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Langmuir : the ACS Journal of Surfaces... Nov 2011Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying...
Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying PEG-triphenylphosphine was prepared for Staudinger ligation with azide-containing biotin, which was conducted in PBS buffer (pH 7.4) at room temperature without a catalyst. Then, immobilization and microarray fabrication of the biotinylated liposome onto a streptavidin-modified glass slide via the specific streptavidin/biotin interaction were investigated by comparing with directly formed biotin-liposome, which was prepared by the conventional liposome formulation of lipid-biotin with all other lipid components. Next, the covalent microarray fabrication of liposome carrying triphenylphosphine onto an azide-modified glass slide and its further glyco-modification with azide-containing carbohydrate were demonstrated for glyco-liposomal microarray fabrication via Staudinger ligation. Fluorescence imaging confirmed the successful immobilization and protein binding of the intact immobilized liposomes and arrayed glyco-liposomes. The azide-reactive liposome provides a facile strategy for membrane-mimetic glyco-array fabrication, which may find important biological and biomedical applications such as studying carbohydrate-protein interactions and toxin and antibody screening.
Topics: Azides; Biomimetic Materials; Biotinylation; Carbohydrates; Coated Materials, Biocompatible; Glass; Liposomes; Microarray Analysis; Microtechnology; Organophosphorus Compounds; Printing; Substrate Specificity; Surface Properties
PubMed: 21928859
DOI: 10.1021/la2032434 -
Journal of Nanobiotechnology Oct 2017Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent...
BACKGROUND
Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled.
RESULTS
Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of "click" reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology.
DISCUSSION
Although previous studies have produced peptides that can both form amyloid fibrils and undergo "click"-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.
Topics: Alkynes; Amino Acid Sequence; Amyloid; Azides; Click Chemistry; Green Fluorescent Proteins; Nanotechnology; Peptides; Prealbumin
PubMed: 28985740
DOI: 10.1186/s12951-017-0300-7 -
Journal of Medicinal Chemistry May 2023Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers...
Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (' ≥ 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and -impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.
Topics: Alkynes; Azides; Catalysis; Copper; Gene Library; Liposomes; Peptides; Pharmacokinetics
PubMed: 37075027
DOI: 10.1021/acs.jmedchem.3c00138 -
Chemistry (Weinheim An Der Bergstrasse,... Feb 2022The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the...
The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.
Topics: Alkynes; Azides; Cycloaddition Reaction; Fluorescent Dyes; Ionophores
PubMed: 34979050
DOI: 10.1002/chem.202104111 -
Bioorganic & Medicinal Chemistry Letters Jan 2018The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a...
The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be prohibitive to some applications. Consequently, we have optimized a copper-free microwave-assisted reaction to alleviate the necessity for the copper catalyst. A small array of triazoles was prepared to examine the scope of this approach, and the methodology was translated to a protein context through the use of unnatural amino acids to demonstrate one of the first microwave-mediated bioconjugations involving a full length protein.
Topics: Alkynes; Azides; Cycloaddition Reaction; Microwaves; Models, Molecular; Molecular Structure; Triazoles
PubMed: 29248298
DOI: 10.1016/j.bmcl.2017.12.007 -
Chembiochem : a European Journal of... Apr 2021Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method...
Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2-diamino-4,5-methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide- and alkene-modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group - not only after addition of ManNAc derivatives to the cell culture. A cyclopropane-modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse-electron-demand Diels-Alder reactions with 1,2,4,5-tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell-surface sialic acid levels are only moderately increased.
Topics: Alkenes; Azides; Cycloaddition Reaction; Fluorescent Dyes; HEK293 Cells; HeLa Cells; Hexosamines; Humans; Metabolic Engineering; Microscopy, Fluorescence; N-Acetylneuraminic Acid
PubMed: 33180370
DOI: 10.1002/cbic.202000715 -
Molecules (Basel, Switzerland) Jun 2013Click chemistry is a powerful chemical reaction with excellent bioorthogonality features: biocompatible, rapid and highly specific in biological environments. For... (Review)
Review
Click chemistry is a powerful chemical reaction with excellent bioorthogonality features: biocompatible, rapid and highly specific in biological environments. For glycobiology, bioorthogonal click chemistry has created a new method for glycan non-invasive imaging in living systems, selective metabolic engineering, and offered an elite chemical handle for biological manipulation and glycomics studies. Especially the [3 + 2] dipolar cycloadditions of azides with strained alkynes and the Staudinger ligation of azides and triarylphosphines have been widely used among the extant click reactions. This review focuses on the azide-based bioorthogonal click chemistry, describing the characteristics and development of these reactions, introducing some recent applications in glycobiology research, especially in glycan metabolic engineering, including glycan non-invasive imaging, glycomics studies and viral surface manipulation for drug discovery as well as other applications like activity-based protein profiling and carbohydrate microarrays.
Topics: Azides; Click Chemistry; Glycomics; Research
PubMed: 23783454
DOI: 10.3390/molecules18067145 -
Accounts of Chemical Research Nov 2010A major challenge in materials science is the ongoing search for coupling agents that are readily synthesized, capable of versatile chemistry, able to easily... (Review)
Review
A major challenge in materials science is the ongoing search for coupling agents that are readily synthesized, capable of versatile chemistry, able to easily functionalize materials and surfaces, and efficient in covalently linking organic and inorganic entities. A decade ago, we began a research program investigating perfluorophenylazides (PFPA) as the coupling agents in surface functionalization and nanomaterial synthesis. The p-substituted PFPAs are attractive heterobifunctional coupling agents because of their two distinct and synthetically distinguishable reactive centers: (i) the fluorinated phenylazide, which is capable of forming stable covalent adducts, and (ii) the functional group R, which can be tailored through synthesis. Two approaches have been undertaken for material synthesis and surface functionalization. The first method involves synthesizing PFPA bearing the first molecule or material with a functional linker R and then attaching the resulting PFPA to the second material by activating the azido group. In the second approach, the material surface is first functionalized with PFPA via functional center R, and coupling of the second molecule or material is achieved with the surface azido groups. In this Account, we review the design and protocols of the two approaches, providing examples in which PFPA derivatives were successfully used in material surface functionalization, ligand conjugation, and the synthesis of hybrid nanomaterials. The methods developed have proved to be general and versatile, and they are applicable to a wide range of materials (especially those that lack reactive functional groups or are difficult to derivatize) and to various substrates of polymers, oxides, carbon materials, and metal films. The coupling chemistry can be initiated by light, heat, and electrons. Patterned structures can be generated by selectively activating the areas of interest. Furthermore, the process is easy to perform, and light activation occurs in minutes, greatly facilitating the efficiency of the reaction. PFPAs indeed demonstrate many benefits as versatile surface coupling agents and offer opportunities for further exploration.
Topics: Azides; Molecular Structure; Nanostructures; Particle Size; Surface Properties
PubMed: 20690606
DOI: 10.1021/ar100066t -
Yakugaku Zasshi : Journal of the... 2018Target identification of screening hit compounds with unknown mechanisms of action obtained from chemical libraries by phenotypic assays has played an important role... (Review)
Review
Target identification of screening hit compounds with unknown mechanisms of action obtained from chemical libraries by phenotypic assays has played an important role in the development of innovative drugs that work based on novel mechanisms. To improve the usability of the target identification based on the photoaffinity labeling method, we have studied the "diazido probe" method, wherein a photoreactive aromatic azido and relatively photostable aliphatic azido groups are sequentially used for photoreaction and introduction of a latent detectable tag via the Staudinger ligation or click reactions, and related synthetic methods that enable expeditious preparation of molecular probes. To facilitate the development of diazido probes, we established short synthetic routes to diazido building blocks with different connectable groups based on sequential iridium-catalyzed C-H borylation and copper-catalyzed azidation of 1,3-disubstituted benzenes, and subsequent diverse functional group transformations leaving the azido groups untouched. To improve the utility of click chemistry for efficient introduction of a latent detectable tag, we developed a transient protection method of cyclooctynes from cycloaddition with an azide by 1 : 1 complexation with a cationic copper(I) salt. Application of this protection method using a cationic copper salt to a cyclooctyne bearing a terminal alkyne allowed for the selective click conjugation with an azide at the terminal alkyne moiety, which rendered functionalized cyclooctyne derivatives easily synthesizable.
Topics: Azides; Click Chemistry; Drug Discovery; Molecular Probes; Photoaffinity Labels
PubMed: 30068846
DOI: 10.1248/yakushi.17-00211-4 -
Journal of Medicinal Chemistry Aug 2022High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that...
High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.
Topics: Alkynes; Azides; Cycloaddition Reaction; High-Throughput Screening Assays; Mass Spectrometry
PubMed: 35912476
DOI: 10.1021/acs.jmedchem.1c02206