheme 81. Synthesis of Electron-Rich−Electron-Deficient Block Copolymers

Scheme 82. Schematic Illustration Showing Polymer Collapse by π-Stacking Interactions

Figure 14. Structure of amphiphilic random copolymers bearing different hydrophobic groups.

monomers as the starting materials for blocks A and C, respectively, was designed and synthesized by RAFT polymerization, with N,N-dimethylacrylamide as the middle block (Scheme 81). The apparent Mn for this triblock copolymer PS30-b-PDMAA150-b-PPFS30 was about 20 kDa (PDI = 1.73). To study the intramolecular self-assembly process (Scheme 82), dilute polymer solutions were prepared (1 mg/mL, 0.04 mM). In aromatic solvents, like benzene or toluene, the quadrupole interactions between the PPFS block and the PS block are inhibited due to the competitive interactions of the

PPFS block with the solvent. In noncompeting solvents, such as DMF, the PS and PPFS blocks interact, reducing the hydrodynamic radius measured. To further characterize the interactions between the copolymer blocks several analytical techniques including DLS, 2D 1H−1H NOESY, and 1H−19F HOESY experiments were conducted. This important work showed how the use of π interactions afforded yet another noncovalent force to impose single-chain collapse. 943


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Scheme 83. Preparation of Amphiphilic Methacrylate Random Copolymers

4.3. Hydrophobic Interactions

interaction between the 2-Np and the Py labels actually occurred at much lower concentrations. These results indicated that although the number of the carbon atoms was basically identical (11 or 12 carbons) the stability of the hydrophobic clusters was very different depending on their geometry, whether linear or cyclic. Following this study, the fluorescence of pyrene is known to be quenched by Tl+ ions due to a “heavy atom effect” in short-range interactions. Indeed, addition of thalium nitrate to the control system (AMPS/Py) resulted in efficient quench of fluorescence. On the other hand, addition of the thalium nitrate to the amphiphilic systems where the Py units were encapsulated within hydrophic domains did not quench fluorescence because the thalium ions could not interact with the pyrene. NMR relaxation and IR experiments supported the assignment of restricted conformations in water. Light scattering experiments for the cD-containing polymer showed a smaller hydrodynamic radius (5.5 nm) than expected relative to the calculated molecular weight (Mw = 51 kDa). In addition, SAXS experiments for 10 wt % solutions were in line with the DLS measurements and suggested that even at such high concentrations the dominant interaction was the intramolecular collapse of the polymer chain. Van De Mark and co-workers also developed a methodology for the formation of SCNPs in water exploiting the folding properties of a hydrophobic polymer backbone with hydrophilic residues.250 The polymer backbone in this case was based on hydrophobic MMA and hydrophilic methacrylic acid (MAA) in 9:1 ratio. The procedure for single-chain folding was to dissolve the polymer chain in a water-miscible organic solvent, e.g., THF, then the acid groups were ionized by addition of ammonium hydroxide followed by the addition of water up to the point where the polymer chain collapsed (60:40 THF:water). The solvent was then stripped off by evaporation, and the SCNPs remained suspended in water. Five polymers were synthesized with Mn between 28 and 122 kDa and PDIs between 1.24 and 1.47. The exact moment where the collapse of the polymer chain occurs is the point where the solution’s viscosity decreases sharply. DLS measurements of the formed SCNP revealed that the size of the SCNPs varied between 3 and 9 nm. Fairly high concentrations were used (100−200 mg/mL depending on polymer size), yet intermolecular aggregation

248

In pioneering work by Morishima et al., the self-associative behavior of random copolymers based on (2-acrylamido)-2methylpropanesulfonate (AMPS) and methacrylamide-based monomers carrying bulky hydrophobic substituents such as lauryl (LA) cyclododecyl (cD), 1-adamantyl (Ad), and 1naphthyl (1-Np) was investigated (Figure 14). All polymers were copolymerized in 1:1 ratio with 1 mol % of pyrene bearing methacrylamide as a fluorescent label. It was known from earlier studies249 that the ratio between the third and the first vibronic bands in pyrene (denoted as I3/I1) decreases with lowering polarity of the microenvironment; thus, it was deemed an effective tool to investigate polymer conformation. The I3/I1 ratio for the model compounds (copolymer of the water-soluble AMPS and 1 mol % of pyrene bearing methacrylamide) was 0.59; in contrast, the I3/ I1 in the (AMPS/LA/Py), (AMPS/cD/Py), and (AMPS/Ad/ Py) systems were 0.80, 0.83, and 0.76, respectively. This indicated that the Py labels were buried inside a more hydrophobic domain. Naphthalene and pyrene labels have been successfully used as singlet donor and acceptor pairs because they have a large spectral overlap and the former can be selectively excited at 290 nm. This technique was applied in this study to check whether the hydrophobic association was an intra- or intermolecular event. For these experiments, LA-, cD-, and Ad-containing polymers labeled with 2-naphthyl groups were prepared. If the polymer chain folds intramolecularly then the naphthyl groups and the Py groups would be buried within different domains and the system should not become fluorescent. On the other hand, if the folding was intermolecular then donor and acceptor may be confined within the same hydrophobic domain, thereby increasing the fluorescence. Aqueous solutions of the 2-Npcontaining polymers and the Py-containing polymers were irradiated at 290 nm, and the fluorescence was monitored at 340 and 395 nm. In the case of the Py−LA system, the fluorescence increased sharply at polymer concentration of 0.2 wt %, meaning that already at this concentration intermolecular association occurred. In contrast, for the cD- and Adcontaining polymers, intermolecular association seemed to predominate only at 7 wt % concentration. In methanol, where the polymers adopted a random coil conformation, the 944


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prepared and characterized by 1H NMR and FTIR spectroscopy. Methylene signals at 4.6 and 5.4 ppm in the 1H NMR spectroscopy that correspond to the PASP (NHCHCH2) and PSI (CCHCH2C) hydrogens confirmed the partial ring closure. Further evidence for succinimide formation was obtained from FTIR spectroscopy that showed stretching vibrations for the carboxylic groups of the amide (1710, 1640, and 1533 cm−1) in PASP and of the imide (1796 cm−1) in PSI. Being amphiphlic, the self-assembly of the star copolymers were investigated by TEM and DLS measurements (Figure 15).

was not observed because of the ionic repulsive interactions between the charged polymer chains. In an extensive report by Sawamoto251 and co-workers, the single-chain folding of amphiphilic random copolymers of PEGMA and hydrophobic methacrylates (RMA) (MMA, n-butyl methacrylate (n-BMA), tert-butyl methacrylate (t-BMA), n-octyl methacrylate (OcMA), dodecyl methacrylate (DMA), and octadecyl methacrylate (ODMA)) via intramolecular hydrophobic interactions was studied. A variety of PEGMA/RMA copolymers were synthesized with DPs of 100 or 200 and PEGMA/RMA ratios of 200/0, 120/80, and 80/20 (Scheme 83). The polymers were fully characterized by SEC, 1H NMR, UV−vis, MALLS, and DLS. MALLS showed that all polymers synthesized with 20% incorporation of RMA formed compact SCNPs in water, determining that the concentration of RMA per chain is a key factor in the folding process. Moreover, plotting the SEC peak-top molecular weight (Mp) (H2O)/Mp (DMF) as a function of the number of carbons in the R chain showed that this ratio clearly decreases with the growing hydrophobicity of the R chain, suggesting that folding becomes more compact with increasing pendant group hydrophobicity. In sharp contrast to the results obtained with the random copolymer, experiments with a block copolymer of PEGMA/DMA showed that large intermolecular aggregates in water (Rh = 110 nm) could be observed. These results clearly indicate that the formation of SCNPs requires a distribution of the hydrophobic groups along the whole polymer sequence and not in block. As a way to study the encapsulation of organic molecules within the SCNP, Reichardt’s dye (RD) was dissolved in water together with the polymer. RD is usually used to measure solvent polarity by virtue of being a solvatochromic compound that induces a hypsochromic (blue) shift with increasing solvent polarity. Due to its lower solubility in water, RD is often encapsulated in hydrophobic domains formed in water. In pure water RD absorbs at λmax = 453 nm and in pure acetone at 675 nm. In the presence of the PEGMA/DMA copolymer (160/40 PEGMA/DMA ratio), the λmax was red shifted to 544 nm, thus indicating that the dye was encapsulated within the hydrophobic domain of the SCNP. A study on the thermosensitivity of this type of polymers was also conducted. PEG-based polymers show lower critical solution temperature phase separation in water. Thus, the cloud point, Cp, was monitored by temperature dependent UV−vis and determined as the point of 50% transmittance at λ = 660 nm. All polymers exhibited reversible phase separation, but their cloud point was increased as the size of the pendant hydrophobic side chain increased. The authors reasonably concluded that hydrophobic moieties encourage the formation of a hydrophobic core and thus stabilize the SCNPs. Sumerlin and co-workers recently demonstrated the use of biodegradable polymeric nanocarriers for responsive delivery in plants.252 For this purpose, the authors synthesized an amphiphilic star copolymer poly(aspartic acid-co-succinimide) (PASP-co-PSI) (Mn = 26.6 kDa, PDI = 1.2) starting from the N-carboxyanhydride of β-benzyl-L-aspartate (Asp(OBzl)NCA) and using the multivalent 2,4,6-triaminopyrimidine as the ring-opening polymerization initiator. Subsequent deprotection of the benzyl esters, followed by the partial ring closing of the resulting star-PASP, delivered the desired amphiphilic star copolymer. Three different copolymers of varying succinimide content (25%, 40%, and 60%) were

Figure 15. (A) Transmission electron microscope (TEM) images of (PASP26-co-PSI17)3 and (B) dynamic light scattering (DLS) size distributions of PASP-co-PSI self-assemblies showing Z-average hydrodynamic diameters of (PASP32-co-PSI11)3, (25%-PSI) = 75 nm (PASP26-co-PSI17)3, (40%-PSI) = 140 nm, and (PASP17-coPSI26)3 (60%-PSI) = 186 nm.

Well-defined nanoparticles with an average size in the range of 30−60 nm, suitable for the delivery in plants, was obtained. Investigation on the controlled release of naphthalene acetic acid (NAA) (a synthetic plant hormone) showed a minimal NAA release at neutral pH, whereas upon raising the pH to 8.5 significant release of the encapsulated molecule was observed (Scheme 84). The authors attributed this behavior to the nanoparticle disassembly via the hydrolysis of hydrophobic PSI units to hydrophilic PASP units. 4.4. Host−Guest Interactions

4.4.1. Cucurbit[n]uril. In an inventive study, Appel et al.253 demonstrated the assembly of SCNPs in water with the assistance of cucurbit[8]uril (CB[8])-based host−guest interactions. The water-soluble polymer chain was constructed by ATRP of N-hydroxyethyl acrylamide; then the hydroxy group was subjected to isocyanate coupling to introduce both the viologen (MV) and the naphthyl guest components required to fill the cucurbituril cavity (Figure 16). Three such polymers were synthesized with molecular weights between 144 and 432 kDa and with low PDI (1.1−1.2). Addition of CB[8] to the polymer chain at high dilution (0.1 mg/mL or below) lead to chain collapse with 2 guest molecules occupying the CB[8] cavity (Scheme 85). Confirmed by DLS measurements, the size of the NP formed was dependent on the concentration of the CB[8] cross-linker. DLS measurements showed that the SCNP size varied between 18 nm for the random coil polymer and 12 nm after addition of 15 mol % of CB[8] for the polymer with a molecular weight of 243 kDa with MV and naphthyl incorporation ratios of 15 mol % each. An ingenious control experiment was conducted using CB[7], which possesses a smaller cavity that can accommodate only one guest molecule. DLS measurements of the polymer with CB[7] showed an 945


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Scheme 84. Synthesis of SCNPs from Amphiphilic Star Copolymer and Synthetic Hormone-Controlled Release Experiments

increase in particle size, indicating that chain collapse does not occur. The increase in size may be due to a more bulky side chain due to host−guest complexation of CB[7]. Mechanistic studies revealed that the viologen is encapsulated first,

followed by naphthyl penetration to the cavity. This greater understanding of the folding process is of utmost importance if controlled architectures in SCNPs are to be formed. The reversibility of the interactions, monitored by the fluorescence 946


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Figure 16. Acrylamide copolymer containing CB[8] guests as pendant units.

Scheme 85. Formation of SCNP via Reversible CB[8] Host−Guest Chemistry

response, was assessed by the addition of a competing guest, 1-adamantaneamine. Unfolding of the collapsed polymer occurred, although relatively high concentrations of about 1 mM were needed to overcome the high effective molarity of the polymer-bound guests. The work on the formation of SCNPs was expanded to another member of the CB[n] family by harnessing the host capacity of nor-seco cucurbit[10]uryl (ns-CB[10]) for MV guests to form 2:1 complexes.254 The polymer chain was constructed as previously described, with only MV substituents. Four polymers were prepared with molecular weights between 170 and 500 kDa. The polymer chain was synthesized with MV loadings of 10% and 30%. The collapse of the polymer chain to form nanostructures was observed upon addition of 5−15% ns-CB[10] to a stirred solution of the polymers at concentrations below 0.1 mg/mL. DLS measurements expectedly showed a decrease in the polymer chain hydrodynamic radius. Control experiments with CB[7] and a competing guest (p-xylenediamine) gave similar results to the previous study. 4.4.2. Cyclodextrins. The Harada group reported the construction of a PEG chain substituted with a cyclodextrin at one end and an azobenzene group on the other 6-AzPEG600-HyCiO-β-CD (Figure 17) and investigated the thermal and photochemical conformation switching of this polymer in water by ROESY-NMR analysis.255,256 After heating at 60 °C for 1 day and then cooling to 30 °C, the 1D 1H NMR showed only the peaks of the trans isomer in 6-trans-Az-PEG600-HyCiO-β-CD, and the 2D ROESY 1H NMR showed a strong correlation between the cyclodextrin protons and both the azobenzene peaks and the hydrocinnamoyl signals, suggesting self-inclusion of the polymer chain, with both trans azobenzene and hydrocinnamoyl protons occupying the cyclodextrin cavity. This conformation of 6-trans-Az-PEG600-HyCiO-β-CD was temperature dependent, and at 1 °C, the ROESY NMR showed only

Figure 17. Structure of 6-Az-PEG600-HyCiO-β-CD.

correlations between the cyclodextrin the hydroxycinnamoyl protons, suggesting a self-threading conformation. However, at 80 °C, no correlations with the cyclodextryin cavity protons were observed at all, meaning probably that the polymer completely unfolded. Upon UV light irradiation (340 nm) the azobenzene moiety photoisomerized to yield 6-cis-AzPEG600-HyCiO-β-CD. A 1 mM sample was examined at 30 °C to reveal a specific correlation between the CD cavity protons to the azobenzene peaks, suggesting a conformation with both aromatic rings of the azobenzene occupying the cyclodextrin cavity. In contrast to the 6-trans-Az-PEG600HyCiO-β-CD this conformation was quite stable and showed no concentration dependency (Scheme 86). On the basis of a similar concept, Barner-Kowollik and coworkers257 demonstrated a the reversible folding of singlepolymer chains in aqueous environment using cyclodextrin− adamantyl host−guest interactions (Scheme 87). The diendfunctionalized N,N-dimethylacrylamide polymer had a cyclodextrin host at one end and an adamantyl group as a highaffinity guest (Kassociation ≈ 105 M−1) at the other. SEC data revealed that the polymer synthesized by RAFT methodology, β-CD-PDMAa-Ada, had an Mn of 11.6 kDa (determined by SEC) with a polydispersity index of 1.19. Due to the tendency of the polymer to form intermolecular aggregates, folding experiments were conducted under high 947


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Scheme 86. Azo-Benzene β-CD α,ω PEG Polymer Conformations in Response to Thermal, Concentration, and Photochemical Stimuli

Scheme 87. Synthesis and Folding of Di-End-Functionalized N,N-Dimethylacrylamide Polymer Using the Host−Guest Interactions between β-CD and Adamantane

Scheme 88. Synthesis of PS-Based Telechelic Triphenylphosphine Macromolecular Ligands

948


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Scheme 89. Formation of Cyclic Polymer by Pd Complexation

Scheme 90. Preparation and Cross-Linking of Pd(II) SCNPs

to a Pd metal center.258 A symmetrical bifunctional ARGET ATRP initiator, ethylene bis(2-bromobutyrate), was utilized to polymerize a styrene monomer backbone to produce polymers with two bromine atoms at the α and ω ends. Subsequent substitution of the bromine atoms by azide followed by CuAAC between the azide groups in the polymer with the alkynes attached to the phosphine ligands produced the desired polymers (Scheme 88). Two macromolecular ligands were synthesized by this strategy with molecular weights of 6600 and 12.8 kDa (SEC). A ligand exchange reaction in DCM under high dilution with the telechelic polymer and Pd(COD)Cl2 led to the formation of what the authors dubbed single-chain metal complexes (SCMCs) (Scheme 89). The self-folding process was readily monitored by 1H NMR and 31P NMR, showing the expected downfield shifts. To verify that complexation leads to the formation of SCNPs; SEC and DLS experiments were conducted. The Dh measured for the macromolecular ligands was 3.8 and 7.1 nm. Upon complexation, the Dh decreased by 26% and 27% to 2.8 and 5.2 nm, respectively, although a small population of higher hydrodynamic radii due to intermolecular aggregation could also be detected. In the

dilution. The concentration limit for achieving single-chain folding was 0.6 mM as determined by DOSY (when the diffusion coefficient starts leveling). At a concentration of 0.57 mM, the number-weighted mean hydrodynamic diameter (Dh) was 7.7 nm; however, upon heating the Dh increased to 12.5 nm due to the reversibility of this interaction. After cooling to room temperature the measured Dh returned to 6.5 nm, which was in good agreement with the initial value. The reversibility of the folding process was also examined by a displacement experiment with a competing guest, 1-adamantamine hydrochloride. A solution of β-CD-PDMAa-Ada was heated to 70 °C with addition of excess of 1-adamantamine hydrochloride. After a 24 h equilibrium period the Dh measurement was 15.8 nm, indicating the displacement of the adamantyl group from the cyclodextrin cavity by the competing guest. 4.5. Metal Complexation

In one of the first works on the use of organometallic bonds to fold polymers, the Barner-Kowollik group reported the collapse of polymer chains by employing telechelic triphenylphosphine macromolecular ligands capable of binding 949


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Scheme 91. Preparation of SCNPs Through Cu(II) Complexation

coupling reaction between 2-bromopyridine and phenylacetylene in diethylamine and CuI as cocatalyst. The results showed 26% conversion for 5 mg of Pd−SCNPs and 45% conversion for double the amount of Pd−SCNPs after 24 h, yet the [Pd(PPh3)2Cl2] complex afforded 75% conversion under similar conditions. Thus, the insertion of the metal within the polymer was not found to be beneficial for this type of catalysis; perhaps the cocatalyst penetration to the polymer environment was not so efficient and hindered reaction progress. Pomposo et al.260 recently disclosed the synthesis and characterization of metallo-folded SCNPs by complexation with Cu(II) ions and their unique catalytic behavior. The random copolymer P1 backbone was constructed by copolymerizing MMA and 30% 2-(acetoacetoxy)ethyl methacrylate (AEMA) (Mw = 375 kDa, PDI = 1.4). The βdiketone moieties serve as the handle for binding Cu(II) ions and folding of the polymer chain. The SCNPs formation was carried out under mild conditions in THF at room temperature by using Cu(OAc)2 at high dilution conditions (1 mg/mL) to avoid intermolecular interactions (Scheme 91). The formation of the folded nanoparticles NP1 was monitored by SEC and MALLS. A significant increase in retention time was observed for NP1 compared to P1 as a result of the reduction in the hydrodynamic volume. MALLS showed that the radius of gyration (Rh) was decreased from 26 nm for P1 to 15 nm for NP1. Evidence for the AEMA βdiketone unit binding to the Cu(II) ion was further obtained via IR spectroscopy by monitoring the characteristic CO stretching band at 1600 and 1515 cm−1. TGA showed that the incorporation of copper was 26 mol % with respect to the AEMA units, and the oxidation state of the copper ions was determined to be Cu2+ by XPS. The catalytic behavior of these SCNPs was examined by a model oxidative crosscoupling reaction of terminal acetylene compounds. Control experiments were carried out with 3 mol % of CuCl2 and 3 mol % of Et3N. The most salient result with the SCNP catalyst was a distinct selectivity toward the homocoupling of a propargylic substrate from a mixture of other terminal alkyne-bearing substrates, while the control experiments

SEC experiments, cyclization manifested itself also in the apparent loss of molecular weight, 15% and 19% with respect to the molecular weight of the macromolecular ligands. After demonstrating the concept of selective point folding for Pd(II) SCNPs, the Barner-Kowollik group extended their work to form Pd (II) coordinated SCNPs in a “repetitive unit approach” where phosphane ligands were embedded along the polymer chain.259 This was realized by random copolymerization of styrene and 4-chloromethylstyrene (CMS). The polymer structure was verified by NMR, indicating 12% incorporation of CMS units. After polymerization and isolation of P1 (10.2 kDa, PDI = 1.17) the phosphine groups were installed by simple and quantitative substitution with 4diphenylphosphino benzoic acid to yield P2 (12.3 kDa, PDI = 1.16). Intramolecular cross-linking of P2 was performed under high-dilution conditions in dichloromethane using [Pd(COD)Cl2] as the Pd (II) source (Scheme 90). An alternative approach for the chain collapse was to add the polymer solution (0.2 M) to a Pd (II) solution in a dropwise manner (1 mL/min). This strategy circumvents the need for high dilution and afforded full cross-linking. SEC data showed a 21% decrease in the apparent mass of the SCNPs relative to the parent polymer’s mass, while DLS showed an expected decrease in the Dh from 8.8 to 5.4 nm. The cross-linking was confirmed also by 1H and 31P NMR and 1H spin−spin relaxation experiments by comparing chemical shifts of the SCNPs to the synthesized 4-methylphenyl diphenylphosphino(L1) and 4-ethylphenyl diphenylphosphino (L2) Pd(II) complexes, [Pd(L1)Cl2] and [Pd(L2)Cl2]. The X-ray diffraction of these complexes revealed a trans configuration in the solid state, and such configuration was also assumed to be present in the Pd−SCNPs. The transition from linear polymer (P2) to Pd−SCNPs was analyzed by lognormal distribution (LND) simulations which fitted perfectly to the experimental data obtained from the SEC chromatograms. To verify the phosphine ligand content of the polymer, XPS studies were conducted and confirmed that the phosphine ligand content was in line with the results seen by NMR analysis. In addition, the catalytic activity of these Pd−SCNP was tested using a benchmark Sonogashira 950


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Scheme 92. Synthesis of Cu-Containing SCNPs by a Cyclotetramerization Reaction

Scheme 93. Synthetic Route toward Poly(1,5-cyclooctadiene) (PCOD) SCNPs

and the polydispersities were between 1.1 and 1.2. The SCNPs were obtained by a cyclotetramerization reaction using CuCl and the phthalonitrile pendant groups in benzyl alcohol (Scheme 92). To minimize intermolecular interactions high-dilution conditions were applied (1.0 mg/mL). The color of the reaction mixture changed from dark brown to bluish green after the macrocyclization. The successful intramolecular chain collapse was confirmed by SEC with the SCNPs exhibiting a lower hydrodynamic volume than the polymer precursor. Furthermore, the formation of the SCNPs could be monitored also using UV−vis as a strong absorption at 600 nm as the reaction proceeds. Also, FTIR was used to study the reaction by monitoring the disappearance of the cyano group at 2230 cm−1 and the appearance of a new peak

afforded diyne mixtures. However, it must be noted that the reactions were run under solvent-free conditions where the SCNP probably undergoes intermolecular aggregation. The very promising specificity achieved in this work certainly deserves further study to continue toward the goal of enzymemimic polymers. Paik and co-workers reported on the incorporation of Cu into SCNPs using copper phthalocyanine (CuPC) structures.261 A styrene monomer containing phthalonitrile (VBOP) was synthesized via Williamson synthesis between 4-vinyl benzyl chloride and 4-hydroxyphthalonitrile. Well-defined copolymers of VBOP and styrene containing different molar feed ratios of the VBOP monomerP1 (3% VBOP), P2 (9% VBOP), and P3 (17% VBOP)were synthesized by RAFT polymerization. The molecular weights of the polymers were similar (41−46 kDa), 951


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corresponding to the imine group at 1720 cm−1. ICP analysis revealed that the SCNP derived from P1 contained 0.68% copper, which is approximately 5 CuPCs per polymer chain or about one copper atom every 10 kDa, although spectroscopic data gave a somewhat lower value of 3 phthalocyanines per chain. The SCNPs derived from P2 and P3 were calculated to have 7 phthalocyanines per chain, which seems to be the limit for the number of macrocycles that may be formed within the collapsed polymer. This new methodology allows for the preparation of organic soluble polymeric nanoparticles with useful phthalocyanines incorporated within them for imaging or catalysis applications.262 Lemcoff et al. recently reported on a convenient and straightforward synthesis of well-defined rhodium-containing SCNPs of ROMP-derived poly(1,5-cyclooctadiene) (PCOD) (Scheme 93).263 The ligand exchange reaction between commercially available chlorobis(ethylene) rhodium(I) dimer and model ligands containing a 1,5-diene motif was shown to be quite facile, and this methodology could be readily extended to PCOD (Mn = 25 kDa, PDI = 1.34) that also contains the 1,5-diene chelating moiety. The polymer scaffold reacted rapidly with 10 mol % of Rh(I) complex (with respect to the diene units in PCOD) in THF at room temperature under high-dilution conditions (10−5 M). SEC and DLS confirmed that the π-bound rhodium(I) indeed lead to chain collapse, with a decrease in the hydrodynamic radius from 8.9 to 6.1 nm after crosslinking and an expected increase in the GPC retention time. Control experiments conducted with polycyclooctene (PCOE), another polymer with a π-rich backbone but lacking the 1,5-diene motif, showed an increase in the hydrodynamic radius under the same reaction conditions, highlighting the importance of the chelating effect for single-chain collapse. TEM analysis showed spherical particles with a diameter of around 20 nm which was in good agreement with the DLS observations. To examine the relationship between the SCNP size and their metal content, a series of SCNPs with varying amount of metal content from 1% to 10% with rhodium(I) was synthesized. The results showed a systematic decrease in the polymer size from 9.3 (the original PCOD size) to 7.3 nm with 10% Rh(I) incorporation. The reversibility of the crosslinking was probed by reacting the collapsed SCNPs with 2(diphenylphosphino) benzaldehyde (PCHO) (Scheme 94).

electrical insulator; however, upon formation of the organometallic nanoparticles a clear semiconductor behavior was observed, showing the unique properties of these special SCNPs. This methodology to induce single-chain collapse to form organometallic nanoparticles was further extended to iridium(I) and nickel (0) metals, including also bimetallic SCNPs, and their catalytic potential was studied.264 The formation of Ir−ONPs using [IrCl(COE)2]2 dimer as crosslinker was verified by NMR, SEC, DLS, and TEM (Figure 18), as previously shown for Rh and by UV−vis spectroscopy as well, where a clear shift in the maximum of the absorbance curve could be seen upon complexation with the polymer.

Figure 18. (a and b) TEM Image of Ir−ONPs bearing 10 mol % Ir(I).

The synthesis of Ni(0) organometallic nanoparticles was somewhat more challenging due to the relative instability of nickel(0) complexes with 1,5-dienes. To overcome this, the Ni(0) organometallic nanoparticles (Ni(0)−ONPs) were prepared in situ by reducing nickel(II) acetylacetonate in the presence of PCOD in THF under high-dilution conditions (Scheme 95). Once again, SEC and DLS analyses provided compelling evidence for successful single-chain collapse. Following the simple complex addition protocol, organobimetallic nanoparticles containing both Rh(I) and Ir(I) metal centers (Rh−Ir−ONPs) were also synthesized with varying ratios of the metals and in indiscriminate commutative order (the order of addition of the metal did not affect the end result) (Scheme 96). Finally, the catalytic potential of the Rh(I) and Ir(I) nanoparticles was investigated and compared to organic transformations previously reported with simple 1,5-dieneligated Rh(I) and Ir(I) metal complexes. For example, allylation of acetophenone with allyl boronic acid smoothly afforded the expected product in 97% yield in the presence of 2 mol % Ir−ONP. Most significantly, after the reaction the Ir−ONPs were precipitated, filtered, and reused 3 times without loss of catalytic activity (filtration of the SCNP afforded the clean product without traces of the catalyst). In another reaction, the Ir−ONPs were successfully used as an Ir reservoir in the Et2SiH2-promoted reduction of benzyl benzamide (100% conversion). A more complex catalytic behavior was observed in the cross-coupling of aromatic aldehydes with phenyl boronic acid. In the presence of 5 mol % commercially available [RhCl(COD)]2 complex, 99% conversion of the starting material to the cross-coupled product was achieved. In contrast, when the same conditions were applied using the Rh−ONPs, only moderate conversions were obtained and the homocoupled biphenyl oxidation product was obtained as the major product. Control experiments hinted that the highly concentrated Rh(I) within

Scheme 94. Unfolding of Rh(I)−ONPs by Competing Ligand

The reaction was monitored by 1H NMR spectroscopy, and formation of the expected hydride complex was observed; moreover, SEC analysis showed that the polymer returned to its original size (9.3 nm), confirming that the Rh(I) crosslinker was stripped away from the PCOD backbone. To determine the electronic properties of such organometallic nanoparticles, an electrical conductivity study was performed. The precursor PCOD was shown to be an 952


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Scheme 95. Schematic Illustration of the Preparation of Ni(0)−ONPs

Scheme 96. Synthesis of Organobimetallic SCNPs with Rhodium(I) and Iridium(I)

macromolecules and assemblies of several nanometers in size is extremely common. Indeed, nature provides a compelling model of the significance of controlling the structure to determine function. Will we ever reach the bioinspired skill to fold polymers in a controlled manner to achieve desired functions? As with many other obstacles in the past, this is the paradigm we face today. The foldamer field has given an important step in this direction,219 but gaining fine control over the collapsed structure of synthetic polymers for specific functions will definitely be a remarkable achievement. Naturally, the emulation of natural enzymes is one of the top aspirations for chemists in the field, but also applications in nanomedicine, optoelectronics, and fundamental material properties can be foreseen to appear in the near future. Regarding the questions asked, we are probably still far away from reaching Nature’s efficiency in the control of macromolecular architecture to achieve desired functions. Nonetheless, the important first steps in this fascinating journey are being taken, and we are beginning to understand at least some of the challenges ahead. Herein, we summarized recent developments in this area, and in juxtaposition to its name, the field of single-chain collapse is certainly expanding rapidly both in structural and in functional capacities.

the limited volume of the SCNP played a part in the unusual outcome of this reaction. Interestingly, this was the first report for homocoupling selectivity under cross-coupling conditions. The authors also showed that the Rh−ONP could be further reacted with strongly coordinating N-heterocyclic carbene (NHC) ligand replacing one metal−diene bond. By doing this, the selectivity of the cross-coupling reaction was restored and 70% conversion could be obtained. Note, introduction of the bulky NHC ligands both disrupted the cross-link and increased the size of the macromolecule. The facile method by which the diene-containing polymers could be collapsed by using different metal ions bodes well for future applications in this field, including expanding the type of metals used and also introducing copolymers with orthogonal cross-linking functionalities.

5. CONCLUSIONS AND OUTLOOK Among some of the greatest challenges faced in materials science this century we find the development of smart materials at the top of the list. The design of complex and highly functional systems requires the appropriate scaffolds, and these can only be invented by novel technologies and creative approaches. Undoubtedly the field of single-chain collapse is rapidly growing. Richard Feynman postulated a fascinating question back in 1959: “What would the properties of materials be if we could really arrange the atoms the way we want them?” Synthetic chemists and materials scientists vie for the construction of distinct molecular architectures of definite sizes and shapes. For traditional synthetic organic chemistry, the dimension limit is up to a few tens of Angstroms. In living systems, however, the production of

AUTHOR INFORMATION Corresponding Author

*E-mail: lemcoff@bgu.ac.il. Notes

The authors declare no competing financial interest. 953


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Biographies

Inbal Berkovich (1984, Israel) received her B.Sc degree in Chemistry from Ben-Gurion University of the Negev in 2008. She then joined Professor Gonen Ashkenasy’s research group in Ben-Gurion University of the Negev as a graduate student, where she received her M.Sc degree in the field of protein-based molecular electronics devices. After working as a process engineer at Intel Electronics Ltd., she returned to Ben-Gurion University of the Negev in 2013 for Ph.D studies under the supervision of Professor N. Gabriel Lemcoff. Her research focuses on the development, characterization, and applications of polymeric organometallic complexes.

Sudheendran Mavila received his M.Sc. degree in Organic Chemistry from Mahatma Gandhi University, India, in 2003. After working as a senior scientist at Syngene International Ltd, India, he was a doctoral student under the supervision of Prof. Dr. Michael R. Buchmeiser at the University of Stuttgart, Germany. He received his Ph.D. degree in 2011 on metathesis-derived functional monolithic media. Since February 2012, he has been working as a postdoctoral fellow in Prof. N. Gabriel Lemcoff’s research group at the Ben-Gurion University of the Negev, Israel, on synthesis and application of organometallic nanoparticles.

N. Gabriel Lemcoff was born in Buenos Aires and immigrated to Israel in 1991. He finished his undergraduate studies at Tel-Aviv University, where he also received his Ph.D. degree in Chemistry in 2002 on novel macromolecular diacetal systems under the supervision of Prof. Benzion Fuchs. He then joined Prof. Steven C. Zimmerman’s group at the University of Illinois at Urbana− Champaign working on molecularly imprinted dendrimers. In the fall of 2004 he joined Ben-Gurion University of the Negev as a Senior Lecturer and was promoted to Associate Professor in 2011. His research deals mainly with the development of novel organometallic catalysts for olefin metathesis and the study of macromolecular architectures. Since 2012 he has been the head of the Chemistry Department at Ben-Gurion University of the Negev.

ACKNOWLEDGMENTS The Israel Science Foundation is gratefully acknowledged for financial support (grant no. 537/14).

Or Eivgi (1987, Israel) received his B.Sc. degree in Chemistry (Cum Laude) from the Ben-Gurion University of the Negev in Beer-Sheva in 2013. He then joined Prof. N. Gabriel Lemcoff’s group for his M.Sc. studies in Organic Chemistry working on selective photochemical reactions and novel redox reactions of aryl thiols. He will begin his Ph.D. studies this fall under the supervision of Prof. Lemcoff.

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NOTE ADDED AFTER ASAP PUBLICATION This paper published ASAP on August 13, 2015. The image for Figure 7 was incorrect. The revised version was reposted on August 18, 2015.

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Intramolecular Cross-Linking Methodologies for the Synthesis of

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