Azide-alkyne Cycloaddition Click Reaction Research Articles

“Clicking” Amphiphilic Block Copolymers onto POSS Core: A General Approach for “Star-like” Polymers with Different Symmetry

“Star-like” macromolecules have been widely studied because of their unique physical and chemical properties, which play a great role in many important application fields. In this study, a library of star-like amphiphilic block copolymers consisting of a polyhedral oligomeric silsesquioxane (POSS) core and polyethylene glycol-bolck-polystyrene (PEGm-b-PSn) diblock copolymer arms with an exact arm number were prepared through a robust copper-catalysed azide-alkyne cycloadditions (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions. Moreover, homo- and mikto-arms star polymers with different compositions were obtained by precisely regulating the central symmetry of the POSS core. The target polymers and intermediate products were characterized by nuclear magnetic resonance (NMR), size exclusion chromatography (SEC), Fourier transform infrared (FT-IR) spectroscopy, and matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry. This study demonstrates the chemical robustness of the novel POSS-based building block and establishes a general and efficient approach to prepare star-like macromolecules, especially for asymmetric heteroarm star-like macromolecules, which serve as important model compounds in the study of their bulk properties as well as self-assembling behaviors.

Towards Aptamer-Targeted Drug Delivery to Brain Tumors: The Synthesis of Ramified Conjugates of an EGFR-Specific Aptamer with MMAE on a Cathepsin B-Cleavable Linker.

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody-drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer-drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer-enzymatically cleavable payload conjugates of various drug-aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer-drug conjugates by means of a stepwise Cu-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer-drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

Application of RET Approach for Ratiometric Response Enhancement of ICT Fluorescent Hg2+ Probe based on Crown-containing Styrylpyridinium Dye.

Styrylpyridinium dye bearing azadithia-15-crown-5 ether receptor group SP and 4-alkoxy-1,8-naphthalimide fluorophore were linked using copper-catalyzed azide-alkyne cycloaddition click reaction to afford dyad compound NI-SP. Chemosensor NI-SP exhibited selective ratiometric fluorescent response to the presence of Hg2+ in aqueous solution due to the interplay between resonance energy transfer (RET) and intramolecular charge transfer (ICT) processes occurred upon excitation. The observed switching of the ratio of emission intensities in the blue and red channels R was higher than in the case of monochromophoric styrylpyridine derivative SP showing ratiometric response based on ICT mechanism only. Biological studies revealed that NI-SP penetrates into human lung adenocarcinoma A549 cells and accumulates in cytoplasm and lysosomes. When cells were pre-incubated with mercury (II) perchlorate, the ratio R was increased 2.6 times, which enables detection of intracellular Hg2+ ions and their quantitative analysis in the 0.7-6.0 μM concentration range.

Discovery of A Synthetic Hub for Regio- and Stereoselective Construction of Triazolyl Vinyl Sulfonyl Fluorides.

A new sulfonyl fluoride reagent 1-bromobut-3-ene-1,3-disulfonyl difluoride (BEDF) was developed. This unique reagent possesses two clickable functionalities to be used for both azide-alkyne cycloaddition click and SuFEx click reactions. This new reagent was applied for the regioselective construction of a class of novel triazolyl vinyl sulfonyl fluorides in which the C-4 position 1H-1,2,3-triazoles were functionalized with vinyl sulfonyl fluorides of exclusively E-configuration.

Synthesis and Photo/Radiation Chemical Characterization of a New Redox-Stable Pyridine-Triazole Ligand.

Photocatalysis using transition-metal complexes is widely considered the future of effective and affordable clean-air technology. In particular, redox-stable, easily accessible ligands are decisive. Here, we report a straightforward and facile synthesis of a new highly stable 2,6-bis(triazolyl)pyridine ligand, containing a nitrile moiety as a masked anchoring group, using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. The reported structure mimics the binding motif of uneasy to synthesize ligands. Pulse radiolysis under oxidizing and reducing conditions provided evidence for the high stability of the formed radical cation and radical anion 2,6-di(1,2,3-triazol-1-yl)-pyridine compound, thus indicating the feasibility of utilizing this as a ligand for redox active metal complexes and the sensitization of metal-oxide semiconductors (e. g., TiO2 nanoparticles or nanotubes).

Development of Novel Immobilized Copper-Ligand Complex for Click Chemistry of Biomolecules.

Copper-catalyzed azide-alkyne cycloaddition click (CuAAC) reaction is widely used to synthesize drug candidates and other biomolecule classes. Homogeneous catalysts, which consist of copper coordinated to a ligand framework, have been optimized for high yield and specificity of the CuAAC reaction, but CuAAC reaction with these catalysts requires the addition of a reducing agent and basic conditions, which can complicate some of the desired syntheses. Additionally, removing copper from the synthesized CuAAC-containing biomolecule is necessary for biological applications but inconvenient and requires additional purification steps. We describe here the design and synthesis of a PNN-type pincer ligand complex with copper (I) that stabilizes the copper (I) and, therefore, can act as a CuAAC catalyst without a reducing agent and base under physiologically relevant conditions. This complex was immobilized on two types of resin, and one of the immobilized catalyst forms worked well under aqueous physiological conditions. Minimal copper leaching was observed from the immobilized catalyst, which allowed its use in multiple reaction cycles without the addition of any reducing agent or base and without recharging with copper ion. The mechanism of the catalytic cycle was rationalized by density functional theory (DFT). This catalyst's utility was demonstrated by synthesizing coumarin derivatives of small molecules such as ferrocene and sugar.

Copper phosphorylated cellulose nanofibers mediated azide-alkyne cycloaddition click reaction in water

Heterogenous copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was performed by using the phosphorylated carbohydrate-based cellulose nanofibers loaded with copper(II) ions. The copper-containing phosphorylated cellulose nanofibers (here after noted Cu(II)-PCNFs) were prepared in two different morphologies, namely the paper and foam ones and characterized by different techniques, including Scanning Electronic Microscopy (SEM), Energy Dispersive X-ray (EDX), Brauner-Emmett-Teller (BET), FT-IR spectroscopy (FTIR), Thermal Gravimetric Analysis (TGA), X-ray Photoelectron spectroscopy (XPS) and Atomic Force Microscopy (AFM). Cu(II)-PCNFs showed high activity in the CuAAC reaction when applied to the ligation of various organic azides and terminal alkynes without any reducing agent, resulting in the regioselective synthesis of 1,4-disubstituted-1,2,3-triazoles in water at room temperature. These nanofibers were recovered and reused with no significant loss of catalytic activity or selectivity. A carbohydrate-based bio-support cellulose as reliable heterogenous catalyst was efficiently developed in view of taking the click chemistry concept to sustainable chemistry.

Click Chemoselective Probe with a Photoswitchable Handle for Highly Sensitive Determination of Steroid Hormones in Food Samples.

Residues of endocrine disrupting steroid hormones in food might cause various diseases like cardiovascular diseases and breast and prostate cancers. Monitoring steroid hormone levels plays a vital role in ensuring food safety and exploring the pathogenic mechanism of steroid hormone-related diseases. Based on the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction, a novel chemoselective probe, Azo-N3, which contains a reactive site N3, an imidazolium salt-based MS tag, and an azobenzene-based photoswitchable handle, was designed and synthesized to label ethynyl-bearing steroid hormones. The probe Azo-N3 was applied for the highly selective and sensitive detection of four ethynyl-bearing steroid hormones in food samples (milk, egg, and pork) by using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The ionization efficiency of the labeled analytes could be increased by 6-105-fold, and such a labeled method exhibited satisfactory detection limits (0.04-0.2 μg/L), recovery (80.6-122.4%), and precision (RSDs% lower than 6.9%). Interestingly, the efficient immobilization of the probe Azo-N3 onto α-cyclodextrin (α-CD)-modified magnetic particles to construct a solid supported chemoselective probe Fe3O4-CD-Azo-N3 and UV light-controlled release of the labeled analytes from a magnetic support can be achieved by taking advantage of the photoswitched host-guest inclusion between the azobenzene unit and α-CD. The potential applications of Fe3O4-CD-Azo-N3 for labeling, capturing, and the photocontrolled release of the labeled steroid hormones were fully investigated by mass spectrometry imaging analysis. This work not only provides a sensitive and accurate method to detect steroid hormones in food but also opens a new avenue in designing solid supported chemoselective probes.

Endogenous Enzyme-Activatable Spherical Nucleic Acids for Spatiotemporally Controlled Signal Amplification Molecular Imaging and Combinational Tumor Therapy.

Due to the adjustable hybridization activity, antinuclease digestion stability, and superior endocytosis, spherical nucleic acids (SNAs) have been actively developed as probes for molecular imaging and the development of noninvasive diagnosis and image-guided surgery. However, since highly expressed biomarkers in tumors are not negligible in normal tissues, an inevitable background signal and the inability to precisely release probes at the chosen region remain a challenge for SNAs. Herein, we proposed a rationally designed, endogenous enzyme-activatable functional SNA (Ep-SNA) for spatiotemporally controlled signal amplification molecular imaging and combinational tumor therapy. The self-assembled amphiphilic polymer micelles (SM-ASO), which were obtained by a simple and rapid copper-free strain-promoted azide-alkyne cycloaddition click reaction between dibenzocyclooctyne-modified antisense oligonucleotide and azide-containing aliphatic polymer polylactic acid, were introduced as the core elements of Ep-SNA. This Ep-SNA was then constructed by connecting two apurinic/apyrimidinic (AP) site-containing trailing DNA hairpins, which could occur via a hybridization chain reaction in the presence of low-abundance survivin mRNA to SM-ASO through complementary base pairing. Notably, the AP site-containing trailing DNA hairpins also empowered the SNA with the feasibility of drug delivery. Once this constructed intelligent Ep-SNA nanoprobe was specifically cleaved by the highly expressed cytoplasmic human apurinic/apyrimidinic endonuclease 1 in tumor cells, three key elements (trailing DNA hairpins, antisense oligonucleotide, and doxorubicin) could be released to enable subsequent high-sensitivity survivin mRNA imaging and combinational cancer therapy (gene silencing and chemotherapy). This strategy shows great application prospects of SNAs as a precise platform for the integration of disease diagnosis and treatment and can contribute to basic biomedical research.

(Bio)Functionalisation of Metal-Organic Polyhedra via Click Chemistry.

The surface chemistry of Metal-Organic Polyhedra (MOPs) is crucial to their physicochemical properties because it governs how they interact with external substances such as solvents, synthetic organic molecules, metal ions, and even biomolecules. Consequently, the advancement of synthetic methods that facilitate the incorporation of diverse functional groups onto MOP surfaces will significantly broaden the range of properties and potential applications for MOPs. Herein, we describe the use of copper(I)-catalysed, azide-alkyne cycloaddition (CuAAC) click reactions to post-synthetically modify the surface of alkyne-functionalised cuboctahedral MOPs. To this end, we synthesised a novel Rh(II)-based MOP with 24available surface alkyne groups. We have demonstrated that each of the 24 alkyne groups on the surface of our "clickable" Rh-MOP can be reacted with azide-containing molecules at room temperature, without compromising the integrity of the MOP. We exploited the wide substrate catalogue and orthogonal nature of CuAAC click chemistry to densely functionalise MOPs with diverse functional groups, including polymers, carboxylic and phosphonic acids, and even biotin moieties, which retained their recognition capabilities once anchored onto the surface of the MOP.

Phosphorus Dendrimers for Metal-Free Ligation: Design of Multivalent Pharmacological Chaperones against Gaucher Disease.

The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes β-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best β-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase β-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100 nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.

Ultrasensitive and Specific Phage@DNAzyme Probe-Triggered Fluorescent Click Chemistry for On-Site Detection of Foodborne Pathogens Using a Smartphone.

Rapid, specific, and on-site detection of virulent foodborne pathogenic strains plays a key role in controlling food safety. In this work, an ultrasensitive and specific Phage@DNAzyme signal probe was designed to detect foodborne pathogens. The proposed sensing probe was composed of the selected phage and functionalized DNAzyme, which realized the specific recognition of target foodborne pathogens at the strain level and the efficient catalysis of copper(II) based azide-alkyne cycloaddition (CuAAC) click reaction with fluorescent signal, respectively. As a proof of concept, the virulent Escherichia coli O157:H7 (E. coli O157:H7) as the representative analyte was first enriched and purified from the complex food samples by a 4-mercaptophenylboronic acid-modified gold slide. Following, the Phage@DNAzyme probes were specifically combined with the captured E. coli O157: H7 and catalyzed the click reaction between 3-azido-7-hydroxycoumarin and 3-butyn-1-ol with the assistance of Cu(II) to generate a visual fluorescent signal. Finally, the corresponding fluorescent signals were measured by a smartphone to quantify the target concentrations. Under optimized conditions, the bioassay exhibited a wide linear range from 102 to 108 CFU/mL and the detection limit was 50 CFU/mL (S/N = 3). It was further extended to the detection of another foodborne pathogen Salmonella typhimurium with satisfying sensing performances. This work gives a new path for developing rapid, specific, and on-site detection methods for trace levels of pathogenic strains in foods.

Thiol-ene photopolymerization meets azide-alkyne click reactions: P/N/Si-containing, dual curable eugenol-based hybrid coatings

The use of bio-based building blocks for the synthesis of polymers is increasing day by day. Among the bio-based building blocks, eugenol is a highly promising monomer for the preparation of thermoset materials. In this study, we combined thiol-ene photopolymerization (TEP) and thermal azide-alkyne cycloaddition click reactions to prepare eugenol-based thermally stable, P-, N-, and Si-containing networks. To this end, we synthesized a P-containing, eugenol-based monomer bearing azide group and a siloxane compound containing an alkyne group. By mixing these monomers with multifunctional thiols and by utilizing a dual-curing strategy, we managed to obtain optically transparent and thermally stable coatings with excellent adhesion to glass substrates. Thermal stability, optical transmittance, pendulum hardness, solvent resistance, and the adhesion performance of the coatings were evaluated. The gel contents of the thermoset materials were found to be over 95%. At 600 °C, the char yields of the dual-cured coatings were found to be over 30%. Coatings were also found to be resistant to acidic and basic conditions as well as solvents.

BODIPY-Labeled Estrogens for Fluorescence Analysis of Environmental Microbial Degradation.

Biodegradation of estrogen hormone micropollutants is a well-established approach toward their remediation. Fluorescently labeled substrates are used extensively for rapid, near-real-time analysis of biological processes and are a potential tool for studying biodegradation processes faster and more efficiently than conventional approaches. However, it is important to understand how the fluorescently tagged surrogates compare with the natural substrate in terms of chemical analysis and the intended application. We derivatized three natural estrogens with BODIPY fluorophores by azide-alkyne cycloaddition click reaction and developed an analytical workflow based on simple liquid-liquid extraction and HPLC-PDA analysis. The developed methods allow for concurrent analysis of both fluorescent and natural estrogens with comparable recovery, accuracy, and precision. We then evaluated the use of BODIPY-labeled estrogens as surrogate substrates for studying biodegradation using a model bacterium for estrogen metabolism. The developed analytical methods were successfully employed to compare the biological transformation of 17β-estradiol (E2), with and without the BODIPY fluorescent tag. Through measuring the complete degradation of E2 and the transformation of BODIPY-estradiol to BODIPY-estrone in the presence of a co-substrate, we found that BODIPY-labeled estrogens are biologically viable surrogates for investigating biodegradation in environmental bacteria.

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis.

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide-alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 hvs 19 h) and decreases in motility (30 µm h-1 vs 15µm h-1 ). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

Selective oligosaccharide recognition with phenylboronic acid through Cu(I)-catalysed click imprinted surfaces

With the emerging role of glycans as potential diagnostic markers of disease, highly selective diagnostic platforms must be developed to realise the clinical value of irregular glycan expression patterns. Overcoming chemical and structural homogeneity between oligosaccharide structures however remains a significant challenge, which largely rests on the capability to develop materials embedding precise carbohydrate recognition. Herein we report a surface imprinting strategy that takes advantage of the high yield Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) click reaction to achieve oligosaccharide imprinted cavities featuring precisely organized phenylboronic acid (PBA) motifs. Central to the construction of the imprinted surface was the choice of a foundation alkyne-terminated monolayer of a long alkyl chain to impart a high surface click efficiency of the PBA motif. By pre-forming and clicking an oligosaccharide:PBA complex onto the alkyne-terminated monolayer and capping the free space, selective oligosaccharide imprinted cavities were formed with low millimolar dissociation constants.

Molecular Diffusivity of Click Reaction Components: The Diffusion Enhancement Question.

Micrometer-sized objects are widely known to exhibit chemically driven motility in systems away from equilibrium. Experimental observation of reaction-induced motility or enhancement in diffusivity at the much shorter length scale of small molecules is, however, still a matter of debate. Here, we investigate the molecular diffusivity of reactants, catalyst, and product of a model reaction, the copper-catalyzed azide–alkyne cycloaddition click reaction, and develop new NMR diffusion approaches that allow the probing of reaction-induced diffusion enhancement in nanosized molecular systems with higher accuracy than the state of the art. Following two different approaches that enable the accounting of time-dependent concentration changes during NMR experiments, we closely monitored the diffusion coefficient of reaction components during the reaction. The reaction components showed distinct changes in the diffusivity: while the two reactants underwent a time-dependent decrease in their diffusivity, the diffusion coefficient of the product gradually increased and the catalyst showed only slight diffusion enhancement within the range expected for reaction-induced sample heating. The decrease in diffusion coefficient of the alkyne, one of the two reactants of click reaction, was not reproduced during its copper coordination when the second reactant, azide, was absent. Our results do not support the catalysis-induced diffusion enhancement of the components of the click reaction and, instead, point to the role of a relatively large intermediate species within the reaction cycle with diffusivity lower than that of both the reactants and product molecule.

Clickable biocompatible brush polymers as a versatile platform toward development of multifunctional drug delivery vehicles

Clickable polymers have been gaining attention in the biomedical field because of their ability to carry various functional moieties, including drugs, imaging agents, and cell targeting ligands made by highly efficient click-reaction chemistry. In particular, brush polymers formed by the conjugation of polyethylene glycol (PEG) to clickable polymers are very promising for developing advanced drug delivery systems because of the enhanced water-solubility, biocompatibility, and so-called “stealth” capabilities. Recently, we developed and patented a clickable brush polymer PEAMO-g-PEG by the ring-opening polymerization of an alkyne-substituted oxetane monomer following the conjugation of PEG via Cu(I)-catalyzed azide-alkyne cycloaddition click reaction. The clickable brush polymer has excellent water solubility and biocompatibility as well as flexibility for coupling therapeutic drugs and other multi-modality entities. A novel chronotherapeutic polymeric drug for brain cancer therapy was then developed using the brush polymer platform as an example of this technology. The anticancer drug camptothecin primarily targeting proliferating cells was coupled to the PEAMO polymer and delivered to mouse brain via intra-tumoral delivery resulting in significantly improved survival. In this perspective, we review and discuss this new drug delivery platform with an in-depth discussion of its structural features, properties, and therapeutic applications. Future directions and possible utilization of this platform for novel formulations are also elaborated upon.

An Overview on the Performance of 1,2,3-Triazole Derivatives as Corrosion Inhibitors for Metal Surfaces.

This review accounts for the most recent and significant research results from the literature on the design and synthesis of 1,2,3-triazole compounds and their usefulness as molecular well-defined corrosion inhibitors for steels, copper, iron, aluminum, and their alloys in several aggressive media. Of particular interest are the 1,4-disubstituted 1,2,3-triazole derivatives prepared in a regioselective manner under copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions. They are easily and straightforwardly prepared compounds, non-toxic, environmentally friendly, and stable products to the hydrolysis under acidic conditions. Moreover, they have shown a good efficiency as corrosion inhibitors for metals and their alloys in different acidic media. The inhibition efficiencies (IEs) are evaluated from electrochemical impedance spectroscopy (EIS) parameters with different concentrations and environmental conditions. Mechanistic aspects of the 1,2,3-triazoles mediated corrosion inhibition in metals and metal alloy materials are also overviewed.

Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase.

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.