Single Molecular Nanomedicines Based on Macrocyclic Carrier-Drug Conjugates for Concentration-Independent Encapsulation and Precise Activation of Drugs.

Nanomedicines often rely on noncovalent self-assembly and encapsulation for drug loading and delivery. However, challenges such as reproducibility issues due to the multicomponent nature, off-target activation caused by premature drug release, and complex pharmacokinetics arising from assembly dissociation have hindered their clinical translation. In this study, we introduce an innovative design concept termed single molecular nanomedicine (SMNM) based on macrocyclic carrier-drug conjugates. Through the covalent linkage of two chemotherapy drugs to a hypoxia-cleavable macrocyclic carrier, azocalix[4]arene, we obtained two self-included complexes to serve as SMNMs. The intramolecular inclusion feature of the SMNMs has not only demonstrated comprehensive shielding and protection for the drugs but also effectively prevented off-target drug leakage, thereby significantly reducing their side effects and enhancing their antitumor therapeutic efficacy. Additionally, the attributes of being a single component and molecularly dispersed confer advantages such as ease of preparation and good reproducibility for SMNMs, which is desirable for clinical applications.

Cellular Uptake of Cell-Penetrating Peptides Activated by Amphiphilic p-Sulfonatocalix[4]arenes.

We report the synthesis of a series of amphiphilic p-sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4-Cn) and their application as efficient counterion activators for membrane transport of cell-penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO-K1, HCT 116, and KTC-1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4-Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure-activity relationships for calixarene-based counterion activators and show that CX4-Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4-C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP-counterion complexes into the cytosol and highlights the potential benefits of CX4-Cn for efficient and direct translocation of CPPs and CPP-conjugated cargo molecules into the cytosol of live cells.

Multivalent Calixarene Complexation of a Designed Pentameric Lectin.

We describe complex formation between a designed pentameric ß-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the ß-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

Polymeric membrane potentiometric antibiotic sensors using computer-aided screening of supramolecular macrocyclic carriers.

Carrier-based polymeric membrane potentiometric sensors are an ideal tool for detecting ionic species. However, in the fabrication of these sensors, the screening of carriers still relies on empirical trial- and error-based optimization, which requires tedious and time-consuming experimental verification. In this work, computer-aided screening of carriers is applied in the preparation of polymeric membrane potentiometric sensors. Molecular docking is used to study the host-guest interactions between receptors and targets. Binding energies are employed as the standard to screen the appropriate carrier. As a proof-of-concept experiment, the antibiotic ciprofloxacin is selected as the target model. A series of supramolecular macrocyclic receptors including cyclodextrins, cucurbiturils and calixarenes are chosen as potential receptors. The proposed sensor based on the receptor calix[4]arene screened by molecular docking shows a lower detection limit of 0.5 µmol L-1 for ciprofloxacin. It can be expected that the proposed computer-aided screening technique of carriers can provide a simple but highly efficient method for the fabrication of carrier-based electrochemical and optical sensors.

Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

2D MOF-enhanced SPR detector based on tunable supramolecular probes for direct and sensitive detection of DOX in serum.

Therapeutic drug monitoring of doxorubicin (DOX) is important to study pharmacokinetics in patients undergoing chemotherapy for reduction of side effects and improve patient survival by rationally controlling the dose of DOX. A fast and ultra-sensitive surface plasmon resonance (SPR) detector without sample pre-handling was developed for DOX monitoring. First, the two-dimensional metal-organic framework was modified on the Au film to enhance SPR, and then, the supramolecular probes with tunable cavity structure were self-assembled at the sensing interface for direct detection of DOX through specific host-guest interactions with a low detection limit of 60.24 pM. The precise monitoring of DOX in serum proved the possibility of clinical application with recoveries in the range 102.86-109.47%. The mechanisms of host-guest interactions between supramolecular and small-molecule drugs were explored in depth through first-principles calculations combined with SPR experiments. The study paves the way for designing facile and sensitive detectors and provides theoretical support and a new methodology for the specific detection of small molecules through calixarene cavity modulation.

Calixarene-Based Supramolecular Sensor Array for Pesticide Discrimination.

The identification and detection of pesticides is crucial to protecting both the environment and human health. However, it can be challenging to conveniently and rapidly differentiate between different types of pesticides. We developed a supramolecular fluorescent sensor array, in which calixarenes with broad-spectrum encapsulation capacity served as recognition receptors. The sensor array exhibits distinct fluorescence change patterns for seven tested pesticides, encompassing herbicides, insecticides, and fungicides. With a reaction time of just three minutes, the sensor array proves to be a rapid and efficient tool for the discrimination of pesticides. Furthermore, this supramolecular sensing approach can be easily extended to enable real-time and on-site visual detection of varying concentrations of imazalil using a smartphone with a color scanning application. This work not only provides a simple and effective method for pesticide identification and quantification, but also offers a versatile and advantageous platform for the recognition of other analytes in relevant fields.

Thiacalixarene Carboxylic Acid Derivatives as Inhibitors of Lysozyme Fibrillation.

Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.

Expanding the Hydrophobic Cavity Surface of Azocalix[4]arene to Enable Biotin/Avidin Affinity with Controlled Release.

The development of artificial receptors that combine ultrahigh-affinity binding and controllable release for active guests holds significant importance in biomedical applications. On one hand, a complex with an exceedingly high binding affinity can resist unwanted dissociation induced by dilution effect and complex interferents within physiological environments. On the other hand, stimulus-responsive release of the guest is essential for precisely activating its function. In this context, we expanded hydrophobic cavity surface of a hypoxia-responsive azocalix[4]arene, affording Naph-SAC4A. This modification significantly enhanced its aqueous binding affinity to 1013 M-1, akin to the naturally occurring strongest recognition pair, biotin/(strept-)avidin. Consequently, Naph-SAC4A emerges as the first artificial receptor to simultaneously integrate ultrahigh recognition affinity and actively controllable release. The markedly enhanced affinity not only improved Naph-SAC4A's sensitivity in detecting rocuronium bromide in serum, but also refined the precision of hypoxia-responsive doxorubicin delivery at the cellular level, demonstrating its immense potential for diverse practical applications.

Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes.

Supramolecular synthetic transporters are crucial to understand and activate the passage across lipid membranes of hydrophilic effector molecules. Herein, we introduce photoswitchable calixarenes for the light-controlled transport activation of cationic peptide cargos across model lipid bilayers and inside living cells. Our approach was based on rationally designed p-sulfonatocalix[4]arene receptors equipped with a hydrophobic azobenzene arm, which recognize cationic peptide sequences at the nM range. Activation of membrane peptide transport is confirmed, in synthetic vesicles and living cells, for calixarene activators featuring the azobenzene arm in the E configuration. Therefore, this method allows the modulation of the transmembrane transport of peptide cargos upon Z-E photoisomerization of functionalized calixarenes using 500 nm visible light. These results showcase the potential of photoswitchable counterion activators for the light-triggered delivery of hydrophilic biomolecules and pave the way for potential applications in remotely controlled membrane transport and photopharmacology applications of hydrophilic functional biomolecules.

Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes.

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. Regioselective C-H functionalization reactions are attractive for preparing alkylated arenes; however, the selectivity of existing methods is modest and primarily governed by the substrate's electronic properties. Here, we demonstrate a biocatalyst-controlled method for the regioselective alkylation of electron-rich and electron-deficient heteroarenes. Starting from an unselective "ene"-reductase (ERED) (GluER-T36A), we evolved a variant that selectively alkylates the C4 position of indole, an elusive position using prior technologies. Mechanistic studies across the evolutionary series indicate that changes to the protein active site alter the electronic character of the charge transfer (CT) complex responsible for radical formation. This resulted in a variant with a significant degree of ground-state CT in the CT complex. Mechanistic studies on a C2-selective ERED suggest that the evolution of GluER-T36A helps disfavor a competing mechanistic pathway. Additional protein engineering campaigns were carried out for a C8-selective quinoline alkylation. This study highlights the opportunity to use enzymes for regioselective radical reactions, where small molecule catalysts struggle to alter selectivity.

Supramolecular Modulation of Tumor Microenvironment through Pillar[5]arene-Based Host-Guest Recognition to Synergize Cancer Immunotherapy.

Despite the tremendous breakthrough of immunotherapy, the low response rate and resistance of immune checkpoint inhibitors (ICIs) toward solid tumors occur frequently. A highly hypoxic tumor microenvironment (TME) provides tumor cells with high concentrations of HIF-1α and polyamines to evade immune cell destruction. Reprogramming of an immunogenic TME has exhibited a brilliant future to boost immunotherapeutic performances. Herein, a supramolecular nanomedicine (TAPP) is developed on the basis of host-guest molecular recognition and metal coordination, showing the capability to remodel the immunosuppressive TME. Tamoxifen (Tmx) and Fe3+ are encapsulated into TAPP to achieve the combination of chemotherapy and chemodynamic therapy (CDT). Tmx directly downregulates HIF-1α, and a pillar[5]arene-based macrocyclic host successfully eliminates polyamines in tumors. Enhanced immunogenic cell death is achieved by Tmx and Fe3+, and the therapeutic efficacy is further synergized by immune checkpoint blockade (ICB) therapy. This supramolecular reprogramming modality encourages cytotoxic T lymphocyte infiltration, achieving pre-eminent immune response and long-term tumor suppression.

Pillararene-Inspired Macrocycles: From Extended Pillar[n]arenes to Geminiarenes.

chemistry since their establishment due to their innate functional features of molecular recognition and complexation. The rapid development of modern supramolecular chemistry has also significantly benefited from creating new macrocycles with distinctive geometries and properties. For instance, pillar[n]arenes (pillarenes), a relatively young generation of star macrocyclic hosts among the well-established ones (e.g., crown ethers, cyclodextrins, cucurbiturils, and calixarenes), promoted a phenomenal research hotspot all over the world in the past decade. Although the synthesis, host-guest properties, and various supramolecular functions of pillarenes have been intensively studied, many objective limitations and challenges still cannot be ignored. For example, high-level pillar[n]arenes (n > 7) usually do not possess applicable large-sized cavities due to structural folding and cannot be synthesized on a large scale because of the uncompetitive cyclization process. Furthermore, two functional groups must be covalently para-connected to each repeating phenylene unit, which severely limits their structural diversity and flexibility. In this context, we have developed a series of pillarene-inspired macrocycles (PIMs) using a versatile and modular synthetic strategy during the past few years, aiming to break through the synthetic limitations in traditional pillarenes and find new opportunities and challenges in supramolecular chemistry and beyond. Specifically, by grafting biphenyl units into the pillarene backbones, extended pillar[n]arenes with rigid and nanometer-sized cavities could be obtained with reasonable synthetic yields by selectively removing hydroxy/alkoxy substitutes on pillarene backbones, leaning pillar[6]arenes and leggero pillar[n]arenes with enhanced structural flexibility and cavity adaptability were obtained. By combining the two types of bridging modes in pillarenes and calixarenes, a smart macrocyclic receptor with two different but interconvertible conformational features, namely geminiarene, was discovered. Benefiting from the synthetic accessibility, facile functionalization, and superior host-guest properties in solution or the solid state, this new family of macrocycles has exhibited a broad range of applications, including but not limited to supramolecular assembly/gelation/polymers, pollutant detection and separation, porous organic polymers, crystalline/amorphous molecular materials, hybrid materials, and controlled drug delivery. Thus, in this Account, we summarize our research efforts on these PIMs. We first present an overview of their design and modular synthesis and a summary of their derivatization strategies. Thereafter, particular attention is paid to their structural features, supramolecular functions, and application exploration. Finally, the remaining challenges and perspectives are outlined for their future development. We hope that this Account and our works can stimulate further advances in synthetic macrocyclic chemistry and supramolecular functional systems, leading to practical applications in various research areas.

Protein-Calixarene Complexation: From Recognition to Assembly.

This Account summarizes the progress in protein-calixarene complexation, tracing the developments from binary recognition to the glue activity of calixarenes and beyond to macrocycle-mediated frameworks. During the past 10 years, we have been tackling the question of protein-calixarene complexation in several ways, mainly by cocrystallization and X-ray structure determination as well as by solution state methods, NMR spectroscopy, isothermal titration calorimetry (ITC), and light scattering. Much of this work benefitted from collaboration, highlighted here. Our first breakthrough was the cocrystallization of cationic cytochrome c with sulfonato-calix[4]arene leading to a crystal structure defining three binding sites. Together with NMR studies, a dynamic complexation was deduced in which the calixarene explores the protein surface. Other cationic proteins were similarly amenable to cocrystallization with sulfonato-calix[4]arene, confirming calixarene-arginine/lysine encapsulation and consequent protein assembly. Calixarenes bearing anionic substituents such as sulfonate or phosphonate, but not carboxylate, have proven useful.Studies with larger calix[n]arenes (n = 6, 8) demonstrated the bigger better binder phenomenon with increased affinities and more interesting assemblies, including solution-state oligomerization and porous frameworks. While the calix[4]arene cavity accommodates a single cationic side chain, the larger macrocycles adopt different conformations, molding to the protein surface and accommodating several residues (hydrophobic, polar, and/or charged) in small cavities. In addition to accommodating protein features, the calixarene can bind exogenous components such as polyethylene glycol (PEG), metal ions, buffer, and additives. Ternary cocrystallization of cytochrome c, sulfonato-calix[8]arene, and spermine resulted in altered framework fabrication due to calixarene encapsulation of the tetraamine. Besides host-guest chemistry with exogenous components, the calixarene can also self-assemble, with numerous instances of macrocycle dimers.Calixarene complexation enables protein encapsulation, not merely side chain encapsulation. Cocrystal structures of sulfonato-calix[8]arene with cytochrome c or Ralstonia solanacearum lectin (RSL) provide evidence of encapsulation, with multiple calixarenes masking the same protein. NMR studies of cytochrome c and sulfonato-calix[8]arene are also consistent with multisite binding. In the case of RSL, a C3 symmetric trimer, up to six calixarenes bind the protein yielding a cubic framework mediated by calixarene dimers. Biomolecular calixarene complexation has evolved from molecular recognition to framework construction. This latter development contributes to the challenge in design and preparation of porous molecular materials. Cytochrome c and sulfonato-calix[8]arene form frameworks with >60% solvent in which the degree of porosity depends on the protein:calixarene ratio and the crystallization conditions. Recent developments with RSL led to three frameworks with varying porosity depending on the crystallization conditions, particularly the pH. NMR studies indicate a pH-triggered assembly in which two acidic residues appear to play key roles. The field of supramolecular protein chemistry is growing, and this Account aims to encourage new developments at the interface between biomolecular and synthetic/supramolecular chemistry.

Biphen[n]arenes: Modular Synthesis, Customizable Cavity Sizes, and Diverse Skeletons.

Macrocyclic compounds are fundamental tools in supramolecular chemistry and have been widely used in molecular recognition, biomedicine, and materials science. The construction of new macrocycles with distinctive structures and properties would unleash new opportunities for supramolecular chemistry. Traditionally popular macrocycles, e.g., cyclodextrins, calixarenes, cucurbiturils, and pillararenes, possess specific cavities that are usually less than 10 Å in diameter; they are normally suitable for accommodating small- or medium-sized guests but cannot engulf giant molecules or structures. Furthermore, the skeletons of traditional macrocycles are impoverished and incapable of being changed; functional substituents can be introduced only on their portals.Thus, it is very challenging to construct macrocycles with customizable cavity sizes and/or diverse backbones. We have developed a versatile and modular strategy for synthesizing macrocycles, namely, biphen[n]arenes (n = 3-8), based on the structure- or function-oriented replacement of reaction modules, functional modules, and linking modules. First, two reaction modules and one functional module are connected by Suzuki-Miyaura coupling to obtain a monomer having two reaction sites. Then Friedel-Crafts alkylation between the monomer and an aldehyde (linking module) serves to afford diversely functionalized macrocycles. Moreover, large macrocycles can be achieved by using long and rigid oligo(para-phenylene) monomers. Because of the modular synthesis and plentiful molecular supplies, the biphen[n]arenes showed interesting recognition properties for both small molecules and large polypeptides. Customizable functional backbones and binding sites endowed this new family of macrocycles with peculiar self-assembly properties and potential applications in gas chromatography, pollutant capture, and physisorptive separation. Biphen[n]arenes would be a promising family of workhorses in supramolecular chemistry.In this Account, we summarize our recent work on the chemistry of biphen[n]arenes. We introduce their design and modular synthesis, including systematic exploration for reaction modules, customizable cavity sizes, skeleton functionalization, pre- and postmodification, and molecular cages. Thereafter, we discuss their host-guest properties, involving the binding for small guests by cationic/anionic/neutral biphen[n]arenes, as well as the complexation of polypeptides by large quaterphen[n]arenes. In addition, we outline the self-assembly and potential applications of this new family of macrocycles. Finally, we forecast their further development. The chemistry of biphen[n]arenes is still in its infancy. Continued exploration will not only further expand the supramolecular toolbox but also open new avenues for the use of biphen[n]arenes in the fields of biology, pharmaceutical science, and materials science.

Selective Metal-ion Complexation of a Biomimetic Calix[6]arene Funnel Cavity Functionalized with Phenol or Quinone.

In the biomimetic context, many studies have evidenced the importance of the 1st and 2nd coordination sphere of a metal ion for controlling its properties. Here, we propose to evaluate a yet poorly explored aspect, which is the nature of the cavity that surrounds the metal labile site. Three calix[6]arene-based aza-ligands are compared, that differ only by the nature of cavity walls, anisole, phenol or quinone (LOMe , LOH and LQ ). Monitoring ligand exchange of their ZnII complexes evidenced important differences in the metal ion relative affinities for nitriles, halides or carboxylates. It also showed a possible sharp kinetic control on both, metal ion binding and ligand exchange. Hence, this study supports the observations reported on biological systems, highlighting that the substitution of an amino-acid residue of the enzyme active site, at remote distance of the metal ion, can have strong impacts on metal ion lability, substrate/product exchange or selectivity.

Probing the dynamical interaction of the para-sulfonato-calix[4]arene with an antifungal protein.

Calixarenes are hallmark molecules in supramolecular chemistry as hosts for small ligands. They have also conversely proved their interest as ligands toward assisted co-crystallization of proteins. These functionalized macrocycles target positively-charged residues, and notably surface-exposed lysines, with a site-selectivity finely characterized experimentally, but that remains to be assessed. Relying on a tailored molecular dynamics simulations protocol, we explore the association of para-sulfonato-calix[4]arenes with an antifungal protein, as a small yet most competitive system with 13 surface-exposed lysines. Our computational approach probes de novo the electrostatically-driven interaction, ruled out by a competition with salt bridges, corroborating the presence of two main binding sites probed by X-ray. The attach-pull-release (APR) method provides a very good assessment of the overall binding free energy measured experimentally (-6.42 ± 0.5 vs. -5.45 kcal mol-1 by isothermal titration calorimetry). This work also probes dynamic modifications upon ligand binding, and our computational protocol could be generalized to situate the supramolecular forces ruling out the calixarene-assisted co-crystallization of proteins.

Calix[4]arene-pyrazole conjugates as potential cancer therapeutics.

Tumor selectivity is yet a challenge in chemotherapy-based cancer treatment. A series of calixarenes derivatized at the lower rim with 3-phenyl-1H-pyrazole units with variable upper-rim substituent and conformations of macrocyclic core, alkyl chain length between heterocycle and core, as well as phenolic monomer (5-(4-tert-butylphenyloxy)methoxy-3-phenyl-1H-pyrazole) have been synthesized and characterized in a range of therapeutically relevant cellular models (M-HeLa, MCF7, A-549, PC3, Chang liver, and Wi38) from different target organs/systems. Specific cytotoxicity for M-HeLa cells has been observed in tert-butylcalix[4]arene pyrazoles in 1,3-alternate (compound 7b) and partial cone (compound 7c) conformations with low mutagenicity and haemotoxicity and in vivo toxicity in mice. Compounds 7b,c have induced mitochondrial pathway of apoptosis of M-HeLa cells through caspase-9 activation preceded by the cell cycle arrest at G0/G1 phase. A concomitant overexpression of DNA damage markers in pyrazole-treated M-HeLa cells suggests that calixarene pyrazoles target DNA, which was supported by the presence of interactions between calixarenes and ctDNA at the air-water interface.

Organo-macrocycle-containing hierarchical metal-organic frameworks and cages: design, structures, and applications.

Developing hierarchical ordered systems is challenging. Using organo-macrocycles to construct metal-organic frameworks (MOFs) and porous coordination cages (PCCs) provides an efficient way to obtain hierarchical assemblies. Macrocycles, such as crown ethers, cyclodextrins, calixarenes, cucurbiturils, and pillararenes, can be incorporated within MOFs/PCCs and they also endow the resultant composites with enhanced properties and functionalities. This review summarizes recent developments of organo-macrocycle-containing hierarchical MOFs/PCCs, emphasizing applications and structure-property relationships of these hierarchically porous materials. This review provides insights for future research on hierarchical self-assembly using macrocycles as building blocks and functional ligands to extend the applications of the composites.

New Carboxytriazolyl Amphiphilic Derivatives of Calix[4]arenes: Aggregation and Use in CuAAC Catalysis.

This work focuses on the synthesis of a new series of amphiphilic derivatives of calix[4]arenes for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The aggregation properties of synthesized calix[4]arenes were studied using various techniques (fluorescence spectroscopy, nanoparticle tracking analysis, and dynamic light scattering). Increasing the length of the alkyl substituent led to stronger hydrophobic interactions, which increased polydispersity in solution. The zwitterionic nature of the synthesized calix[4]arenes was established using different types of dyes (Eosin Y for anionic structures and Rhodamine 6G for cationic structures). The synthesized calix[4]arenes were used as organic stabilizers for CuI. The catalytic efficiency of CuI-calix[4]arene was compared with that of the phase transfer catalyst tetrabutylammonium bromide (TBAB) and the surfactant sodium dodecyl sulfate (SDS). For all calixarenes, the selectivity in the CuAAC reaction was higher than that observed when TBAB and SDS were estimated.