Copper-free Click Chemistry Research Articles

Ordered and kinetically discrete sequential protein release from biodegradable thin films.

Multidrug regimens can sometimes treat recalcitrant diseases when single-drug therapies fail. Recapitulating complex multidrug administration from controlled release films for localized delivery remains challenging because their release kinetics are frequently intertwined, and an initial burst release of each drug is usually uncontrollable. Kinetic control over protein release is demonstrated by cross-linking layer-by-layer films during the assembly process. We used biodegradable and naturally derived components and relied on copper-free click chemistry for bioorthogonal covalent cross-links throughout the film that entrap but do not modify the embedded protein. We found that this strategy restricted the interdiffusion of protein while maintaining its activity. By depositing a barrier layer and a second protein-containing layer atop this construct, we generated well-defined sequential protein release with minimal overlap that follows their spatial distribution within the film.

Antibody quantum dot conjugates developed via copper-free click chemistry for rapid analysis of biological samples using a microfluidic microsphere array system.

Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features toward realization of this goal such as high photostability, brightness, and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation, and stability issues. We report a facile yet effective strategy to conjugate anti-epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin-streptavidin schemes. The Fc and Fab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/mL. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients, and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood.

Parallel synthesis and screening of peptide conjugates.

Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.

Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles.

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry.

Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis.

The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.

A Homologation Approach to the Synthesis of Difluorinated Cycloalkynes

Difluorinated cyclooctynes are important reagents for labeling azido-biomolecules through copper-free click chemistry. Here, a safe, scalable synthesis of a difluorinated cyclooctyne is reported, which involves a key homologation/ring-expansion reaction. Sequential ring expansions were also employed to synthesize and study a novel difluorinated cyclononyne.

Masthead: Macromol. Rapid Commun. 6/2014

Copper-free azide-alkyne click chemistry is utilized to covalently modify polyvinyl chloride(PVC). Phthalate plasticizer mimics di(2-ethylhexyl)-1H-triazole-4,5 dicarboxylate (DEHT), di(nbutyl)-1H-1,2,3-triazole-4,5-dicarboxylate (DBT), and dimethyl-1H-triazole-4,5-dicarboxylate(DMT) are covalently attached to PVC. DEHT, DBT, and DMT have similar chemical structures to traditional plasticizers di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), and dimethyl phthalate (DMP), but pose no danger of leaching from the polymer matrix and forming small endocrine disrupting chemicals. The synthesis of these covalent plasticizers is expected to be scalable, providing a viable alternative to the use of phthalates, thus mitigating dangers to human health and the environment.

Chemical Tumor-Targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry

Tumor-targeting strategies for nanoparticles have been predominantly based on optimization of physical properties or conjugation with biological ligands. However, their tumor-targeting abilities remain limited and insufficient. Furthermore, traditional biological binding molecules have intrinsic limitations originating from the limited amount of cellular receptors and the heterogeneity of tumor cells. Our two-step in vivo tumor-targeting strategy for nanoparticles is based on metabolic glycoengineering and click chemistry. First, an intravenous injection of precursor-loaded glycol chitosan nanoparticles generates azide groups on tumor tissue specifically by the enhanced permeation and retention (EPR) effect followed by metabolic glycoengineering. These 'receptor-like' chemical groups then enhance the tumor-targeting ability of drug-containing nanoparticles by copper-free click chemistry in vivo during a second intravenous injection. The advantage of this protocol over traditional binding molecules is that there are significantly more binding molecules on the surface of most tumor cells regardless of cell type. The subsequent enhanced tumor-targeting ability can significantly enhance the cancer therapeutic efficacy in animal studies.

Production of Site-Specific Antibody–Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.

Investigating the cellular fate of a DNA-targeted platinum-based anticancer agent by orthogonal double-click chemistry

Confocal fluorescence microscopy was used to study a platinum-based anticancer agent in intact NCI-H460 lung cancer cells. Orthogonal copper-catalyzed azide-alkyne cycloaddition (click) reactions were used to simultaneously determine the cell-cycle-specific localization of the azide-functionalized platinum-acridine agent 1 and monitor its effects on nucleic acid metabolism. Copper-catalyzed postlabeling showed advantages over copper-free click chemistry using a dibenzocyclooctyne (DIBO)-modified reporter dye, which produced high background levels in microscopic images and failed to efficiently label platinum adducts in chromatin. Compound 1 was successfully labeled with the fluorophore DIBO to yield 1* (characterized by in-line high-performance liquid chromatography/electrospray mass spectrometry). 1 and 1* show a high degree of colocalization in the confocal images, but the ability of 1* to target the (compacted) chromatin was markedly reduced, most likely owing to the steric bulk introduced by the DIBO tag. Nuclear platinum levels correlated inversely with the ability of the cells to synthesize DNA and cause cell cycle arrest, as confirmed by bivariate flow cytometry analysis. In addition, a decrease in the level of cellular transcription, shrinkage of the nucleolar regions, and redistribution of RNA into the cytosol were observed. Postlabeling in conjunction with colocalization experiments is a useful tool for studying the cell killing mechanism of this type of DNA-targeted agent.

In situ-forming robust chitosan-poly(ethylene glycol) hydrogels prepared by copper-free azide-alkyne click reaction for tissue engineering.

A water-soluble azide-functionalised chitosan was crosslinked with propiolic acid ester-functional poly(ethylene glycol) using copper-free click chemistry. The resultant hydrogel materials were formed within 5-60 min at 37 °C and resulted in mechanically robust materials with tuneable properties such as swelling, mechanical strength and degradation. Importantly, the hydrogels supported mesenchymal stem cell attachment and proliferation and were also non-toxic to encapsulated cells. As such these studies indicate that the hydrogels have potential to be used as injectable biomaterials for tissue engineering.

Monovalent maleimide functionalization of gold nanoparticles via copper-free click chemistry.

A single maleimide was installed onto the self-assembled monolayer of gold nanoparticles by copper-free click chemistry. Simple covalent biofunctionalisation is demonstrated by coupling fibroblast growth factor 2 and an oligosaccharide in a 1 : 1 stoichiometry by thiol-Michael addition.

DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection.

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

Copper-free azide–alkyne cycloaddition of targeting peptides to porous silicon nanoparticles for intracellular drug uptake

Porous silicon (PSi) has been demonstrated as a promising drug delivery vector for poorly water-soluble drugs. Here, a simple and efficient method based on copper-free click chemistry was used to introduce targeting moieties to PSi nanoparticles in order to enhance the intracellular uptake and tumor specific targeting hydrophobic drug delivery. Two RGD derivatives (RGDS and iRGD) with azide-terminated groups were conjugated to bicyclononyne-functionalized PSi nanoparticles via copper-free azide–alkyne cycloaddition. The surface functionalization was performed in aqueous solution at 37 °C for 30 min resulting in conjugation efficiencies of 15.2 and 3.4% (molar ratios) and the nanoparticle size increased from 165.6 nm to 179.6 and 188.8 nm for RGDS and iRGD, respectively. The peptides modification enhanced the cell uptake efficiency of PSi nanoparticles in EA.hy926 cells. PSi-RGDS and PSi-iRGD nanoparticles loaded with sorafenib showed a similar trend for the in vitro antiproliferation activity compared to sorafenib dissolved in dimethyl sulfoxide. Furthermore, sorafenib-loaded PSi-RGDS deliver the drug intracellulary efficiently due to the higher surface conjugation ratio, resulting in enhanced in vitro antiproliferation effect. Our results highlight the surface functionalization methodology for PSi nanoparticles applied here as a universal method to introduce functional moieties onto the surface of PSi nanoparticles and demonstrate their potential active targeting properties for anticancer drug delivery.

Oligonucleotide tagging for copper-free click conjugation.

Copper-free click chemistry between cyclooctynes and azide is a mild, fast and selective technology for conjugation of oligonucleotides. However, technology for site-specific introduction of the requisite probes by automated protocols is scarce, while the reported cyclooctynes are large and hydrophobic. In this work, it is demonstrated that the introduction of bicyclo[6.1.0]nonyne (BCN) into synthetic oligonucleotides is feasible by standard solid-phase phosphoramidite chemistry. A range of phosphoramidite building blocks is presented for incoporation of BCN or azide, either on-support or in solution. The usefulness of the approach is demonstrated by the straightforward and high-yielding conjugation of the resulting oligonucleotides, including biotinylation, fluorescent labeling, dimerization and attachment to polymer.

A general approach to DNA-programmable atom equivalents

Nanoparticles can be combined with nucleic acids to programme the formation of three-dimensional colloidal crystals where the particles' size, shape, composition and position can be independently controlled. However, the diversity of the types of material that can be used is limited by the lack of a general method for preparing the basic DNA-functionalized building blocks needed to bond nanoparticles of different chemical compositions into lattices in a controllable manner. Here we show that by coating nanoparticles protected with aliphatic ligands with an azide-bearing amphiphilic polymer, followed by the coupling of DNA to the polymer using strain-promoted azide-alkyne cycloaddition (also known as copper-free azide-alkyne click chemistry), nanoparticles bearing a high-density shell of nucleic acids can be created regardless of nanoparticle composition. This method provides a route to a virtually endless class of programmable atom equivalents for DNA-based colloidal crystallization.

Cell surface clustering of heparan sulfate proteoglycans by amphipathic cell-penetrating peptides does not contribute to uptake

For arginine-rich cell-penetrating peptides (CPPs), an association with heparan sulfate (HS) chains is considered the first step in the stimulation of uptake for many cells. Much less is known about the role of HS chains in the cell-association and internalization of arginine-free amphipathic CPP such as transportan-10 (TP10). Here, we report that various TP10 analogs differ in their capacity to accumulate on HS-rich plasma membranes in an HS-dependent manner. No accumulation was observed on HS-poor plasma membranes or when HS was removed by enzymatic cleavage. The TP10 analog that strongly clustered on the cell surface, also showed a pronounced capacity to form clusters with HS chains in solution. However, aggregation occurred in a thermodynamically different way compared to the interaction of arginine-rich CPP with HS. To monitor the impact of the peptide on the aggregation of the glycocalyx by time-lapse microscopy, sialic acids were visualized by metabolic labeling using copper-free click chemistry to attach fluorophores to metabolically incorporated azido sugars. Strikingly, a highly enhanced HS-mediated accumulation on the plasma membrane of a particular TP10 analog did not correlate with a better uptake. These findings illustrate that the mode of interaction between cell-penetrating peptides and HS chains has important functional consequences regarding peptide internalization and that there is no direct coupling of interaction, accumulation and uptake.

Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group

We developed a novel method for capturing proteins that interact with a target protein. This method utilizes a protein containing a site-specifically incorporated 3-azidotyrosine (N3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N3-Y at position 72 and conjugated it to FG beads by copper-free click chemistry. From the Ca(2+)/CaM-binding proteins captured from mouse brain cell lysate and analysis by mass spectrometry, we identified six proteins: alpha-enolase (ENOA), glucose-6-phosphate isomerase (GPI), annexin A5 (ANXA5), malate dehydrogenase 1 (MDH1), glyceraldehyde-3-phosphate dehydrogenase and the well-known CaM-binding protein phosphoglycerate kinase 1 (PGK1). The presence of photo-crosslinking products via N3-Y for all the captured proteins except GPI indicated that they bound directly to CaM. In this study, ENOA, ANXA5 and MDH1 were identified as novel CaM-binding proteins, and PGK1 was bound to Ca(2+)/CaM and also Ca(2+)-free CaM. This method should prove useful for capturing novel interacting proteins and serve as a useful tool for proteomic analyses.

Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging

An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with (64)Cu via a strain-promoted azide-alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with (64)Cu radionuclide. Following incubation with the (64)Cu-radiolabeled DBCO complex (DBCO-PEG4-Lys-DOTA-(64)Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with (64)Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the (64)Cu-radiolabeled CNPs ((64)Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After (64)Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, in vivo biodistribution and tumor-targeting ability of (64)Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles.

Design considerations for PAMAM dendrimer therapeutics

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer–ester MTX conjugates were much more cytotoxic than the dendrimer–amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.