Synthesis of Single-Chain Nanoparticles for Catalysis and Sensing

Synthesis of Single-Chain Nanoparticles for Catalysis and Sensing Author: Jokin Pinacho Olaciregui Supervisors: Prof. José A. Pomposo and Prof. Daniel Taton Donostia – San Sebastián, 2024 (cc) 2025 Jokin Pinacho Olaciregui (cc by-nc-nd 4.0) CONTENTS ABSTRACT .............................................................................................................................................. 7 RESUMEN ............................................................................................................................................. 11 RÉSUMÉ ............................................................................................................................................... 16 1. INTRODUCTION ................................................................................................................................ 21 1.1. NANOSCIENCE AND SOFT MATTER ........................................................................................................... 23 1.2. SINGLE-CHAIN NANOPARTICLES .............................................................................................................. 25 1.2.1. Design and synthesis of SCNPs ................................................................................................ 26 1.2.1.1. Precursor synthesis .......................................................................................................................... 27 1.2.1.2. Precursor functionalization .............................................................................................................. 29 1.2.1.3. Intrachain Folding/Collapse ............................................................................................................. 30 1.2.1.4. Morphology of Single-Chain Nanoparticles (SCNPs) in Solution ...................................................... 36 1.3. APPLICATIONS OF SCNPS ...................................................................................................................... 38 1.3.1. Catalysis .................................................................................................................................. 39 1.3.2. Sensing .................................................................................................................................... 46 1.3.3. Nanomedicine ......................................................................................................................... 48 1.3.4. Other applications ................................................................................................................... 51 1.5. REFERENCES ....................................................................................................................................... 52 2. OUTLINE AND OBJECTIVES OF THE THESIS ........................................................................................ 61 3. LANTHANIDE-BASED SINGLE-CHAIN NANOPARTICLES AS “VISUAL” PASS/FAIL SENSORS OF MAXIMUM PERMISSIBLE CONCENTRATION OF CU2+ IONS IN DRINKING WATER .................................. 65 3.1 MOTIVATION ........................................................................................................................................ 67 3.2 INTRODUCTION ..................................................................................................................................... 67 3.3. MATERIALS, TECHNIQUES AND METHODS .................................................................................................. 69 3.3.1. Materials ................................................................................................................................. 69 3.3.2. Techniques ............................................................................................................................... 70 3.3.3.Methods ................................................................................................................................... 71 3.4. RESULTS AND DISCUSSION ...................................................................................................................... 72 2.4.1. Eu-SCNPs as m.p.c.(Cu2+) “visual” pass/fail sensors ................................................................ 72 3.4.2. Tb-SCNPs as m.p.c.(Cu2+) “visual” pass/fail sensors ................................................................ 84 3.4.3. Dy-SCNPs as m.p.c.(Cu2+) “visual” pass/fail sensors .............................................................. 93 3.5. CONCLUSIONS ................................................................................................................................... 100 3.6. REFERENCES ...................................................................................................................................... 101 4. GOLD NANOCLUSTERS SYNTHESIZED WITHIN SINGLE-CHAIN NANOPARTICLES AS CATALYTIC NANOREACTORS IN WATER ................................................................................................................ 105 4.1. MOTIVATION ..................................................................................................................................... 107 4.2. INTRODUCTION .................................................................................................................................. 107 4.3. MATERIALS AND METHODS .................................................................................................................. 109 4.3.1. Materials ............................................................................................................................... 109 4.3.2. Techniques ............................................................................................................................. 109 4.4. PROCEDURES ..................................................................................................................................... 110 4.4.1. Procedure for the Synthesis of Poly(OEGMA-co-AEMA) ........................................................ 110 4.4.2. Procedure for the Synthesis of Gold Nanoclusters (Au-NCs) within Poly(OEGMA-co-AEMA) Single-Chain Nanoparticles (SCNPs) ................................................................................................ 111 4.4.3. Procedure for the Reduction of 4-Nitrophenol Catalyzed by AuNCs/SCNPs .......................... 111 4.4.4. Procedure for the Reduction of Nitrobenzene Catalyzed by AuNCs/SCNPs ........................... 112 4.4.5. Procedure for the Reduction of 3-(4-Nitrophenyl)-1,3-oxazolidin-2one Catalyzed by AuNCs/SCNPs ....................................................................................................................................... 112 4.5. RESULTS AND DISCUSSION .................................................................................................................... 113 4.5.1. Synthesis of Gold Nanoclusters within Single-Chain Nanoparticles (Au-NCs/SCNPs) ............ 113 4.5.2. Gold Nanoclusters within Single-Chain Nanoparticles (AuNCs/SCNPs) as Catalytic Nanoreactors .................................................................................................................................. 118 4.5.2.1. Reduction of 4-Nitrophenol to 4-Aminophenol Catalyzed by Au-NCs/SCNPs ................................ 118 4.5.2.2. Reduction of Nitrobenzene to Aniline Catalyzed by Au-NCs/SCNPs .............................................. 125 4.5.2.3. Reduction of 3-(4-Nitrophenyl)-1,3-oxazolidin-2-one to 3-(4-Aminophenyl)-1,3-oxazolidin-2-one Catalyzed by Au-NCs/SCNPs ....................................................................................................................... 132 4.6. CONCLUSIONS ................................................................................................................................... 138 4.7. REFERENCES ...................................................................................................................................... 140 5. CONSECUTIVE ONE-POT ALKYNE SEMIHYDROGENATION/ ALKENE DIOXYGENATION REACTIONS BY PT(II)/CU(II) SINGLE-CHAIN NANOPARTICLES IN GREEN SOLVENT ...................................................... 143 5.1. MOTIVATION ..................................................................................................................................... 145 5.2. INTRODUCTION .................................................................................................................................. 145 5.3. MATERIALS AND METHODS ................................................................................................................... 147 5.3.1. Materials ............................................................................................................................... 147 5.3.2. Techniques ............................................................................................................................. 148 5.4. PROCEDURES ..................................................................................................................................... 149 5.4.1. Synthesis of P0 ....................................................................................................................... 149 5.4.2. Synthesis of P1 ....................................................................................................................... 149 5.4.3. Synthesis of P1-SCNPs (as a control) ..................................................................................... 150 5.4.4. Synthesis of Pt(II)-SCNPs ........................................................................................................ 150 5.4.5. Synthesis of Pt(II)/Cu(II)-SCNPs .............................................................................................. 150 5.4.6. Synthesis of Cu(II)-SCNPs (as a control). ................................................................................ 151 5.4.7. General procedure for the consecutive one-pot alkyne semihydrogenation/alkene dioxygenation reactions catalysed by ............................................................................................. 151 Pt(II)/Cu(II)-SCNPs in NBP at r.t. ...................................................................................................... 151 5.5. RESULTS AND DISCUSSION .................................................................................................................... 152 5.6. CONCLUSIONS ................................................................................................................................... 169 5.7. REFERENCES ...................................................................................................................................... 170 6. CONCLUSIONS ................................................................................................................................ 173 7. PUBLICATIONS ................................................................................................................................ 177 8. EXPERIMENTAL TECHNIQUES .......................................................................................................... 181 SIZE EXCLUSION CHROMATOGRAPHY (SEC) .................................................................................................... 183 DYNAMIC LIGHT SCATTERING (DLS) ............................................................................................................. 186 NUCLEAR MAGNETIC RESONANCE (NMR) ...................................................................................................... 187 FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) .................................................................................... 188 ULTRAVIOLET−VISIBLE SPECTROSCOPY (UV-VIS) .............................................................................................. 189 FLUORESCENCE SPECTROSCOPY .................................................................................................................... 190 ELEMENTAL ANALYSIS (EA) .......................................................................................................................... 191 INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS) ....................................................................... 192 UV IRRADIATION ....................................................................................................................................... 192 TRANSMISSION ELECTRON MICROSCOPY (TEM) .............................................................................................. 193 REFERENCES............................................................................................................................................. 194 AGRADECIMIENTOS/ACKNOWLEDGMENTS ........................................................................................ 197 7 Abstract This thesis dives into the world of Single-Chain Nanoparticles (SCNPs), from design to synthesis, by combining different metallic elements designed for applications in sensing and catalysis. Single-Chain Polymer Nanoparticles (SCNPs) are nanoscale particles formed by the folding and intramolecular cross-linking of single polymer chains. They exhibit unique properties such as high surface area, tuneable size, and the ability to encapsulate various substances, making them promising for applications in nanomedicine, catalysis, and material science. Chapter 1 of this Thesis serves as an introduction to the topic of SCNPs, detailing their formation, properties, and potential applications. This chapter aims to provide a comprehensive overview of the state of the art in SCNP research, allowing the progress made in this Thesis to be understood in the context of existing knowledge and technological advancements at the outset. Against this background, this work tries to further tailor the capabilities of SCNPs over and above the conventional functionalities with the help of a review that attempted to explore the most unique chemical and physical properties displayed by some metals, such as lanthanides (Eu, Tb and Dy), gold, platinum, and copper. Indeed, integration of metals within the SCNP architecture enhances their catalytic performance not only through their electronic and surface properties but also considers specific features of these metals' interaction with light and molecules to enhance their sensing properties. For example, gold's outstanding electron transfer characteristics or platinum's resistance to a myriad of conditions, have been used as a catalyst toward a variety of chemical transformations with a unique preciseness and ruggedness. On the other hand, the luminescence lanthanides are employed in the preparation of SCNPs for use as highly sensitive and selective sensors for environmental analytes. In the conjunction of knowledge comprising chemistry, material science, and nanotechnology, this is an interdisciplinary approach giving a comprehensive study over the synthesis of metal incorporated SCNPs. This thesis brings a series of well-designed experiments that not only contribute to the basic knowledge of SCNP assembly and strategies for metal integration but also open the door to new possibilities in applications in catalysis and 8 sensing technologies, highlighting the potential of SCNPs as a versatile and powerful tool in nanotechnological and materials scientific applications. The present study in Chapter 2 is aimed at synthesis and characterization of a new class of water-soluble lanthanide-containing SCNPs that have potential for visual sensing of Cu2+ ions for up to their maximum permissible concentration (m.p.c.) in drinking water. This novel sensing mechanism explores the characteristic properties of lanthanides and the structural benefits of SCNPs to design an easy, sensitive, and selective tool for monitoring copper ions, an important environmental and health concern. Even if copper is an important micro-element, it has the potential to turn into a toxic element at high concentrations, posing alarming situations both for the environment and human health. In this work, we developed europium (Eu), terbium (Tb), and dysprosium (Dy) integrated SCNPs that exhibit distinct fluorescence color changes under ultraviolet light upon reacting with Cu2+ ions, facilitating a straightforward "visual" pass/fail test. The approach of this process lies in the development of an amphiphilic random copolymer decorated with beta-ketoester functional groups, able to complex lanthanide ions, so as to obtain water-soluble lanthanide-containing SCNPs. This method does intrachain complexation of β-ketoester/lanthanide, which is significance for the formation and function of these nanoparticles. The characterization of SCNPs has been made by different approaches: size-exclusion chromatography (SEC), dynamic light scattering (DLS), inductively coupled plasma mass spectrometry (ICP-MS), and fluorescence spectroscopy. SCNPs have been successfully formed and are able to sensitize copper ions (Cu2+). The results seem to clearly indicate that all three Eu-, Tband Dy-based SCNPs can be very useful for a direct, visual detection of an excess Cu2+ concentration in water by the naked eye, due to the respective red-to-transparent, green-to-transparent and yellowtotransparent color changes under UV light. In summary, this work provides an important advance in environmental monitoring technology with the presentation of lanthanidebased SCNPs as effective, easy-to-use sensors for an important task: providing safe 9 drinking water. This approach will not only give help towards public health and environmental protection but opens the avenue for new development in terms of similar sensors for other contaminants. Chapter 3 was based on developing a novel idea comprising the synthesis of gold nanoclusters (Au-NCs) within single-chain nanoparticles (SCNPs) and to apply the former as a nanoreactor in aqueous catalysis. The efficiency and selectivity of metalloenzymes in cellular environments have provoked interest in the present study, which needs to be mimicked by natural catalysts using simplified metalloenzyme-mimetic nano-objects. As opposed to most metal-containing SCNPs reported so far, wherein the metal ions are complexed, in this work, the objective was the encapsulation of metal nanoclusters, more precisely Au-NCs with sizes smaller than 5 nm, within the SCNPs, in order to exploit their emergent catalytic properties. For the synthesis of Au-NCs/SCNPs, an amphiphilic random copolymer of poly (OEGMAco-AEMA) was used to self-assemble in water and form SCNPs, reducing agent Au(III) ions to Au(0). The beta-ketoester groups located in the AEMA units along the copolymer chain acted as very effective reductants and stabilizing agents for the Au-NCs. On the other hand, characterizations with TEM, DLS, and UV-Vis spectroscopy did establish a good conformation of formed and stable Au-NCs/SCNPs. The study revealed new utilities of these nanostructures as catalytic nanoreactors for the reduction of 4-nitrophenol, nitrobenzene, and 3-(4-nitrophenyl)-1,3-oxazolidin2one by borohydride (BH4−) in water at r.t. These reactions well represent benchmarks for the assessment of the catalytic activity of the synthesized Au-NCs/SCNPs in water and, therefore, open up an entirely new scope for potential applications regarding use in catalysis, biomedicine, energy, and electronics. These results, therefore, open new avenues for the synthesis and incorporation of metal nanoclusters within SCNPs for advanced catalysis in aqueous media. The fact that polymer and metal nanocluster-based systems actually manage to emulate 16 Résumé Cette thèse explore le monde des nanoparticules à chaîne unique (SCNPs), de leur conception à leur synthèse, en combinant différents éléments métalliques destinés à des applications dans la détection et la catalyse. Les nanoparticules polymères à chaîne unique (SCNPs) sont des particules à l'échelle nanométrique formées par le repliement et la réticulation intramoléculaire de chaînes polymères uniques. Elles présentent des propriétés uniques telles qu'une grande surface, une taille modulable et la capacité d'encapsuler diverses substances, ce qui les rend prometteuses pour des applications en nanomédecine, catalyse et science des matériaux. Le chapitre 1 de cette thèse sert d'introduction au sujet des SCNPs, détaillant leur formation, leurs propriétés et leurs applications potentielles. Ce chapitre vise à fournir un aperçu complet de l'état de l'art dans la recherche sur les SCNPs, permettant de comprendre les progrès réalisés dans cette thèse dans le contexte des connaissances existantes et des avancées technologiques. Dans ce contexte, ce travail tente de développer davantage les capacités des SCNPs audelà des fonctionnalités conventionnelles en s'appuyant sur une revue qui a tenté d'explorer les propriétés chimiques et physiques les plus uniques affichées par certains métaux, tels que les lanthanides (Eu, Tb et Dy), l'or, le platine et le cuivre. En effet, l'intégration des métaux dans l'architecture des SCNPs améliore leurs performances catalytiques non seulement grâce à leurs propriétés électroniques et de surface, mais prend également en compte les caractéristiques spécifiques de l'interaction de ces métaux avec la lumière et les molécules pour améliorer leurs propriétés de détection. Par exemple, les caractéristiques exceptionnelles de transfert d'électrons de l'or ou la résistance du platine à une myriade de conditions ont été utilisées comme catalyseur pour diverses transformations chimiques avec une précision et une robustesse uniques. D'autre part, les lanthanides luminescents sont utilisés dans la préparation des SCNPs pour servir de capteurs hautement sensibles et sélectifs pour les analytes environnementaux. En combinant des connaissances en chimie, science des matériaux et nanotechnologie, il s'agit d'une approche interdisciplinaire offrant une étude 17 complète sur la synthèse des SCNPs incorporant des métaux. Cette thèse présente une série d'expériences bien conçues qui non seulement contribuent aux connaissances de base sur l'assemblage des SCNPs et les stratégies d'intégration des métaux, mais ouvrent également la voie à de nouvelles possibilités d'applications dans les technologies de catalyse et de détection, mettant en lumière le potentiel des SCNPs comme outil polyvalent et puissant dans les applications nanotechnologiques et scientifiques des matériaux. L'étude présentée dans le chapitre 2 vise la synthèse et la caractérisation d'une nouvelle classe de SCNPs contenant des lanthanides solubles dans l'eau, ayant le potentiel de détecter visuellement les ions Cu2+ jusqu'à leur concentration maximale admissible (m.p.c.) dans l'eau potable. Ce nouveau mécanisme de détection explore les propriétés caractéristiques des lanthanides et les avantages structurels des SCNPs pour concevoir un outil facile, sensible et sélectif pour la surveillance des ions cuivre, une préoccupation importante pour l'environnement et la santé. Bien que le cuivre soit un micro-élément important, il peut devenir un élément toxique à des concentrations élevées, posant des situations alarmantes tant pour l'environnement que pour la santé humaine. Dans ce travail, nous avons développé des SCNPs intégrant de l'europium (Eu), du terbium (Tb) et du dysprosium (Dy) qui présentent des changements de couleur distincts sous lumière ultraviolette lors de la réaction avec les ions Cu2+, facilitant un test visuel simple "pass/fail". L'approche de ce processus réside dans le développement d'un copolymère aléatoire amphiphile décoré de groupes fonctionnels bêta-cétoester, capable de complexer les ions lanthanides, afin d'obtenir des SCNPs contenant des lanthanides solubles dans l'eau. Cette méthode réalise une complexation intrachaîne du complexe βcétoester/lanthanide, ce qui est significatif pour la formation et la fonction de ces nanoparticules. La caractérisation des SCNPs a été réalisée par différentes approches : chromatographie d'exclusion de taille (SEC), diffusion dynamique de la lumière (DLS), spectrométrie de masse à plasma à couplage inductif (ICP-MS) et spectroscopie de fluorescence. Les SCNPs ont été formés avec succès et sont capables de sensibiliser les ions cuivre (Cu2+). 18 Les résultats semblent indiquer clairement que les SCNPs à base de Eu, Tb et Dy peuvent être très utiles pour une détection directe et visuelle d'un excès de concentration de Cu2+ dans l'eau à l'œil nu, en raison des changements de couleur respectifs de rouge à transparent, vert à transparent et jaune à transparent sous lumière UV. En résumé, ce travail apporte une avancée importante dans la technologie de surveillance environnementale avec la présentation de SCNPs à base de lanthanides comme capteurs efficaces et faciles à utiliser pour une tâche importante : fournir de l'eau potable sûre. Cette approche contribuera non seulement à la santé publique et à la protection de l'environnement, mais ouvrira également la voie à de nouveaux développements en termes de capteurs similaires pour d'autres contaminants. Le chapitre 3 est basé sur le développement d'une idée novatrice comprenant la synthèse de nanoclusters d'or (Au-NCs) au sein de nanoparticules à chaîne unique (SCNPs) et l'application de ces derniers comme nanoréacteur en catalyse aqueuse. L'efficacité et la sélectivité des métalloenzymes dans les environnements cellulaires ont suscité l'intérêt de la présente étude, qui doit être imitée par des catalyseurs naturels utilisant des nano-objets mimétiques de métalloenzymes simplifiés. Contrairement à la plupart des SCNPs contenant des métaux rapportés jusqu'à présent, où les ions métalliques sont complexés, dans ce travail, l'objectif était l'encapsulation de nanoclusters métalliques, plus précisément des Au-NCs de taille inférieure à 5 nm, au sein des SCNPs, afin d'exploiter leurs propriétés catalytiques émergentes. Pour la synthèse des Au-NCs/SCNPs, un copolymère aléatoire amphiphile de poly (OEGMA-co-AEMA) a été utilisé pour s'auto-assembler dans l'eau et former des SCNPs, réduisant les ions Au(III) en Au(0). Les groupes bêta-cétoester situés dans les unités AEMA le long de la chaîne du copolymère ont agi comme réducteurs et agents stabilisants très efficaces pour les Au-NCs. D'autre part, des caractérisations par TEM, DLS et spectroscopie UV-Vis ont établi une bonne conformation des Au-NCs/SCNPs formés et stables. L'étude a révélé de nouvelles utilités de ces nanostructures en tant que nanoréacteurs catalytiques pour la réduction de 4-nitrophénol, nitrobenzène et 3-(4-nitrophényl)-1,3- 19 oxazolidin-2-one par borohydrure (BH4−) dans l'eau à température ambiante. Ces réactions représentent bien des points de référence pour l'évaluation de l'activité catalytique des Au-NCs/SCNPs synthétisés dans l'eau et ouvrent donc une nouvelle perspective d'applications potentielles en catalyse, biomédecine, énergie et électronique. Ces résultats ouvrent ainsi de nouvelles voies pour la synthèse et l'incorporation de nanoclusters métalliques au sein des SCNPs pour une catalyse avancée en milieu aqueux. Le fait que les systèmes à base de polymères et de nanoclusters métalliques parviennent effectivement à imiter l'activité des métalloenzymes ouvre donc des promesses de développement de systèmes catalytiques efficaces, stables et sélectifs dans un large champ d'applications. Le chapitre 4 introduit une nouvelle approche de la catalyse avec des nanoparticules polymères à chaîne unique hétérobimétalliques Pt(II)/Cu(II) (SCNPs), introduisant stratégiquement des groupes fonctionnels α-diazo-β-cétoester et β-cétoester nus dans la structure des SCNPs. Cela implique la génération de carbène photoactivée avec une lumière de longueur d'onde de 365 nm et des ions Pt(II), en sélectionnant le dichloro(1,5cyclooctadiène)Pt(II) pour son affinité avec les groupements carbène générés. Par la suite, le Cu(II) est introduit par des réactions avec l'acétate de Cu(II), résultant en une structure polymérique de complexes Cu(II)-(β-cétoester)2. Cette incorporation duale de métaux est essentielle pour le repliement du précurseur polymérique en la structure nanoscopique finale, comme en témoignent la spectroscopie infrarouge, la chromatographie d'exclusion de taille et la diffusion dynamique de la lumière, indiquant la formation de SCNPs Pt(II)/Cu(II) bien définis. La nouveauté de cette investigation réside dans le développement et l'application de ces nouveaux SCNPs. 1. Introduction 1. Introduction 23 1.1. Nanoscience and Soft Matter Since the advent of nanoscience and nanotechnology, as pioneered by Nobel laureate Richard P. Feynman in his seminal 1959 lecture, "There's Plenty of Room at the Bottom”,1the scientific community has witnessed a plethora of transformative developments across the disciplines of physics, chemistry, and biology. These advancements have materialized Feynman's visionary concepts of manipulating matter at the most diminutive scale, specifically at the molecular and atomic levels, herein referred to as the nanoscale. The term "nanotechnology," albeit subject to varied interpretations across different scientific domains and geographical regions, is frequently employed as an umbrella term to encapsulate technologies that operate on an exceedingly minute scale. Nonetheless, nanotechnology is principally delineated as the scientific endeavor encompassing the comprehensive understanding, precise manipulation, and strategic restructuring of matter at dimensions on the order of nanometers (notably, less than 100 nm).2 Whitesides (2004) highlights that at this scale, the properties of materials undergo a fundamental shift, distinguishing them markedly from their bulk counterparts.3 This field has witnessed an exponential growth over recent decades, leading to the emergence of groundbreaking disciplines such as nanomedicine, as elucidated by Mishra (2013),4 nanoelectronics, described by Puers et al. (2017),5 and nanocatalysis, as discussed by Bahadur-Singh and Kumar-Tandon (2014).6 The surge in nanoscience research and investment is well-documented by Velmurugan and Radhakrishnan (2016).7 Solid-state physics and electronics have ventured into the realm of nanostructures through the employment of lithography and etching processes, denoted as the "topdown" approach. This methodology facilitates the creation of structures with dimensions not less than approximately 20 nanometers. Conversely, nature exemplifies an alternative paradigm for the assembly of diminutive structures. In this context, 1. Introduction 24 individual molecules are amalgamated into larger functional entities and intricate structural hierarchies through a process of self-organization, a strategy referred to as the "bottom-up" approach.8 In nature, dynamic individual biomacromolecules self-assemble into a variety of functional nanoentities on various scales,9 giving rise to complex materials like viruses or DNA. Seen from this angle, the natural world serves as a model for the design of small buildings. Fundamental to the creation of nanostructures is the nanoparticle, a fundamental unit that is larger than atoms or simple molecules, which are subject to quantum mechanics, but much smaller than macroscopic objects governed by Newtonian mechanics.10 Nanoparticles are classified into two varieties based on composition: "hard nanoparticles", which are usually made of inorganic materials, and "soft nanoparticles", which are made of organic components. Exceptional control over size and shape has been achieved in the creation of hard nanoparticles, such as metal oxide nanoparticles, quantum dots, and gold nanoclusters. Macromolecular architecture modification has made tremendous strides in the last several decades in the field of artificial soft nano-objects. The development of effective Controlled Radical Polymerization (CRP) methods is primarily responsible for these improvements. Many complicated macromolecular structures, including star polymers, comb-like copolymers, and hyperbranched macromolecules, have been developed by using CRP methodologies.11, 12 The materials produced as a result of these processes differ significantly from their linear analogs with similar molecular weights in terms of their characteristics (Figure 1). Within this spectrum of artificial soft nano-objects, a notable category is represented by Single-Chain Nanoparticles (SCNPs). 1. Introduction 25 Figure 1. Different examples of hard-nanoparticles and soft-nanoparticles classified by type and morphology. 1.2. Single-Chain Nanoparticles The development of Single-Chain Polymer Nanoparticles (SCNPs) constitutes a vibrant area within the realm of macromolecular chemistry, aimed at replicating the precision of natural polymeric models. Analogous to the configuration of proteins, SCNPs are crafted through the intramolecular folding of synthetic linear polymer precursors (Figure 2). This innovative approach positions SCNPs as viable candidates for a wide array of applications across diverse fields such as catalysis,13,14microelectronics,15 nanomedicine,16 DNA delivery mechanisms,17 sensory technologies16 or imaging agents.18 Figure 2. Schematic illustration of a linear polymer precursor and a single-chain nanoparticle (SCNP) obtained through intra-chain folding/collapse of individual polymer chains at very dilute conditions. 1. Introduction 32 Figure 7. Illustration of cross-linker induced collapse technique employed for the construction of SCNPs. Regarding the bonding interactions utilized, single-chain nanoparticles are synthesized through intrachain covalent bonds (yielding irreversible SCNPs) or via intrachain noncovalent and dynamic-covalent bonds (yielding reversible SCNPs). A) Irreversible Single-Chain Polymer Nanoparticle Systems Covalent bonding The synthesis of irreversible single-chain nanoparticles (SCNPs) is facilitated through covalent bonding, utilizing both conventional organic and "click" chemistry reactions for the effective creation of robust unimolecular soft nanoparticles. It's imperative to note that the covalent stabilization of polymer geometry negates the dynamic nature of single-chain entities, thus restricting their application in dynamic biomimetic systems, such as protein folding/unfolding mechanisms. Initial disclosures of irreversible single-chain nanoparticle synthesis via the intrachain homocoupling method under highly diluted conditions by Mecerreyes et al. utilized poly(styrene)-, poly(alkyl methacrylate)-, and poly(ε-caprolactone)-based precursors with vinyl reactive groups.19 Jiang et al. and Cherian et al. respectively utilized unsaturated groups for poly(4-N-Boc-aminostyrene)- and poly(carbonate)-based singlechain nanoparticles.35 Subsequently, Harth et al. synthesized benzosulfone-decorated precursors for individual unimolecular nanoparticle creation via quinodimethane 1. Introduction 33 formation under similar conditions.36 Poly(methyl methacrylate)-based SCNPs were obtained by Zhu et al. through intramolecular Bergman cyclization at lower temperatures (150 ºC).37 The synthesis of poly(styrene) and poly(alkyl methacrylate)based unimolecular nanoparticles via intramolecular cross-linking of sulfonyl azide-38 and benzoxazine-39 functionalized polymers required elevated temperatures (190–200 ºC). Additionally, Dirlam et al. recently prepared poly(styrene)- based unimolecular nanoparticles via intrachain crosslinking through oxidative polymerization of 3,4propylenedioxythiophene functional groups at 50 ºC.40 Figure 8. Scheme of the Michael synthesis route followed by Sanchez-Sanchez et al.41 for the formation of the SCNPs. Colmenero et al. presented a study using small-angle neutron scattering (SANS) and neutron spin echo (NSE) techniques to explore the structure and dynamics of irreversible single-chain nanoparticles (SCNPs) in a dilute solution. These SCNPs were obtained through Michael addition-mediated multidirectional self-assembly (Figure 8). Trimethylolpropane triacrylate acted as the intrachain cross-linking agent. Precursors were random copolymers of methyl methacrylate (MMA) and (2-acetoacetoxy)ethyl methacrylate (AEMA).41 The intrachain heterocoupling technique has been employed for the fabrication of irrevesible single-chain nanoparticles using efficient azide-alkyne "click" chemistry (i.e., copper-catalyzed [3+2] cycloaddition of alkynes and azides (CuAAC)).42 Ruiz de Luzuriaga et al. synthesized biofunctionalized poly(methyl methacrylate) unimolecular 1. Introduction 34 nanoparticles at room temperature and high yield utilizing azideand protected alkynedecorated polymer precursors.43 Pomposo et al. simplified this technique, employing copolymers with protected alkyne and chloromethyl groups, converting them to azidomethyl groups via a straightforward substitution reaction with sodium azide.44 The intrachain hetero-coupling method has also facilitated the creation of thermoresponsive single-chain nanoparticles.45 Nitrile imine mediated tetrazole-ene cycloaddition (NITEC) has been applied for the synthesis of well-defined fluorescent single-chain nanoparticles.18 The crosslinker-mediated collapse of polymer chains represents another efficient methodology for obtaining SCNPs. Poly(γ-glutamic acid)-based single-chain nanoparticles were produced through the crosslinker-induced collapse method using a biosynthetic poly(γ-glutamic acid) precursor and 2,2´-(ethylenedioxy)diethylamine as the bifunctional crosslinker in the presence of carbodiimide.46 B) Reversible Single-Chain Polymer Nanoparticle Systems Two principal types of reversible interactions, i.e., non-covalent interactions and dynamic covalent bonds, enable the synthesis of responsive, structurally dynamic singlechain nanoparticles (SCNPs). Non-covalent Interactions The concept of non-covalent bonds has captivated researchers across various fields since Linus Pauling's seminal work on hydrogen bonding in the 1930s.47 Jean Marie Lehn's introduction of “supramolecular chemistry”48 has facilitated the exploration of non-covalent bonds in constructing advanced artificial architectures, including supramolecular polymers. The susceptibility of main-chain supramolecular polymers to environmental variations, manifesting in solvent polarity and concentration-dependent molecular weights, underscores the influence of factors such as temperature, pressure, and concentration on non-covalent bond strengths and the equilibrium between bonded and unbonded species. 1. Introduction 35 Non-covalent, or supramolecular, interactions are categorized into three classes based on bond strength: i) weak interactions (0-15 kcal/mol), including van der Waals forces, hydrophobic interactions, π-π stacking interactions, and hydrogen bonds; ii) medium strength interactions (15-60 kcal/mol), such as multiple hydrogen bonds and weaker metal coordination complexes; and iii) strong interactions (>60 kcal/mol), encompassing ionic interactions, host-guest interactions, and robust metal coordination or chelate complexation (Figure 9). The strength of non-covalent bonds is contingent on external factors like solvent type, temperature, and concentration, precluding a strict categorization.49 Non-covalent or supramolecular interactions have facilitated the creation of complex architectures involving side-chain supramolecular polymers,50 multi-block supramolecular polymers,51 and multi-arm self-assembled stars.52 Figure 9. Organization of non-covalent interactions by bond strength.49 Dynamic Covalent Bonds Dynamic covalent chemistry encompasses reversible covalent reactions that enable the exchange of molecular components to reach the system's thermodynamic equilibrium.53 Dynamic covalent bonds, exhibiting the robustness of covalent bonds, are only disruptable and reformable under specific external conditions (e.g., catalyst presence). Furthermore, dynamic covalent chemistry has successfully contributed to dynamic combinatorial libraries,54 drug discovery,55 and controlled fragrance delivery.56 1. Introduction 36 Hawker, Kim, and their colleagues' pioneering efforts in 2008 demonstrated an efficient approach to supramolecular single-chain nanoparticles via benzamide dimerization, forming quadruple hydrogen bonds between benzamide motifs.57 Subsequent extensive research by Meijer, Palmans, and their teams advanced the field of metastable noncovalent bonded single-chain nanoparticles through the introduction of orthogonal techniques based on ureidopyrimidinone (UPy)58 and benzene-1,3,5-tricarboxamide (BTA)59 hydrogen bonding motifs. Meijer's group recently showcased sequential supramolecular nanoparticle synthesis through complementary self-assembly of UPy and BTA units.60 Host-guest interactions involving cucurbit[n]uril complexation61 and hydrophobic L-phenylalanine Phe-Phe interactions62 have been leveraged to fabricate water-borne supramolecular single-chain nanoparticles. 1.2.1.4. Morphology of Single-Chain Nanoparticles (SCNPs) in Solution Understanding and controlling the morphology of SCNPs overall is a necessity to properly utilize their properties in a wide variety of applications including drug delivery, sensing, and the development of new nanomaterials. Being able to extract the precise SCNPs out of precursor polymer synthesis will be critical in the future of how we can effectively predict the final morphology type for a well-designed SCNPs for a desired application. As illustrated in Figure 10, it all begins in the synthetic selection of the precursor: choosing block copolymers and enabling mostly Janus SCNPs with generally selfassembly behaviors enabling complex multi-molecular structures such as micelles and lamellae, or random copolymers with mostly isolated/sparse or globular morphologies without self-assembly behaviors in general. 1. Introduction 37 Figure 10. Scheme of the different morphologies of SCNPs from block copolymer precursor and from random copolymer precursor. It showed some self-assembly structures formed by Janus SCNPs and the similarity of morphology between sparse SCNPs and intrinsically disordered proteins (IDPs), and globular SCNPs and enzymes. Amphiphilic block copolymers self-assemble into nanostructures due to block incompatibility. Janus Single-Chain Nanoparticles (SCNPs) 63 have bifacial properties, leading to twin or tadpole morphologies, and can form micelles, lamellae, or vesicles. 64 1. Introduction 38 Random copolymers, in contrast, are formed from two or more monomers polymerized in a random sequence, resulting in a statistical distribution of monomers along the polymer chain. These random copolymers can give rise to either sparse or globular SCNPs. Sparse SCNPs are morphologically analogous to Intrinsically Disordered Proteins (IDPs), while globular SCNPs resemble enzymes. In good solvents, sparse SCNPs exhibit a morphology with extended linear sections and compact regions, driven by the interplay between intra-chain cross-linking and a preference for short-range loop formation, as demonstrated by Molecular Dynamics (MD) simulations.65, 66 Globular SCNPs, on the other hand, possess a homogeneous coreshell structure akin to that of native proteins, such as enzymes. The synthesis of globular SCNPs often involves complex synthetic routes. Techniques such as the utilization of long cross-linkers and bifunctional groups have been employed to enhance loop formation and compaction within these structures. 67, 68 1.3. Applications of SCNPs The versatility of single-chain nanoparticles (SCNPs) encompasses a broad spectrum of applications, including catalysis, sensing, and nanomedicine, as depicted in Figure 11. The efficacy of SCNP technology across these domains stems primarily from the nanoparticles' minute dimensions and their adaptability tailored to specific functionalities. Although research endeavors pertaining to SCNPs have predominantly delved into fundamental investigations, these materials have manifested practical utility in various contexts. Intrinsically, the interior composition of the nanoparticle furnishes a conducive chemical milieu, characterized by tunable hydrophobic or hydrophilic properties, alongside controlled size modulation achievable through manipulation of intra-chain cross-linking densities and the molecular weight of the precursor polymer. Moreover, the integration of selective functional sites within the nanoparticle structure is facilitated by controlled polymerization methodologies and post-polymerization modification techniques. Subsequent sections encapsulate recent applied research endeavors elucidating the diverse applications of SCNPs. 1. Introduction 39 Figure 11. Illustration of some examples of potential applications of SCNPs. 1.3.1. Catalysis The development of catalytically active single-chain nanoparticles is considered an essential approach in polymer chemistry, creating new ways for the design of catalysts seamlessly integrating the advantages of both homogeneous and heterogeneous catalysis systems. The PhD thesis regards the concept of polymer chains serving as spatially demanding supports for metal-ions in catalytic systems and the effects of such configurations on the catalytic efficiency. Substitution of molecular ligands by polymer chains in organometallic catalysts is an event that inflicts large changes which may influence catalytic activity either by diffusional limitations or because of dense catalytic pockets, which hinder substrate access. This phenomenon noted by the group led by Pomposo underscores the challenge for ensuring the free diffusion within the SCNP nanoreactors in order to maintain activity but, at the same time, recognizing that selective substrate access should sharpen reaction specificity. 1. Introduction 40 The initial development of SCNPs focused on structural models, using intramolecular crosslinking to induce chain collapse into nanoparticle form. Earlier studies on such systems were aimed at understanding changes in intrinsic viscosity and the radius of gyration upon chain collapse.69 This era of discovery offered us critical knowledge of the physical behaviour of SCNPs, but at that time, did not yet leverage their catalytic potential. Significant advancements came in the early 2010s with new polymer synthesis and crosslinking methodologies, reviving interest in SCNPs and paving the way for their application in catalysis.70 The concept of SCNPs as functional nanoreactors began to take shape, leveraging the idea of intramolecular crosslinking to fold polymer chains into discrete, stable nanostructures. These nanostructures could be tailored to incorporate catalytic sites, mimicking the active sites found in natural enzymes. This approach allowed for the development of SCNPs with highly specific substrate binding and catalytic efficiency, similar to that of enzymes. One pioneering study in this area was conducted by Pomposo et al., who explored the catalytic site distribution within 'clickase' SCNPs. They show that SCNPs with clustered catalytic sites had significantly higher activity than those with homogeneous catalytic site distribution, which suggested that precise internal morphology control can give rise to enhancements in catalytic performance to a much greater extent than was realized at the beginning of the discoveries about SCNPs.71 This discovery underscored the potential of SCNPs to achieve enzyme-like efficiency through synthetic means. The incorporation of metal ions into SCNPs was particularly fruitful. Barner-Kowollik and Roesky teams reported bimetallic configurations in SCNPs. Platinum and europium metals were combined, leading to the development of multifunctional nanoreactors. Such SCNPs offered high catalytic activity, ease of recoverability, and reusability—the two limitations highly relevant for conventional homogeneous catalysts.72 In an illustrative example, Knöfel et al. reported the synthesis of phosphine-functionalized polymer chain-bearing SCNPs and their use as homogeneous catalysts in the amination 1. Introduction 41 of allyl alcohol. Pt(II)-SCNPs, depending on the polarity of the solvent, could be separated from the reaction mixture and recycled in additional cycles with good potential in terms of high catalytic efficiency and ease of recoverability. They also worked on the synthesis and the catalytic applications of SCNPs that contain both Au and Y. The researchers synthesized SCNPs chemically by trapping Au(I) and Y(III) ions into the polymer matrix. Such NPs were observed to exhibit recyclable and very high catalytic behavior. Research gave much attention to the recovery and reusing of SCNPs over several catalytic cycles by keeping the efficiency and stability of the same. This alluded to the possible uses of the new heterobimetallic SCNPs in sustainable catalysis, toughness, and application to chemical reactions.73 Further advancements were made adding additional functionalities into SCNPs. Maag et al. developed SCNPs that exhibit fluorescence, facilitating easy tracking and recycling of the catalysts. This novelty highlighted the possibility to combine catalytic function with optical properties in SCNPs, meaning a broader potential application field.74 A library of zinc porphyrin cores was prepared based on poly (methyl methacrylate-co-anthracene methacrylate) polymers by Patenaude, Berda, and Pazicni. These SCNPs were equipped with different functional groups, and showed enhanced reactivity and substrate specificity, emphasizing the importance of secondary coordination spheres in SCNP catalysis.75 The structural design of SCNPs was inspired by natural enzymes, particularly their ability to create a specified microenvironment for the catalytic reaction. Researchers have imitated those structural attributes of natural enzymes during the design of SCNPs to attain high catalytic efficiency and substrate specificity. For example, Palmans and Meijer developed a concept of ruthenium-based catalysts, dispersed in amphiphilic block copolymers, to synthesize SCNPs with central hydrophobic cores similar to enzyme active sites. Such strategy provided an improvement not only in terms of substrate binding but also in the protection of the catalytic sites from deactivation. As a result, operational life of the catalyst was extended in this design.76 1. Introduction 48 changes in fluorescence changed SCNPs potential activity, making them applicable to high-sensitivity and stable fluorescent detection of trace levels of H2O2.90 SCNPs have been developed with outstanding sensitivity, selectivity, and versatility as sensing systems. The development of SCNPs for metal ion detection, protein sensing, and hydrogen peroxide detection symbolizes the broad applicability and innovative potential of these nanostructures. With inevitable progress in research, SCNPs will likely find their applications broadened even further and firmly claim their place in advanced sensing technologies. 1.3.3. Nanomedicine Nanomedicine is a discipline leveraging the particular characteristics of nanoparticles for purposes of diagnosis, monitoring, therapy, and control of biological systems. The use of SCNPs in conveying all such purposes outplays those played by traditional small molecules and other nanoparticle systems due to more prolonged circulation times, lower degradation, and facility of targeting of specific tissues or cellular markers to enhance delivery and therapeutic agent efficacy.91 Cytotoxicity Studies Biocompatibility is one of the most critical aspects a nanomaterial should possess when it has been designed for biomedical applications SCNPs. Biocompatible SCNPs were synthesized using the strategy of organo-catalyzed ring-opening polymerization by Qiao et al.92 The synthesized SCNPs showed low toxicity to human embryonic kidney cells (HEK293T). Their study revealed that these SCNPs, covalently cross-linked by biodegradable polyester linkages, were nontoxic at concentrations up to 100 µg/mL, showing promise for safe biomedical applications. Further investigations by Lemcoff and Zimmerman explored the influence of molecular weight on cell viability using fluorescein-containing SCNPs. They found that SCNPs with molecular weights ranging from 50 to 100 kDa exhibited high HeLa cell viability (≥80%) at a concentration of 10 µM. This study highlighted the potential of SCNPs to serve as 1. Introduction 49 non-toxic carriers for imaging agents or therapeutic drugs.93 Additional research by Loinaz et al. (2016), combined in vitro and in vivo toxicity research toward SCNPs from poly(methacrylic acid) and they observed minimal toxicity in pancreatic adenocarcinoma cell lines at concentrations up to 50 µg/mL and no acute toxicity in mice at a dose of 100 mg/kg, underscoring the safety of SCNPs for potential clinical use.94 Controlled Drug Delivery Systems SCNPs have been extensively studied as nanocarriers for the controlled delivery of therapeutic agents, including chiral amino acid derivatives, peptides, vitamins, and small molecule drugs. The precise control over SCNP morphology and functionality enables tailored drug release profiles, enhancing therapeutic efficacy while minimizing side effects. For instance, Hamilton and Hard developed SCNP-based nanocarriers for the intracellular delivery of peptide therapeutics. These SCNPs were decorated with dendritic molecular transporters and fluorescent probes, facilitating efficient cellular uptake and controlled peptide release while preserving the biological activity of the peptides.95 This approach has significant potential for delivering therapeutic peptides to intracellular targets, which are often challenging to reach with conventional delivery systems. Pomposo et al. synthesized SCNPs designed for the dermal delivery of vitamin B9. These SCNPs showed a progressive and controlled release of the vitamin following a Fickian diffusion mechanism, with complete release observed within six hours.96 In a subsequent study, they explored the simultaneous delivery of hinokitiol and vitamin B9, achieving efficient release profiles at different pH levels. This dual-delivery system demonstrated the versatility of SCNPs in administering multiple therapeutic agents with distinct release kinetics. Cheng and Zimmerman developed stimuli-responsive SCNPs capable of encapsulating the anticancer drug 5-fluorouracil (5-FU). These SCNPs exhibited excellent thermo/pH- 1. Introduction 50 responsive behavior, with the highest drug release observed under conditions mimicking the tumor microenvironment (lower pH and higher temperature). This specificity reduces the risk of premature drug release and enhances the therapeutic targeting of cancer cells.97 Image Contrast Agents SCNPs have also been evaluated as potential agents for various imaging techniques, including magnetic resonance imaging (MRI), single photon emission computerized tomography (SPECT), and fluorescence imaging. These applications leverage the unique optical and magnetic properties of SCNPs, enhancing the sensitivity and specificity of imaging modalities. For example, Loinaz and Odriozola synthesized gadolinium (Gd3+)-decorated SCNPs that demonstrated enhanced relaxivity compared to traditional gadolinium chelates. These SCNPs, with an average relaxivity value of 6.8 mM-1s-1, offered improved contrast in MRI scans, making them promising candidates for advanced diagnostic imaging.98 Similarly, Harth et al. developed water-soluble SCNPs with dual MRI and fluorescence imaging capabilities, enhancing their multifunctionality for diagnostic applications.99 Fluorescent SCNPs have also been developed with reduced photobleaching and enhanced emission properties. Zimmerman et al. demonstrated the use of fluorescein-loaded SCNPs for long-term bioimaging, showing strong fluorescence signals inside HeLa cells.100 As a conclusion, The potential of SCNPs in nanomedicine is vast, with promising applications in drug delivery, imaging, and sensing. Their unique properties, such as controlled morphology, biocompatibility, and the ability to functionalize with various therapeutic and diagnostic agents, position SCNPs as versatile tools in the advancement of nanomedicine. Future research is expected to further explore and optimize these applications, paving the way for innovative therapeutic and diagnostic solutions. The development of SCNPs offers a glimpse into the future of personalized medicine, where treatments are tailored to individual patients' needs, enhancing efficacy and reducing side effects. 1. Introduction 51 1.3.4. Other applications Beyond the primary applications previously delineated, Single-Chain Nanoparticles (SCNPs) are poised to revolutionize the creation of new materials endowed with tailored properties. By strategically engineering polymer chain architectures, it is now possible to synthesize materials demonstrating customized mechanical, thermal, or electrical characteristics. These materials are destined to find application in everything from standard coatings to advanced composite materials. While these represent more specialized applications currently under investigation, SCNPs have been used to engineer nanostructures,101enhance oil recovery processes,102 and synthesize plasmonic nanoparticles.103 The use of these materials has also been expanded to design hydrophobic coatings104 and realize hydrophobic cotton with greatly improved resistance,105 ,106 underscoring the pervasive utility of SCNPs in the field of material science and engineering. 1. Introduction 52 1.5. References 1. Feynman, R. P. Plenty of Room at the Bottom. 2. THE NATIONAL NANOTECHNOLOGY INITIATIVE Strategic Plan. www.nano.gov. (2007). 3. Whitesides, G. M. Nanoscience, Nanotechnology, and Chemistry. Small 1, 172– 179 (2005). 4. Mishra, A. K. Nanomedicine for Drug Delivery and Therapeutics. (John Wiley & Sons, 2013). 5. Front Matter. in Nanoelectronics i–xlviii (2017). doi:https://doi.org/10.1002/9783527800728.fmatter. 6. Singh, S. & Tandon, P. Catalysis: A brief review on Nano-Catalyst. 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Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 67 3.1 Motivation The maximum permissible concentration (m.p.c.) of Cu2+ ions in drinking water, as set by the World Health Organization (WHO) is m.p.c.(Cu2+)WHO = 30 µM, whereas the US Environmental Protection Agency (EPA) establishes a more restrictive value of m.p.c.(Cu2+)EPA = 20 µM. Herein, we develop -for the first time evera family of m.p.c.(Cu2+) “visual” pass/fail sensors based on water-soluble lanthanide-containing single-chain nanoparticles (SCNPs) exhibiting an average hydrodynamic diameter below 10 nm. Both europium (Eu)- and terbium (Tb)-based SCNPs allow excessive Cu2+ concentration to be readily detected in water, as can be naked-eye monitored by red-totransparent and green-to-transparent color changes under ultra-violet (UV) light irradiation, respectively, taking place at ca. 30 µM of Cu2+ ions in both cases. Complementary, dysprosium (Dy)-based SCNPs show a yellow-to-transparent color transition under UV light irradiation at ca.15 µM of Cu2+ ions. Eu-, Tband Dy-containing SCNPs prove to be selective for Cu2+ ions as they do not respond against other metal ions, such as Fe. These new m.p.c.(Cu2+) “visual” pass/fail sensors are thoroughly characterized by a combination of techniques including size exclusion chromatography (SEC), dynamic light scattering (DLS), inductively coupled plasma mass spectrometry (ICP-MS), infra-red (IR), UV and fluorescence spectroscopy. 3.2 Introduction Copper is an essential trace element for animals and plants.1 In fact, Cu2+ -the 3th most important metal ion present in biological systemis involved as cofactor of numerous metalloenzymes.2,3 Cu2+ ions are also essential for the human body and defect of this crucial micronutrient can impart cytopenia (a reduction in the number of mature blood cells) and profound neurological deficits.4,5 On the contrary, an excessive Cu2+ intake from occupational exposure or contaminated water can cause gastrointestinal problems, liver and kidney damage, hemolytic anemia and impaired immune function, among other effects.6,7 Thus, Cu2+ ions act as one of the environment pollutants to control due 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 68 to their increased use at several levels (home, industrial, and agricultural operations), as well as environmental persistency.8 Different regulatory Organisms have established the maximum permissible concentration (m.p.c.) of Cu2+ ions in drinking water. The m.p.c. of Cu2+ ions in drinking water by the World Health Organization (WHO) is m.p.c.(Cu2+)WHO = 30 µM, whereas the US Environmental Protection Agency (EPA) establishes a more restrictive value of m.p.c.(Cu2+)EPA = 20 µM.9,10 Although different analytical techniques are available to accurately determine the concentration of Cu2+ ions in drinking water (e.g., atomic absorption spectroscopy, ion chromatography, inductively coupled plasma mass spectrometry), these techniques often involve sophisticated instrumentation and require skilled users. Consequently, alternative methods based on “visual” pass/fail sensors providing operational simplicity, sensitivity and selectivity are highly desirable. To tackle that challenge, we envisioned to design single-chain polymer nanoparticles (SCNPs) incorporating lanthanide metal ions as a versatile platform towards a new generation of m.p.c.(Cu2+) operating as “visual” pass/fail sensors. SCNPs are individual polymeric chains that are intramolecularly folded through intra-chain interactions.11 The folding of discrete synthetic polymer chains into SCNPs attempts to mimic the natural folding of biomacromolecules, such as proteins.12 The chain folding process leads to locally compact domains within SCNPs, which can be of practical use to bind active species (e.g., metal ions,13 luminophores,14 drugs15). Consequently, SCNPs offer interesting opportunities for the development of improved sensors16, innovative drug delivery vehicles17 and biomimetic catalysts,18 among other practical applications. On the other hand, a variety of complexes based on lanthanide ions19-22 have been investigated as fluorescent “turn-off” sensors of Cu2+ ions. However, most of these molecular sensors are not directly operative in water, but instead, in organic solvent/water mixtures. We hypothesized that, by using an amphiphilic random copolymer featuring beta-ketoester functional groups able to complex with lanthanide ions, water-soluble lanthanide containing SCNPs could be 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 69 achieved via intra-chain beta-ketoester/lanthanide complexation. The resulting lanthanide based SCNPs were thus employed as innovative m.p.c.(Cu2+) “visual” pass/fail sensors in drinking water. Here we report the rational design of europium (Eu)-, terbium (Tb)- and dysprosium (Dy)-based SCNPs, as well as their use -for the first time everas efficient “visual” pass/fail sensors of maximum permissible concentration of Cu2+ ions in water. 3.3. Materials, techniques and methods 3.3.1. Materials Oligo(ethylene glycol) methyl ether methacrylate (OEGMA) (average Mn 300 g/mol), (2-acetoacetoxy)ethyl methacrylate (AEMA) (95%), 2,2-azobis(2-methylpropionitrile) (AIBN) (≥98%), triethylamine (Et3N) (>99%), methyl acetoacetate 99%, 1,4-dioxane (anhydrous, 99.8%), hexane (anhydrous, 95 %), 4-cyano-4-(thiobenzoylthio)pentanoic acid (≥ 97%), europium trichloride hexahydrate (EuCl3 x 6 H2O) (99.99% trace metals basis), terbium trichloride hexahydrate (TbCl3 x 6 H2O) (99.99% trace metals basis), dysprosium trichloride hexahydrate (DyCl3 x 6 H2O) (99.99% trace metals basis), copper (II) acetate (98%), iron (II) acetate (95%), cobalt (II) acetate tetrahydrate (99%), barium acetate (99%), nickel (II) acetate tetrahydrate (99%), mercury (II) acetate (≥ 98.0%), lead (II) acetate tetrahydrate (99%) zinc (II) acetate dehydrate (99%), iron (III) acetylacetonate (97%), calcium acetate monohydrate (99%), manganese (II) acetylacetone (98%), magnesium chloride (98%) and chromium(III) acetate (98%) were supplied by Merk (SigmaAldrich). Potassium hydroxide (KOH) (≥ 85%, pellets) was supplied by PanReac AppliChem (ITW Reagents). Silver(I) acetate (99%) was supplied by ITW Reagents (Acros Organics). Deionized water was obtained from a Thermo Scientific apparatus (Barnstead TII Pure Water System). Tetrahydrofuran (THF) was supplied by Scharlab. Deuterated chloroform (CDCl3) (99.8 atom % D) was supplied by Merk. 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 70 3.3.2. Techniques 1H nuclear magnetic resonance (NMR) spectra were obtained at room temperature (r.t.) using a Bruker spectrometer operating at 400 MHz with CDCl3 as the solvent. Size exclusion chromatography (SEC) measurements were conducted at 30 °C in an Agilent 1200 system that was equipped with PLgel 5 μm Guard and PLgel 5 μm MIXED-C columns. The measurements employed a triple detection system, which included a differential refractive index detector (Optilab Rex, Wyatt), a multi-angle laser light scattering (MALLS) detector (MiniDawn Treos, Wyatt), and a viscosimetric (VIS) detector (ViscoStar-II, Wyatt). SEC data were analysed using Wyatt's ASTRA Software (version 6.1). Tetrahydrofuran (THF) was used as the eluent with a flow rate of 1 mL/min. For both the precursor and the single-chain nanoparticles, a value of dn/dc = 0.1150 was applied. Dynamic Light Scattering (DLS) measurements were carried out at r.t. on a Malvern Zetasizer Nano ZS apparatus. Metal content in the single-chain nanoparticles was determined by inductively coupled plasma mass spectrometry (ICP-MS). Fourier transform infrared (FTIR) spectra were recorded at r.t. on a JASCO 3600 FTIR spectrometer. UV spectroscopy was carried out in an Agilent 8453A spectrometer. Photoluminescence (PL) spectra were recorded at r.t. on an Agilent Cary Eclipse spectrometer. Horiba Laquatwin-pH-33 compact pH-meter was used for pH measurements. Scheme 1. Schematic illustration of the synthesis of lanthanide (Ln)-based single-chain polymer nanoparticles (SCNPs) using an amphiphilic random copolymer decorated with beta-ketoester functional groups, namely, poly(OEGMA-co-AEMA). 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 71 3.3.3.Methods Synthesis of amphiphilic random copolymer featuring beta-ketoester functional groups OEGMA (1.54 mL, 5.6 mmol), AEMA (0.26 mL, 1.38 mmol), 4-cyano-4-(thiobenzoylthio) pentanoic acid (18.3 mg, 0.065 mmol), and AIBN (2.15 mg, 0.013 mmol) were dissolved at r.t. in 1,4-dioxane (3 mL). The resulting mixture was degassed by purging with argon for 15 min. Then, the mixture was subjected to reversible addition fragmentation chaintransfer (RAFT) copolymerization at 70 °C for 24 h. The resulting poly(OEGMAco-AEMA) copolymer, a pink oil, was isolated by precipitation in hexane. Subsequently, the product was dissolved in a minimal amount of THF and added to an excess of hexane (twice). After removal of volatile organic solvents, further drying was carried out at r.t. under vacuum and corfirmed by 1H NMR (Figure 1). Figure 1. 1H NMR spectrum of poly(OEGMA-co-AEMA). 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 72 Synthesis of lanthanide-based single-chain polymer nanoparticles (SCNPs) Poly(OEGMA-co-AEMA) (15 mg, 0.03 mmol) and the corresponding lanthanide trichloride hexahydrate (LnCl3 x 6 H2O; Ln = Eu, Tb, Dy) (30 μM) were dissolved in water (15 mL) at r.t. and pH = 7.5 for 24 h. The resulting lanthanide-containing SCNPs were purified by dialysis against deionized water. ICP-MS data about the lanthanide content in the SCNPs are provided in Table 1. Successful formation of SCNPs was confirmed by SEC and DLS measurements, according to well-established literature procedures.11 To mitigate possible interference from molecular oxygen, all lanthanide-based SCNPs were subjected to an extensive degassing (N2) process before performing the fluorescence measurements. Table 1. SEC and ICP-MS data of Ln-SCNPs. Ln-SCNPs Mw(MALLS) (kDa) Ɖ Ln3+ content (µg/mg)a Ln3+ content (µg/mg)b Eu-SCNPs 85.0 1.12 4.6 2.7 Tb-SCNPs 88.9 1.36 4.8 2.0 Dy-SCNPs - - 4.9 - aTheoretical value. bExperimental value determined from ICP-MS measurements. 3.4. Results and discussion 2.4.1. Eu-SCNPs as m.p.c.(Cu2+) “visual” pass/fail sensors Europium-based single-chain nanoparticles (Eu-SCNPs) were prepared at a concentration of 1 mg/mL, following the procedure depicted in Scheme 1. A linear amphiphilic random copolymer, namely, poly(OEGMA-co-AEMA) was first synthesized by 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 73 RAFT copolymerization of the hydrophilic monomer OEGMA and the hydrophobic monomer AEMA. Poly(OEGMA-co-AEMA) was found to contain 35 mol% of betaketoester functional groups, as determined by 1H NMR spectroscopy (Figure 1). Poly(OEGMA-co-AEMA) showed a weight-average molecular weight of Mw = 80.7 kDa and relatively low dispersity, Ɖ = 1.11 (Table 1). Eu-SCNPs resulted from the complexation of Eu3+ ions in solution by beta-ketoester functional groups of poly(OEGMA-co-AEMA) (see Scheme 1). 7.5 8 8.5 9 9.5 Eu-SCNPs poly(OEGMA-co-AEMA) SEC retention time (min) Figure 2. SEC traces of the precursor, poly(OEGMA-co-AEMA), and the Eu-SCNPs The formation of Ln3+/beta-ketoester complexes involving low molecular weight organic compounds is well documented.23-25 To the best of our knowledge, however, there are no precedents of single-chain polymer nanoparticle formation via intra-chain lanthanide/beta-ketoester complex formation. Successful preparation of Eu-SCNPs was confirmed by a combination of structural and size characterization techniques. Hence, SEC results confirmed a shift of the SEC elution time at peak maximum towards longer retention time and, hence, smaller hydrodynamic size for Eu-SCNPs when compared to 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 80 Figure 11. FTIR spectra of poly(OEGMA-co-AEMA) (blue colour) and Eu-SCNPs (red colour) Figure 12 shows the PL spectra of Eu-SCNPs in water in the presence of increasing amounts of Cu2+ ions (λexc = 254 nm). As shown in Figure 13, Eu-SCNPs can be used as “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water, according to the WHO criterion (m.p.c.(Cu2+)WHO = 30 µM).9 A clear red-to-transparent colour change under UV light (λexc = 254 nm) is indeed observed also in Figure 13 on passing from [Cu2+] ≤ 27.5 µM to [Cu2+] = 30 µM. 100020003000 Poly(OEGMA-co-AEMA) Eu-SCNPs Wavenumber [cm-1] 1632.4 cm-1 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 81 Figure 12. PL spectra of Eu-SCNPs in water in the presence of increasing amounts of Cu2+ ions. Figure 13. (e) demonstration of the utility of EuSCNPs as “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water according to the WHO criterion (i: 5 µM, ii: 10 µM, iii: 15 µM, iv: 22.5 µM, v: 25 µM, vi: 27.5 µM and vii: 30 µM). Analysis of the data in Figure 14 using the Stern-Volmer equation (I0/I = 1+ KSV[Cu2+]) provided a value of KSV = 1.4 x 105 M-1 and a squared coefficient of linear regression of R2 = 0.99. 0 10 20 30 40 50 60 70 80 580 590 600 610 620 630 640 650 Eu-SCNPs [Cu2+] 5 µM [Cu2+] 7.5 µM [Cu2+] 10 µM [Cu2+] 12.5 µM [Cu2+] 15 µM [Cu2+] 17.5 µM [Cu2+] 20 µM [Cu2+] 22.5 µM [Cu2+] 25 µM [Cu2+] 27.5 µM [Cu2+] 30 µM Intensity (a.u.) Wavelenght (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 82 Figure 14. Stern-Volmer plot (I0/I =1+ KSV[Cu2+]) of Eu-SCNPs (error bars estimated from triple measurements) Remarkably, Eu-SCNPs proved highly selective for Cu2+ ions against other metal ions, such as Fe2+, Ag+, Co2+, Ba2+, Ni2+, Hg2+, Pb2+, Zn2+, Fe3+, Ca2+, Mn2+, Mg2+ and Cr3+. From the bar chart (Figure 15), it's clear that the response of Eu-SCNPs to Cu2+ is significantly higher than to other metal ions, indicated by the towering peak for Cu2+. This suggests that Eu-SCNPs have a high selectivity for Cu2+ ions. The line graph (Figure 16) shows the photoluminescence (PL) spectra of Eu-SCNPs in the presence of different metal ions, including Cu2+. The spectra for Cu2+ shows a pronounced quenching of Eu3+ emission, which is not observed with other metal ions. This quenching is attributed to energy transfer from Eu3+ to Cu2+ ions, followed by nonradiative relaxation to the ground state of the Cu2+ ions,27,28 confirming the selectivity of Eu-SCNPs for Cu2+ ions over others. This property is particularly valuable for the development of selective sensors for Cu2+ in various applications. 1 2 3 4 5 6 0 5 10 15 20 25 30 I0/I [Cu2+] µM KSV = 1.4 x 105 M-1 R2 = 0.99 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 83 Figure 15. PL intensity of Eu-SCNPs in the presence of different metal ions at a concentration of 27.5 µM. Figure 16. Selectivity of Eu-SCNPs for Cu2+ ions against other metal ions. 0 1 2 3 4 5 6Eu-SCNPs Fe2+ Ag+ Co2+ Ba2+ Cu2+ Ni2+ Hg2+ Pb2+ Zn2+ Fe3+ Ca2+ Mn2+ Mg2+ Cr3+ I0/I Metal ions 0 10 20 30 40 50 60 70 80 580 590 600 610 620 630 640 650 Eu-SCNPs Fe2+ Ag+ Co2+ Ba2+ Cu2+ Ni2+ Hg2+ Pb2+ Zn2+ Fe3+ Ca2+ Mn2+ Mg2+ Cr3+ Intensity (a.u.) Wavelenght (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 84 3.4.2. Tb-SCNPs as m.p.c.(Cu2+) “visual” pass/fail sensors Tb-SCNPs were obtained from the complexation of Tb3+ ions in solution by betaketoester functional groups of poly(OEGMA-co-AEMA), as displayed in Scheme 1. The average hydrodynamic diameter of the Tb-SCNPs was 7.2 nm (Figure 17), a value smaller than that of the poly(OEGMA-co-AEMA) precursor (8.3 nm). The SEC trace of Tb-SCNPs (Mw = 88.9 kDa, Ɖ = 1.36, see ESI) confirmed the absence of multi-chain aggregates, in accordance with DLS results. Figure 17. DLS size distribution of the precursor, poly(OEGMA-co-AEMA), and the Tb-SCNPs. The Tb-based SCNPs were visualized as greenish fluorescent nanomaterials under UV light irradiation (λexc = 254 nm) in a range of pH between 7.5 and 9 (see Figure 18). Under strong acidic conditions the fluorescence disappeared due to the disruption of Tb3+/beta-ketoester complexes, whereas under high basic pH a decrease in fluorescence was noted, as a consequence of aggregation of the Tb-SCNPs. In this way, the bright 0 5 10 15 20 25 30 110 100 Poly(OEGMA-co-AEMA) Tb-SCNPs Intensity (%) Hydrodynamic diameter (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 85 green glow of the Tb-SCNPs under UV irradiation was clearly visible in Figure 19, indicating that they could be used in sensing within a pH range of 7.5-9. 0 0.2 0.4 0.6 0.8 1 1.2 2 4 6 8 10 12 14 Relative PL intensity pH Figure 18. Relative PL intensity vs. pH for Tb-SCNPs. Figure 19. Illustration of the greenish fluorescent Tb-SCNPs under UV light irradiation (λexc = 254 nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 86 Shown in Figure 20 is the "Stokes shift" (Δλ). This is how the wavelength contrast between the peaks of the absorbance and emission spectra are defined. For fluorescent materials, this is an all-important shift because the larger it is, the more energy is lost from light absorption to emission and the less emitted light will be re-absorbed. As a result, fluorescence efficiency is increased. What you are looking at is a peak for absorbance and a sharper peak for emission. The Stokes shift is obviously the difference in wavelength between those two peaks: 302 nm. Figure 20. Absorbance (dotted) vs emission (continuous) and Stokes shift (Δλ) of Tb-SCNPs. Tb-SCNP fluorescence in 3D and 2D excitation-emission landscapes is shown in Figure 21 and 22. 3D fluorescence analysis (Figure 21) offers an intuitive presentation. Emission intensities are shown as peaks and interpretation through one height is possible at the different excitation wavelengths. A 2D contour plot (Figure 22) provides a correlated projection on a plane and simplifies the identification of the wavelengths where TbSCNPs display peak fluorescence. These visualization tools are essential for the 0 0,2 0,4 0,6 0,8 1 1,2 0 200 400 600 800 1000 1200 240 320 400 480 560 640 Absorbance (a.u.) Emission (a.u.) Wavelength (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 87 identification of the excitation-emission pair most suitable for Tb-SCNPs’ analytical applications. Figure 21. 3D study of excitation vs. emission for Tb-SCNPs. Figure 22. 2D study of excitation vs. emission for Tb-SCNPs. 200 250 300 350 400 450 300 400 500 600 700 Emition (nm) Exitation (nm) -0.5000 12.06 24.63 37.19 49.75 62.31 74.88 87.44 100.0 Intensity 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 88 As in the case of Tb-SCNPs, the stability results closely aligned with the precious findings. The study focused on the fluorescence intensity of Tb-SCNPs at intervals of 10 minutes, 1 hour, 1 day, and 1 month to assess the nanoparticles' stability. The nearly identical curves from both sets of experiments indicate that the Tb-SCNPs consistently maintain their fluorescence intensity over the examined period (see Figure 23). Figure 23. Stability of the fluorescence intensity over time for Tb-SCNPs. The pH adjustment coral test is shown in Figure 24, which demonstrates the variation of the photoluminescence (PL) intensity of Tb-SCNPs with pH alterations between neutral (pH 7.5) and acidic (pH 4) environments. The marked distinctions in PL intensity as a function of pH show the pH-dependency of the nanoparticles' fluorescence. Their fluorescence intensity is far greater at pH 7.5 than at pH 4, where a striking reduction in fluorescence is evident. In addition, as shown in Figure 24, Tb-SCNPs endure 3-4 cycles of pH alterations until their fluorescence entirely disappears. 0 200 400 600 800 1000 1200 520 530 540 550 560 570 10 min 1h 1 day 1 month Intensity (a.u.) Wavelength (nm) 5.72 % 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 89 Figure 24. Evolution of the PL intensity of Tb-SCNPs upon consecutive pH changes between 7.5 and 4. Figure 25. FTIR spectra of poly(OEGMA-co-AEMA) (blue colour) and Tb-SCNPs (green colour). 0 100 200 300 400 500 0 2 4 6 8 10 pH=7.5 pH=4 Intensity Cycles 100020003000 Poly(OEGMA-co-AEMA) Tb-SCNPs Wavenumber [cm-1] 1637.3 cm-1 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 96 undergo significant degradation or quenching that would otherwise affect their fluorescence. Figure 35. Stability of the fluorescence intensity over time for Dy-SCNPs. The pH adjustment coral test is shown in Figure 36, which demonstrates the variation of the photoluminescence (PL) intensity of Eu-SCNPs with pH alterations between neutral (pH 7.5) and acidic (pH 4) environments. The marked distinctions in PL intensity as a function of pH show the pH-dependency of the nanoparticles' fluorescence. Their fluorescence intensity is far greater at pH 7.5 than at pH 4, where a striking reduction in fluorescence is evident. In addition, as shown in Figure 36, Eu-SCNPs endure 3-4 cycles of pH alterations until their fluorescence entirely disappears. 0 50 100 150 200 250 550 560 570 580 590 600 10 min 1h 1 day 1 month Intensity (a.u.) Wavelenght (nm) 6.71 % 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 97 Figure 36. Evolution of the PL intensity of Dy-SCNPs upon consecutive pH changes between 7.5 and 4. In the FTIR spectrum of the Dy-based SCNPs, the stretching vibration of the enol tautomer of the beta-ketoester groups bonded to Dy3+ was observed at 1642.1 cm-1, which could be assigned to the stretching vibration of the enol tautomer of the betaketoester groups bonded to Dy3+ (Figure 37). Figure 37. FTIR spectra of poly(OEGMA-co-AEMA) (blue colour) and Dy-SCNPs (yellow colour) 0 50 100 150 200 250 300 350 0 2 4 6 8 10 pH=7.5 pH=4 Relative PL intensity Cycles 100020003000 Poly(OEGMA-co-AEMA) Dy-SCNPs Wavenumber [cm-1] 1642.1 cm-1 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 98 Dy-SCNPs show a yellow-to-transparent color transition under UV light irradiation at ca. 15 µM of Cu2+ ions (Figure 38) -very close to the US-EPA criterion (m.p.c.(Cu2+)EPA = 20 µM)- making these Dy-based SCNPs practical “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water according to the EPA regulation. Figure 38. PL spectra of Dy-SCNPs in water in the presence of increasing amounts of Cu2+ ions. Figure 39. Demonstration of the utility of Dy-SCNPs as “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water according to the EPA criterion (i: 1 µM, ii: 2.5 µM, iii: 10 µM, iv: 15 µM, v: 20 µM, and vi: 30 µM), 0 50 100 150 200 250 550 560 570 580 590 600 Dy SCNPs [Cu2+] 5 µM [Cu2+] 7.5 µM [Cu2+] 10 µM [Cu2+] 12.5 µM [Cu2+] 15 µM [Cu2+] 17.5 µM [Cu2+] 20 µM [Cu2+] 22.5 µM [Cu2+] 25 µM [Cu2+] 27.5 µM [Cu2+] 30 µM Intensity (a.u.) Wavelength (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 99 A Stern-Volmer plot for Dy-SCNPs provided KSV = 2.9 x 105 M-1 and R2 = 0.99 (see Figure 40). Similarly, to Eu-based and Tb-based SCNPs, Dy-based SCNPs proved selective for Cu2+ ions against other metal ions in solution (see Figure 41 and 42). Figure 40. Stern-Volmer plot of Dy-SCNPs (error bars estimated from triple measurements) Figure 41. Selectivity of Dy-SCNPs for Cu2+ ions against other metal ions. 1 2 3 4 5 6 0 2 4 6 8 10 12 14 16 I0/I [Cu2+] µM KSV = 2.9 x 105 M-1 R2 = 0.99 0 1 2 3 4 5 6 Dy-SCNPs Fe2+ Ag+ Co2+ Ba2+ Cu2+ Ni2+ Hg2+ Pb2+ Zn2+ Fe3+ Ca2+ Mn2+ Mg2+ Cr3+ I0/I Metal ions 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 100 Figure 42. PL intensity of Dy-SCNPs in the presence of different metal ions at a concentration of 15 µM. 3.5. Conclusions We introduced a new generation of m.p.c.(Cu2+) “visual” pass/fail sensors based on water-soluble lanthanide (Eu, Tb, or Dy)-containing SCNPs of sub-10 nm size range. Under UV light irradiation at λexc = 254 nm, Eu-SCNPs and Tb-SCNPs showed a naked-eye red-to-transparent and green-to-transparent colour transitions, respectively, near [Cu2+] = 30 µM in water at pH = 7.5. As for Dy-based SCNPs, they displayed a yellowtotransparent colour change at ca. [Cu2+] = 15 µM. The characteristic FTIR stretching vibration of the enol tautomer of the beta-ketoester groups bonded to Eu3+, Tb3+ and Dy3+ ions in Eu-SCNPs, Tb-SCNPs and Dy-SCNPs was located at 1632.4, 1637.3 and 1642.1 cm-1, respectively. These “visual” pass/fail sensors show high selectivity towards Cu2+ ions against a variety of other metal ions (Fe2+, Ag+, Co2+, Ba2+, Ni2+, Hg2+, Pb2+, Zn2+, Fe3+, Ca2+, Mn2+, Mg2+ and Cr3+). Consequently, Eu-SCNPs and Tb-SCNPs can be used as “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water according to the WHO criterion (m.p.c.(Cu2+)WHO = 30 µM). Complementary, Dy-SCNPs can be useful as “visual” pass/fail sensors of m.p.c. of Cu2+ ions in water according to the EPA regulation (m.p.c.(Cu2+)EPA = 20 µM). 0 50 100 150 200 250 300 550 560 570 580 590 600 Dy-SCNPs Fe2+ Ag+ Co2+ Ba2+ Cu2+ Ni2+ Hg2+ Pb2+ Zn2+ Fe3+ Ca2+ Mn2+ Mg2+ Cr3+ Intensity (a.u.) Wavelength (nm) 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 101 3.6. References 1. R. A. Muttkowski, Copper in Animals and Plants, Science, 1921, 53, 453-454. 2. W. Kaim and J. Rall, J. Copper - A“Modern” Bioelement, Angew. Chem. Int. Ed., 1996, 35, 43-60. 3. B.-E. Kim, T. Nevitt and D. J. Thiele, Mechanisms for copper acquisition, distribution and regulation, Nat. Chem. Biol., 2008, 4, 176-185. 4. T. R. Halfdanarson, N. 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Huang, Stimuliresponsive single-chain polymeric nanoparticles towards the development of efficient drug delivery systems, Polym. Chem., 2016, 7, 6164-6169. 16. A. Latorre-Sánchez and J. A. Pomposo, A simple, fast and highly sensitive colorimetric detection of zein in aqueous ethanol via zein-pyridine-gold interactions, Chem. Commun., 2015, 51, 15736-15738. 17. N. M.Hamelmann, J. W. D. Paats, Y. Avalos-Padilla, E. Lantero, I. Siden-Kiamos, L. Spanos, X. Fernandez-Busquets and J. M. J. Paulusse, Single-Chain Polymer Nanoparticles Targeting the Ookinete Stage of Malaria Parasites, ACS Infect. Dis., 2023, 9, 56-64. 18. S. Garmendia, S. B. Lawrenson, M. C. Arno, R. K. O'Reilly, D. Taton and A. P. Dove, Catalytically Active N-Heterocyclic Carbene Release from Single-Chain Nanoparticles Following a Thermolysis-Driven Unfolding Strategy, Macromol. Rapid Commun., 2019, 40, 1900071. 19. T. Gunnlaugsson, J. P. Leonard, K. Sénéchal and A. J. Harte, Eu(III)–cyclen–phen conjugate as a luminescent copper sensor: the formation of mixed polymetallic macrocyclic complexes in water, Chem. Commun., 2004, 782-783. 20. A. Nonat, A. J. Harte, K. Senechal-David, J. P. Leonard and T. Gunnlaugsson, Luminescent sensing and formation of mixed f–d metal ion complexes between a Eu(III)cyclen-phen conjugate and Cu(II), Fe(II), and Co(II) in buffered aqueous solution, Dalton Trans., 2009, 4703-4711. 21. Z. Ekmekci, Highly selective fluorescence ‘turn-off’ sensors for Cu2+ in aqueous environments, Tetrahedron Lett., 2015, 56, 1878-1881. 22. L. M. Aroua, R. Ali, A. E. A. E. Albadri, S. Messaoudi, F. M. Alminderej and S. M. Saleh, A New, Extremely Sensitive, TurnOff Optical Sensor Utilizing Schiff Base for Fast Detection of Cu(II), Biosensors, 2023, 13, 359. 3. Lanthanide-based Single-Chain Nanoparticles as “Visual” Pass/Fail Sensors of Maximum Permissible Concentration of Cu2+ Ions in Drinking Water 103 23. B.S. Sankha and R.N. Kapoor, Organic compounds of samarium. II. Reactions of samarium isopropoxide with ethyl acetoacetate, Can. J. Chem., 1966, 44, 13691372. 24. N.K. Dutt and S. Rahut, Chemistry of lanthanons-XXIII. The formation constants of the ethyl acetoacetate complexes of rare earth, J. Inorg. Nucl. Chem. 1969, 31, 3177-3179. 25. A. M. Mishchenko, E. K. Trunova and A. S. Berezhnytska, Lanthanide Complexes with Allyl Acetoacetate in Mixed Water–Organic Media: Formation, Stability and Bonding, J. Solution Chem., 2015, 44, 2117-2128. 26. O. Stern and M. Volmer, Über die Abklingzeit der Fluoreszenz, Zeitschrift für Physik, 1919, 20, 183-188. 27. M. L. Aulsebrook, B. Graham, M. R. Grace and K. L. Tuck, Lanthanide complexes for luminescence-based sensing of low molecular weight analytes, Coord. Chem. Rev., 2018, 375, 191220. 28. K. Szyszka, S. Targońska, A. Lewińska, A. Watras and R. J. Wiglusz, Quenching of the Eu3+ Luminescence by Cu2+ Ions in the Nanosized Hydroxyapatite Designed for Future BioDetection, Nanomaterials, 2021, 11, 464. 4. Gold Nanoclusters Synthesized within SingleChain Nanoparticles as Catalytic Nanoreactors in Water 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 112 μL of 4-nitrophenol and 30.35 mg of NaBH4 were sequentially added under argon into a vial at 0 C. The 4-nitrophenol reduction reaction was then monitored via UV-Vis spectrophotometry. Absorbance was recorded by taking 1 μL of crude at a given reaction time, which was then diluted in 2 mL of deionized water. After reaction, 4-aminophenol was purified via preparative thin-layer chromatography (TLC) (n-hexane/ethyl acetate 1:1), with a yield of 95%. 1H NMR (400 MHz, DMSO-d6, ppm): δ 4.36 (s, 2H), 6.42–6.46 (m, 4H), 8.34 (s, 1H). 4.4.4. Procedure for the Reduction of Nitrobenzene Catalyzed by AuNCs/SCNPs The Au-NC/SCNPs were used as catalysts for the reduction at r.t. of nitrobenzene (0.2 mmol) to aniline in the presence of NaBH4 (0.8 mmol). The same procedure described in Section 3.4.3. was followed. Absorbance was recorded by taking 2 μL of crude at a given reaction time, which was then diluted in 4 mL of deionized water. After reaction, aniline was purified via preparative TLC (n-hexane/ethyl acetate 1:1), with a yield of 96%. 1H NMR (400 MHz, DMSO-d6, ppm): δ 3.65 (s, 2H), 6.67–6.70 (m, 2H), 6.73–6.77 (m, 1H), 7.13–7.17 (m, 2H). 4.4.5. Procedure for the Reduction of 3-(4-Nitrophenyl)-1,3-oxazolidin2one Catalyzed by Au-NCs/SCNPs The Au-NC/SCNPs were used as catalysts for the reduction at r.t. of 3-(4-nitrophenyl)- 1,3-oxazolidin-2-one (0.2 mmol) to 3-(4-aminophenyl)-1,3-oxazolidin-2-one in the presence of NaBH4 (0.8 mmol). The same procedure described in Section 3.4.3. was followed. Absorbance was recorded by taking 2 μL of crude at a given reaction time, which was then diluted in 10 mL of deionized water. It is worth noting that due to the heterogenous character of the reaction and to the vigorous generation of molecular hydrogen gas, aliquots for analysis were prepared ensuring only the aqueous phase was taken. The product, 3-(4-aminophenyl)oxazolidin-2-one, was purified via preparative TCL 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 113 (n-hexane/ethyl acetate 1:1), with a yield of 89%. 1H NMR (400 MHz, CDCl3), ppm): δ 4.63 (s, 2H), 3.97–4.01 (dd, 2H), 4.43–4.47 (dd, 2H), 6.68–6.71 (d, 2H), 7.27–7.30 (d, 2H). 4.5. Results and Discussion 4.5.1. Synthesis of Gold Nanoclusters within Single-Chain Nanoparticles (Au-NCs/SCNPs) We targeted an amphiphilic poly(OEGMA-co-AEMA) random copolymer featuring hydrophilic oligo(ethyleneglycol) methyl ether methacrylate (OEGMA) and hydrophobic (2-acetoacetoxy)ethyl methacrylate (AEMA) units as a template for the synthesis of AuNCs. Based on the literature data of low-molecular-weight ligands,24 we surmised that the beta-ketoester group of AEMA could be used as a reductant of Au(III) ions, as well as a stabilizing and structure-directing agent, to generate the Au-NCs. Poly(OEGMAcoAEMA) was synthesized by means of reversible addition fragmentation chain-transfer (RAFT) polymerization (see Figure 44). Figure 44. Synthesis of an amphiphilic poly(OEGMA-co-AEMA) random copolymer featuring hydrophilic oligo(ethyleneglycol) methyl ether methacrylate (OEGMA) and hydrophobic (2- 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 114 acetoacetoxy)ethyl methacrylate (AEMA) repeat units via reversible addition fragmentation chaintransfer (RAFT) polymerization. Schematic illustration of the synthesis of Au-NCs/SCNPs from poly(OEGMA-co-AEMA) self-assembled in water in the form of SCNPs. The copolymer showed a weight-average molecular weight (Mw) of 80.7 kDa and a low dispersity (D) value of 1.12, as determined by SEC. The content of AEMA units in the copolymer was 35 mol%, as estimated from 1H NMR spectroscopy. It is well-known from previous works that amphiphilic poly(OEGMA-co-AEMA) random copolymers with this AEMA content are able to self-assemble intramolecularly in water at high dilution (1 mg/mL) into discrete core–shell-like SCNPs.25,26 In agreement with previous results, DLS measurements of poly(OEGMA-co-AEMA) in water at 1 mg/mL revealed an average hydrodynamic diameter of Dh = 11.0 nm corresponding to discrete, individual SCNPs without any sign of the presence of multi-chain aggregates (see Table 2 and Figure 44). Table 2. Evolution of average nanoparticle sizes during the synthesis of Au-NCs/SCNPs. Material Type Reaction Time, t DLS Hydrodynamic Diameter, Dh (nm) 2 Poly(OEGMA-co-AEMA) 1 0 min. 11.0 Au-NCs/SCNPs 1 min. 11.0 Au-NCs/SCNPs 15 min. 10.7 Au-NCs/SCNPs 1 h 11.3 Au-NCs/SCNPs 1 day 11.2 Au-NCs/SCNPs 1 week 11.6 1 Self-assembled in water in the form of core–shell-like SCNPs. 2 Standard deviation ca. ± 0.3 nm. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 115 Figure 44. DLS size distribution of poly(OEGMA-co-AEMA) SCNPs and Au-NCs/SCNPs. Figure 45. Comparison of the UV-Vis spectra of Au-NPs synthesized in the presence of poly(OEGMA-co-AEMA) SCNPs at a (Au(III))/(beta-ketoester (AEMA)) ratio of 1 vs Au-NCs synthesized within poly(OEGMA-co-AEMA) SCNPs at a (Au(III))/(beta-ketoester (AEMA)) ratio of 0.08. 0 0.1 0.2 0.3 0.4 0.5 200 300 400 500 600 700 800 Poly (OEGMA-co-AEMA) Au-NPs Au-NCs Intensity (%) Wavelength (nm) 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 116 For the synthesis of Au-NCs within the SCNPs through beta-ketoester-mediated Au(III) reduction (Figure 45), we found control of the (Au(III)/(beta-ketoester (AEMA)) ratio to be critical. Hence, by using a (Au(III))/(beta-ketoester (AEMA)) ratio of 1, we obtained gold nanoparticles (Au-NPs) instead of Au-NCs, as revealed by the intense localized surface plasmon resonance (LSPR) UV-Vis absorbance signal characteristic of Au-NPs (Figure 45). Interestingly, by lowering the (Au(III))/(beta-ketoester (AEMA)) ratio to 0.08, we observed via TEM the presence of Au-NCs (diameter < 5 nm) within individual poly(OEGMA-coAEMA) SCNPs (Figure 46), as well as the total absence of the LSPR band typical of larger Au-NPs (Figure 45). No significant differences were found between the IR spectra of neat poly(OEGMA-co-AEMA) and the Au-NCs/SCNPs, as illustrated in Figure 47, which we attribute to the relatively low (Au(III))/(beta-ketoester (AEMA)) ratio employed. Notably, the size of the Au-NCs/SCNPs was found to be very stable over time, as illustrated in Table 2 and Figure 44, showing the notorious stabilizing effect of the SCNPs against Au-NCs aggregation over time. Conversely, by using a (Au(III))/(betaketoester (AEMA)) ratio of 1, the diameter of the resulting Au-NPs was found to grow with time, as illustrated in Figure 48 by the associated color changes. After 1 day, the DLS hydrodynamic diameter of the Au-NPs was found to be 67.1 nm. (a) (b) Figure 46. (a) TEM picture of Au-NCs synthesized within poly(OEGMA-co-AEMA) SCNPs at a (Au(III))/(beta-ketoester (AEMA)) ratio of 0.08. (b) Illustration of the presence of (darker) AuNCs with a diameter < 5 nm within a representative single-chain nanoparticle of ca. 11 nm in diameter. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 117 Figure 47.IR spectra of neat poly(OEGMA-co-AEMA) SCNPs (blue color) and Au-NCs/SCNPs (red color). Figure 48. Au-NPs synthesized in the presence of poly(OEGMA-co-AEMA) SCNPs at a (Au(III))/(beta-ketoester (AEMA)) ratio of 1 vs Au-NCs synthesized at a ratio of 0.08. 800100012001400160018002000 Poly(OEGMA-co-AEMA) Au-NCs Wavenumber [cm-1] 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 118 Taken together, the above results demonstrate the efficient synthesis of stabilized AuNCs with size < 5 nm within discrete poly(OEGMA-co-AEMA) SCNPs of ca. 11 nm in diameter. Key to the access to stabilized Au-NCs within SCNPs, instead of to larger AuNPs that grow in size over time, is the control of the (Au(III))/(beta-ketoester (AEMA)) ratio employed during the synthesis. 4.5.2. Gold Nanoclusters within Single-Chain Nanoparticles (AuNCs/SCNPs) as Catalytic Nanoreactors We report herein the results of the use of Au-NCs within SCNPs as catalytic nanoreactors for the reduction of 4-nitrophenol, nitrobenzene, and 3-(4-nitrophenyl)- 1,3-oxazolidin2one by borohydride (BH4−) in water at r.t. 4.5.2.1. Reduction of 4-Nitrophenol to 4-Aminophenol Catalyzed by AuNCs/SCNPs Paracetamol (a popular analgesic and antipyretic agent used to treat fever and mild to moderate pain) can be typically synthesized from 4-nitrophenol as an intermediate via its reduction to 4-aminophenol and subsequent acetylation with acetic anhydride. 4nitrophenol is highly soluble in water (11.6 mg/mL at 20 ◦C). We investigated the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by BH4− in water at r.t. by using the Au-NCs/SCNPs as highly efficient catalytic nanoreactors. This transformation has emerged as an important model reaction for assessing the catalytic activity of metallic nanoparticles in water. Section 3.4.3. details the experimental procedure that followed, which is depicted schematically in Figure 49. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 119 Figure 49. (a) Reduction of 4-nitrophenol to 4-aminophenol in water at r.t. by using the AuNCs/SCNPs as highly efficient catalytic nanoreactors. (b) Color changes observed during the reduction of 4-nitrophenol (λmax ≈ 400 nm) to 4-aminophenol (λmax ≈ 300 nm) catalyzed by AuNCs/SCNPs. The conversion of 4-aminophenol to 4-aminophenol was followed by UV-Vis spectrometry due to the different, well-separated absorption bands of the reactant and product, as illustrated in Figure 49(b) and Figure 50. Interestingly, under our reaction conditions, an isosbestic point located at ca. λ = 325 nm was observed in the UV-Vis spectra during the reduction of 4-aminophenol (λmax ≈ 400 nm) to 4-aminophenol (λmax ≈ 300 nm). This fact suggests the major involvement of a direct route mechanisms in the reduction of 4nitrophenol to 4-aminophenol in water at r.t. with Au-NCs/SCNPs.27 No reaction was observed in a model experiment lacking the Au-NCs/SCNPs catalyst. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 120 Figure 50. Kinetics of the reduction of 4-nitrophenol to 4-aminophenol catalyzed by AuNCs/SCNPs as determined by UV-Vis spectroscopy. The purified product’s chemical structure was confirmed using 1H nuclear magnetic resonance spectroscopy (1H NMR). The spectrum shows numerous proton environments within the molecule. A singlet at δ 4.36 (s, 2H) suggests the presence of a pair of equivalent protons, the amino group. The multiplet from δ 6.42-6.46 (m, 4H) signifies an aromatic system with protons which experience slightly different electronic environments, likely due to the influence of an adjacent substituent, such as the -NH2 and -OH groups. Finally, the singlet at δ 8.34 for the remaining proton (s, 1H) is typical of an aromatic proton that is deshielded, typically due to an electronegative group (such as a nitro or carbonyl) nearby or, in this case, this singlet signifies the influence of a hydroxyl group in -para to the amino group. This spectrum indicates a clean product with no significant impurities, in that only the expected signals are observed. 0 0.2 0.4 0.6 0.8 1 1.2 300 400 0 min 2 min 3min 4min 5min 7min 9min 12min 15min Wavelength (nm) Absortion (a.u.) 4-NP 4-AP 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 121 Figure 51. 1H MNR spectrum after isolation via preparative TCL of the 4-aminophenol product. Meticulously constructed calibration curves were done for all compounds via UV-visible spectroscopy. The goal of this effort was to determine the molar extinction coefficients for each compound. This parameter is critical for quantitating the ability of a compound to absorb light at a particular wavelength. The process of creating a calibration curve involves making a series of standard solutions with accurately known concentrations of the target compound. Each set of standards is then subjected to UV-vis spectroscopy, where we record the absorbance at the wavelength where the compound exhibits maximum absorption. The relationship between absorbance and concentration for these standards should be linear, according to the Beer-Lambert law, over the range of concentrations used. The molar extinction coefficient, ε, refers to the absorbance of a one molar (1 M) solution of the compound in a cuvette of cross-section 1 cm. Plotting the absorbance values versus the concentrations of the standards generates a calibration curve. The slope of 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 128 product. Figure 59. Experimental data point to the involvement of both the direct route (upper) and the condensation route (bottom) mechanisms. It was imperative to quantify the molar extinction coefficients so that we could understand how the system evolves in terms of the molar fractions of the constituent species as well as the mechanistic progression and the kinetics of the reaction. Once again, the molar extinction coefficients were deduced through UV-visible spectroscopic analysis (Figure 60,61 and 62) where calibration curves were constructed. Molar extinction coefficients furnished a crucial foundation for our kinetic analysis, allowing us to quantify the molar fractions of the reactants, intermediates, and products throughout the course of the reaction, thus providing a window into the intricate molecular interactions and transformations that dictate the course of the chemistry of the reaction under study. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 129 Figure 60. Calibration curve for determination of the UV-Vis molar extinction coefficient of nitrobenzene in water at r.t.: ε (λmax = 265 nm) ≈ 16632 M-1cm-1. Figure 61. Calibration curve for determination of the UV-Vis molar extinction coefficient of azobenzene in water at r.t.: ε (λmax = 230 nm) ≈ 16566 M-1cm-1. 0 0.1 0.2 0.3 0.4 0.5 0 5 10-6 1 10-5 1.5 10-5 2 10-5 2.5 10-5 y = 0.0096729 + 16632x R2= 0.99641 Absorbance (a.u.) [C] (mol/L) 0 0.2 0.4 0.6 0.8 1 1.2 1.75 10-5 3.5 10-5 5.25 10-5 7 10-5 y = -0.094268 + 16566x R2= 0.99279 Absorbance (a.u.) [C] (mol/L) 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 130 Figure 62. Calibration curve for determination of the UV-Vis molar extinction coefficient of aniline in water at r.t.: ε (λmax = 230 nm) ≈ 20239 M-1cm-1. Figure 63 shows the evolution of the concentration of the reactant and the product over time as estimated from data in Figure 56. A reaction yield of 96% was achieved in 90 min. of reaction time, where the amount of nitrobenzene was totally consumed. As illustrated in Figure 55, the apparent kinetic constant (kapp) of the reaction was estimated to be kapp = 3.8 × 10−4 s−1. 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 5 10-6 1 10-5 1.5 10-5 2 10-5 2.5 10-5 y = 0.026859 + 17171x R2= 0.99692 Absorbance (a.u.) [C] (mol/L) 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 131 Figure 63. Evolution of the concentration of nitrobenzene (blue), cis-azobenzene (red) and aniline (green) over time during the reduction of nitrobenzene to aniline catalyzed by AuNCs/SCNPs. Figure 64. Apparent kinetic constant (kapp) of the reduction of nitrobenzene to aniline catalyzed by AuNCs/SCNPs. 0 0.2 0.4 0.6 0.8 1 020 40 60 80 Nitrobenzene cis-azobenzene Aniline Molar fraction t(min) -2.5 -2 -1.5 -1 -0.5 0 0 1000 2000 3000 4000 5000 6000 ln(A/A0) t(s) kapp = 3.8 x 10-4 s-1 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 132 4.5.2.3. Reduction of 3-(4-Nitrophenyl)-1,3-oxazolidin-2-one to 3-(4Aminophenyl)-1,3-oxazolidin-2-one Catalyzed by Au-NCs/SCNPs Motivated by the high conversion observed during the Au-NCs/SCNPs-catalyzed reduction of both 4-nitrophenol and nitrobenzene, we additionally investigated the reduction of 3-(4-nitrophenyl)-1,3-oxazolidin-2-one to 3-(4-aminophenyl)-1,3oxazolidin2-one catalyzed by Au-NCs/SCNPs. In this sense, the aminophenyl– oxazolidinone fragment is a common motif contained in several drugs like Rivaroxaban, Sutezolid, and Linezolid (see Figure 65). Figure 65. Illustration of the reduction of 3-(4-nitrophenyl)-1,3-oxazolidin-2-one to 3- (4aminophenyl)- 1,3-oxazolidin-2-one by BH4 − in water at r.t. catalyzed by Au-NCs/SCNPs. The aminophenyl– oxazolidinone fragment is a common motif contained in several drugs like Rivaroxaban, Linezolid, and Sutezolid. We found the Au-NCs/SCNPs-catalyzed reduction of 3-(4-nitrophenyl)-1,3oxazolidin2one to proceed in two steps (fast and slow, respectively), as summarized in Figure 66. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 133 Figure 66. Reduction of 3-(4-nitrophenyl)-1,3-oxazolidin-2-one (in blue) to 3-(4aminophenyl)1,3oxazolidin-2-one (in green) catalyzed by Au-NCs/SCNPs. The fast step corresponds to the formation of the diazene intermediate (in red), and the slow step to the generation of the aminophenyloxazolidinone product. Figure 67 shows the UV-Vis spectra recorded at different reaction times corresponding to the fast step. We observed the complete disappearance of the UV-Vis absorption peak of 3-(4-nitrophenyl)-1,3-oxazolidin-2-one (λmax ≈ 320 nm) in 15 min. and then the appearance of a new UV-Vis absorption peak (λmax ≈ 360 nm), which can be attributed to the generation of the diazene intermediate ((Z)-3,3ʹ-(diazene-1,2diylbis(4,1phenylene))bis(oxazolidin2-one)) in 3 h of reaction time. The intensity of this new band was stable after 3 h of reaction time, and it did not change until 9 h of additional reaction time. Then, during the slow step, a progressive disappearance of the UV-Vis peak of the diazene intermediate at λmax ≈ 360 nm was found, as well as the concomitant appearance of a new UV-Vis absorption band at λmax ≈ 250 nm corresponding to the 3(4-aminophenyl)-1,3oxazolidin-2-one product (see Figure 67). Figure 73 illustrates the evolution of the concentration of 3-(4-nitrophenyl)-1,3oxazolidin-2-one, (Z)-3,3ʹ(diazene-1,2-diylbis(4,1phenylene))bis(oxazolidin-2-one), and 3-(4-aminophenyl)-1,3oxazolidin-2-one over time, as estimated from data in Figure 67, cis (Z) = 91 % trans (E) = 9 % FAST STEP SLOW STEP Yield: 89% 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 134 and the corresponding calibration curves are provided in the Supporting Information. After 20 h of reaction time, the reaction yield was 89%. a b Figure 67. Kinetics of the reduction of 3-(4-nitrophenyl)-1,3-oxazolidin-2-one to 3- (4aminophenyl)- 1,3-oxazolidin-2-one catalyzed by Au-NCs/SCNPs as determined by UV-Vis spectroscopy corresponding to the fast step (a) and the slow step (b) of the reaction. 0 0.2 0.4 0.6 0.8 1 260 280 300 320 340 360 380 400 0 min 5min 15min 30min 1h 3h - 12h Absorbance (a.u.) Wavelength (nm) 0 0.2 0.4 0.6 0.8 1 200 250 300 350 400 450 12h 13h 15h 16h 17h 18h 20h Absorbance (a.u.) Wavelength (nm) 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 135 In this case, a clear intermediate was observed again in the reaction, so as in the Reduction of nitrobenzene to aniline, both the intermediate and the final product were characterized by 1H NMR as can be seen in Figure 68 and Figure 69. Figure 68. 1H MNR spectrum after isolation via preparative TCL of the (Z)-3,3'-(diazene-1,2diylbis(4,1-phenylene))bis(oxazolidin-2-one) intermediate species. Figure 69. 1H MNR spectrum after isolation via preparative TCL of the 3-(4-aminophenyl)- 1,3oxazolidin-2-one product. 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 136 Accordingly, in order to ensure that our monitoring was reliable and accurate, calibration curves were constructed to obtain molar extinction coefficients for the substances involved in our reaction. This was necessary in our case because the molar extinction coefficient is highly specific to each chemical species and depends on a number of parameters that are directly related to the experimental conditions. This monitoring is crucial for being able to obtain the full picture of what is happening during a chemical reaction. It can show us the reaction kinetics, so we can optimize the conditions in which we perform the reaction for the greatest yield, and also to maintain the consistency and quality of product from batch to batch. Figure 70. Calibration curve for determination of the UV-Vis molar extinction coefficient of 3-(4nitrophenyl)-1,3-oxazolidin-2-one in water at r.t.: ε (λmax = 320 nm) ≈ 2074 M-1cm-1. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 10-4 2 10-4 3 10-4 4 10-4 5 10-4 y = -0.020172 + 2074.4x R2= 0.99287 Absorbance (a.u.) [C] (mol/L) 4. Gold Nanocluster Synthesized within Single-Chain Nanoparticles as Catalytic Nanoreactors in Water 137 Figure 71. Calibration curve for determination of the UV-Vis molar extinction coefficient of (Z)3,3'- (diazene-1,2-diylbis(4,1-phenylene))bis(oxazolidin-2-one) in water at r.t.: ε (λmax = 360 nm) ≈ 3060 M1cm-1. Figure 72. Calibration curve for determination of the UV-Vis molar extinction coefficient of 3- (4aminophenyl)-1,3-oxazolidin-2-one in water at r.t.: ε (λmax = 250 nm) ≈ 2801 M-1cm-1. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 5 10-5 1 10-4 1.5 10-4 2 10-4 2.5 10-4 3 10-4 y = 0.001723 + 3060.4x R2= 0.99859 Absorbance (a.u.) [C] (mol/L) 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1 10-4 2 10-4 3 10-4 y = 0.1775 + 2800.9x R2= 0.99369 Absorbance (a.u.) [C] (mol/L) 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 145 5.1. Motivation This work was sparked by the pressing need forsustainable progresses in catalysis, particularly for the design of nanocatalysts that can carry out multi-step chemical reactions all in one reaction vessel. Realizing these environmental and economic drawbacks of using toxic solvents and the difficulties that are accompanied by purifying processes in the classical multi-step synthesis, we could focus on overcoming these challenges. Here, we developed a facile methodology that afforded heterobimetallic Pt(II)/Cu(II) single-chain polymer nanoparticles (SCNPs) in a sequential manner, capitalizing both on metal-ligand coordination for intramolecular folding and their synergistic catalytic activity. Thus, this strategy is greener in N-butylpyrrolidone use as a nontoxic solvent and displays the efficiency and selectivity of such bimetallic SCNPs in consecutive one-pot alkyne semihydrogenation and alkene dioxygenation reactions. This work, through this contribution, tries to make a successful attempt at making its contribution to the larger goal of sustainable chemistry, as the above-said complex multimetallic SCNPs may open new vistas in the synthesis and application of these highly versatile and multi-functional nanocatalysts. 5.2. Introduction Metal-ligand coordination allows intramolecularly folding of ligand-decorated discrete synthetic polymer chains to metallofolded single-chain polymer nanoparticles (SCNPs).1 SCNPs are intra-chain cross-linked single polymer chains with manifold promising applications, mainly as catalysts, nanosensors and drug nanocarriers.2 In general, intramolecular folding of the isolated synthetic chains generates locally compact zones within the SCNPs for efficient immobilization of catalytic active species, luminophores or drugs.3 To some extent, the intrachain folding of discrete synthetic polymer chains to SCNPs resembles the folding of certain proteins to their precise functional conformation (i.e., native state).4 In particular, metallo-folded SCNPs leverage the dual role played by the metal: as a folding element via intra-chain metal-ligand coordination, and as an immobilized functional center for subsequent catalysis.5 The number and catalytic 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 146 applications of metallo-folded SCNPs as enzyme-mimetic nanoentities have grown significantly in recent years.6,7 In a seminal work, Terashima et al. reported Rucontaining amphiphilic SCNPs to catalyse the reduction of cyclohexanone to cyclohexanol in water.8 Pomposo et al. pioneered the introduction of metallo-folded Cu(II)-containing SCNPs showing catalytic selectivity in alkyne homocoupling reactions,5 and single-chain globules mimicking the morphology and polymerase activity of metalloenzymes.9 He et al. prepared metallo-folded SCNPs containing Ni-thiolate complexes showing excellent thermal stability under aerobic conditions and excellent activity and selectivity during the photocatalytic reduction of CO2 to CO.10 Zimmerman et al. developed “clickase” SCNPs displaying enzyme-like “click” catalysis in vivo and enabling efficient cell surface glycan editing.11 Taton et al. reported SCNPs containing Ag(I)-N-heterocyclic carbene (NHC) linkages as NHC pre-catalysts for the benzoin condensation reaction.12 Yang et al. synthesized metal-containing SCNPs in concentrated solutions at room temperature (r.t.) by introducing electrostatic repulsion and intra-chain crosslinking by coordination with Cu(II) or Fe(II) or Fe(III) ions.13 Tan et al. synthesized enzyme-mimetic SCNPs with chiral Fe(II)-oxazoline complexes for efficient asymmetric sulfa-Michael addition of thiols to α,β-unsaturated ketones in water at r.t.14 More recently, Pomposo et al. developed a method to upcycling poly(vinyl chloride) (PVC) waste to efficient catalytic Cu(II)- containing SCNPs.15 Current advances in catalysis utilizing SCNPs have been recently reviewed by the Barner-Kowollik team.16 However, introduction of at least two distinct metal species in SCNPs although highly desirable is still synthetically challenging. In a pioneering work, Lemcoff et al. synthesized Rh(I)/Ir(I) SCNPs although their catalytic properties were not evaluated.17 Subsequently, Barner-Kowollik et al. synthesized heterobimetallic Eu(III)/Pt(II)-SCNPs18 and Au(I)/Y(III)- SCNPs19 in which only one of the two metal ions, Pt(II) or Au(I), was used for catalysis, while the other was employed for sensing or intrachain folding (Eu(III) or Y(III), respectively). More recently, the same group decorated SCNPs folded through ferrocene units with Pd(II) atoms, which proved to be an active catalyst for the intramolecular hydroamination of an aminoalkyne.20 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 147 Advanced heterobimetallic nanocatalysts for carrying out multistep chemical processes in a single reaction vessel are currently of great interest in academia and industry.21 Often, however, major costs of many consumer products synthesized in multistep processes are incurred in the purification and isolation of intermediates.22 Additionally, replacement of toxic organic solvents by green solvents has attracted significant interest.23 To the best of our knowledge, bimetallic SCNPs that would allow consecutive one-pot reactions to be performed in a green solvent have not been reported. To fill this gap, we report herein the proof of concept of heterobimetallic SCNPs through the synthesis of Pt(II)/Cu(II)-SCNPs. We show that these can serve as advanced soft nanocatalysts to perform consecutive one-pot alkyne semihydrogenation / alkene dioxygenation reactions in N-butylpyrrolidone (NBP) as a green solvent. 5.3. Materials and methods 5.3.1. Materials Methyl methacrylate (MMA) (99%), (2-acetoacetoxy)ethyl methacrylate (AEMA) (95%), 2.2ʹ-azo(2-methylpropionitrile) (AIBN) (≥98%), p-carboxybenzenesulfonazide (p-CBSA) (97%),triethylamine (Et3N) (>99%), dichloromethane (CH2Cl2) (anhydrous, ≥99.8%), ethyl acetate (EtOAc) (anhydrous, 99.8%), diethyl ether (Et2O) (ACS reagent, anhydrous, >99.0%), deuterated chloroform (CDCl3) (99.96 atom % D, containing 0.03% (v/v) tetramethylsilane), N,Ndimethylformamide (DMF) (≥ 99.9%), n-hexane (Hex) (>95%), Nbutylpyrrolidinone (NBP) (≥99.5%), (1,5-cyclooctadiene)platinum(II) dichloride (Pt(COD)Cl2), copper (II) acetate (Cu(OAc)2) (98%), magnesium sulphate (Mg2SO4) (anhydrous, ≥99.5%), phenylacetylene (1a) (98%) were purchased from Sigma-Aldrich and used, unless specified, as received. 2-Cyanoprop-2-yldithiobenzoate (CPDB) (≥97%) was purchased from Strem Chemicals. Methanol (MeOH) (synthesis grade) and THF (HPLC grade) were purchased from Scharlab. 1-Ethynyl-4-methoxybenzene (1b) (>98.0% by GC), 3-bromophenylacetylene (1c) (>98.0% by GC), 4-ethynylbenzotrifluoride (1d) (>98.0% by GC), 4-Pyridylacetylene (1e) (>98.0% by GC) were purchased form TCI Chemicals. N-Hydroxyphtalimide (NHPI) (98%) was purchased from BLDPharm. Purified 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 148 water was obtained from a Thermo Scientific apparatus (Barnstead TII Pure Water System). AIBN was recrystallized from methanol. MMA was purified by distillation before use. AEMA was purified by passing through alumina. 1a 1b 1c 1d 1e NBP 5.3.2. Techniques 1H nuclear magnetic resonance (NMR) spectra were obtained at room temperature (r.t.) using a Bruker spectrometer operating at 400 MHz with CDCl3 as the solvent. Size exclusion chromatography (SEC) data were acquired using a PL-GPC 50 instrument from Agilent Technologies, which integrates differential refractive index (DRI) and multi-angle light scattering (MALS) detectors. DMF containing 0.1% of LiBr with a flow of 1.0 mL/min was used as an eluent. A PLgel MIXED, 7.5 x 50 mmm, 10 μm, guard column, and a PLgel 500 Å, 7.5 x 300 mm, 5 μm, or a PL PolarGel-M, 7.5 x 300 mm, 8 μm, HPLC columns were used. The molecular weights of the different samples were determined using a dn/dc value of 0.0608. SEC data were analysed using Wyatt's ASTRA Software (version 8.1). Dynamic light scattering (DLS) measurements were carried out at r.t. on a Malvern 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 149 Zetasizer Nano ZS apparatus. Metal content in the single-chain nanoparticles was determined by inductively coupled plasma-mass spectrometry (ICP-MS). Fourier transform infrared (IR) spectra were recorded at r.t. on a JASCO 3600 FTIR spectrometer. Elemental analysis (EA) were performed in a Euro EA3000 elemental analyzer (CHNS). 5.4. Procedures 5.4.1. Synthesis of P0 In a typical procedure, MMA (3 mL, 28.2 mmol), AEMA (2.63 mL, 15.58 mmol), CPDB (22.2 mg, 0.1 mmol) and AIBN (16.5 mg, 1 mmol) were dissolved in EtOAc (10.45 mL). The solution was degassed by bubbling N2 for 15 min. The copolymerization reaction was carried at 65 °C for 24 h. After isolation of the resulting copolymer P0 by precipitation in MeOH, it was dried under dynamic vacuum until constant weight. Yield: 94.0%. βketoester content (1H NMR): 30 mol%. Mw (SEC) = 73.6 kDa, Ɖ (SEC) = 1.17. 5.4.2. Synthesis of P1 The synthesis of P1 was conducted under light-protected conditions. The process involved dissolving 300 mg (0.47 mmol) of P0 in 10 mL of CH2Cl2 at r.t. Next, p-CBSA (12.25 mg, 0.35 eq.) and Et3N (0.4 mL, 2.87 mmol) were added to the solution, which was then stirred for 24 h protected from light. Upon completion of the reaction, the resulting solution was concentrated and precipitated in MeOH, and the functionalized copolymer, P1, was dried in a vacuum oven in the absence of light. Yield: 76%. α-diazoβ-ketoester content (EA): 19 mol%. Mw (SEC) = 75.3 kDa, Ɖ (SEC) = 1.15, Rh (DLS) = 12.5 nm. Table 3. Elemental analysis (EA) results of P0 and P1 Sample C (%) H (%) N (%) P0 57.41 7.51 - P1 55.62 6.65 4.16 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 150 5.4.3. Synthesis of P1-SCNPs (as a control) The synthesis of P1-SCNPs from P1 was achieved via irradiation with ultraviolet light. 15 mg (0.07 mmol) of P1 were dissolved in 15 mL of DMF and the solution was exposed to UV light irradiation (365 nm) for 1 h. Subsequently, the reaction was quenched by the addition of liquid nitrogen. The resulting P1-SCNPs were precipitated with diethyl ether and dried under dynamic vacuum. Yield: 92%. Mw (SEC) = 75.6 kDa, Ɖ (SEC) = 1.11, Rh (DLS) = 10.0 nm. 5.4.4. Synthesis of Pt(II)-SCNPs The synthesis of Pt(II)-SCNPs from P1 was achieved via irradiation with ultraviolet light. Specifically, 15 mg (0.07 mmol) of P1 and 1.96 mg (0.00525 mmol) Pt(COD)Cl2 were dissolved in 15 mL of DMF and the solution was exposed to UV light irradiation (365 nm) for 1 h. Subsequently, the reaction was quenched by the addition of liquid nitrogen. The resulting Pt(II)-SCNPs were precipitated with diethyl ether and dried under dynamic vacuum. Yield: 86%. Pt(II) content (ICP-MS): 0.13 mol%. Mw (SEC) = 78.4 kDa, Ɖ (SEC) = 1.15, Rh (DLS) = 9.9 nm. 5.4.5. Synthesis of Pt(II)/Cu(II)-SCNPs P1 (15 mg, 0.07 mmol) and Pt(COD)Cl2 (1.96 mg, 0.00525 mmol) were dissolved in 15 mL of DMF and the solution was exposed to UV light irradiation (365 nm) for 1 h. The reaction was promptly quenched by the addition of liquid nitrogen. A concentrated solution of Cu(OAc)2 in DMF (0.954 mg, 0.00525 mmol of Cu) was then added dropwise and the reaction mixture was stirred for 24 h. The resulting Pt(II)/Cu(II)-SCNPs were precipitated with diethyl ether and dried under dynamic vacuum. Yield: 79%. Pt(II) content (ICP-MS): 0.14 mol%. Cu(II) content (ICP-MS): 0.43 mol%. Mw (SEC) = 81.6 kDa, Ɖ (SEC) = 1.18, Rh (DLS) = 8.6 nm. 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 151 Table 4. Inductively coupled plasma-mass spectrometry (ICP-MS) results of Pt(II)-SCNPs, Cu(II)SCNPs and Pt(II)/Cu(II)-SCNPs Sample Pt(II) content (g/mg) Cu(II) content (g/mg) Pt(II)-SCNPs 9.29 - Cu(II)-SCNPs - 9.11 Pt(II)/Cu(II)-SCNPs 9.73 9.54 5.4.6. Synthesis of Cu(II)-SCNPs (as a control). P1-SCNPs (15 mg, 0.07 mmol) was dissolved in DMF (15 ml) at r.t. A solution of Cu(OAc)2 in DMF (0.954 mg, 0.00525 mmol) was then added dropwise, and the reaction mixture was stirred for 24 h. The resulting Cu(II)-SCNPs were precipitated with diethyl ether and dried under dynamic vacuum. Yield: 95%. Cu(II) content (ICP-MS): 0.41 mol%. Mw (SEC) = 77.1 kDa, Ɖ (SEC) = 1.21, Rh (DLS) = 10.0 nm. 5.4.7. General procedure for the consecutive one-pot alkyne semihydrogenation/alkene dioxygenation reactions catalysed by Pt(II)/Cu(II)-SCNPs in NBP at r.t. In a 10 mL round bottomed, oven-dried flask, which was previously equipped of magnetic stir bar, 2 mL of Pt(II)/Cu(II)-SCNPs solution in NBP ([polymer] = 1 mg mL-1) were added, followed by 0.07 mmol of the alkyne compound (1a-1e) and the system was sealed with rubber septum. The solution was then degassed by purging argon flow for 5 min. After displacement of atmospheric oxygen, the flask was equipped of a security elastic balloon to ensure both no extra-pressure in the system and, simultaneously, to reduce volatility of solvents and substrates. A low-pressure molecular hydrogen flow was then bubbled in the reaction mixture at r.t. at definite interval of times (10 min bubbling each 20 min until reaction completion). The progress of the reaction was monitored by thin layer chromatography (TLC) using EtOAc and Hex as eluents. After the semihydrogenation reaction was complete, 13.68 mg (0.084 mmol) of Nhydroxyphthalimide was added to the solution at r.t. and under air (open flask). The 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 152 progress of the reaction was monitored by TLC using EtOAc and n-hexane as eluents. After reaction completion, 10 mL of water was added to the solution. The resultant mixture was extracted with EtOAc (3 x 5 mL) and successively washed with water (3 x 5 mL). The organic solution was dried over anhydrous Mg2SO4, filtered and evaporated to give a residue that was purified on silica gel column chromatography using Hex and EtOAc as eluents to afford the corresponding β-Keto-N-alkoxyphthalimide product (3a3e). 5.5. Results and discussion The synthetic procedure followed in this work to produce Pt(II)/Cu(II)-SCNPs is depicted schematically in Figure 74. Initially, the monomers (2-acetoacetoxy)ethyl methacrylate (AEMA) and methyl methacrylate (MMA), which functions as a spacer, were copolymerized via reversible addition fragmentation chain-transfer (RAFT) polymerization5 yielding the random copolymer P0 (Figure 74A). Subsequently, P0 with a content of β-ketoester functional groups of 30 mol%, a weight-average molecular weight of Mw =73.6 kDa and a narrow dispersity of Ɖ = 1.17 was decorated with 19 mol% of α-diazo-β-ketoester functional groups -using p-carboxybenzenesulfonazide (p-CBSA) as the diazo transfer reagent-23 leaving 11 mol% β-ketoester functional groups unreacted (see Figure 74B). 5. Consecutive One-Pot Alkyne Semihydrogenation/Alkene Dioxygenation Reactions by Pt(II)/Cu(II) Single-Chain Nanoparticles in Green Solvent 153 Figure 74A. Preparation of a random copolymer P0 containing naked β-ketoester functional groups (30 mol%) via reversible addition fragmentation chain-transfer (RAFT) polymerization (MMA = methyl methacrylate; AEMA = (2acetoacetoxy)ethyl methacrylate; AIBN = azobisisobutyronitrile; CPDB = 2-cyanoprop-2-yldithiobenzoate; EtOAc = ethyl acetate). Figure 74B. Decoration of P0 with α-diazo-β-ketoester functional groups (19 mol%) to give polymeric precursor P1 (p-CBSA = p-carboxybenzenesulfonazide; Et3N = trimethylamine; CH2Cl2 = dichloromethane).