Analysis and Optimization of Copper‐Catalyzed Azide–Alkyne Cycloaddition for Bioconjugation

Hong, Vu; Presolski, Stanislav I.; Ma, Celia; Finn, M. G.

doi:10.1002/ange.200905087

Cited by 325 publications

(334 citation statements)

References 52 publications

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“…To achieve this goal, we employed copper-catalyzed azide-alkyne cyclo-addition click chemistry to attach active compounds to surfaces 33 . We chose polyethylene glycol (PEG) polymers decorated with azide groups as the surface linkers because PEG is flexible, hydrophilic, and non-bulky, and we used glass as a model surface material.…”

Section: Resultsmentioning

confidence: 99%

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Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

et al. 2017

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Bacteria use a process called quorum sensing to communicate and orchestrate collective behaviors including virulence factor secretion and biofilm formation. Quorum sensing relies on production, release, accumulation, and population-wide detection of signal molecules called autoinducers. Here, we develop concepts to coat surfaces with quorum-sensing-manipulation molecules as a method to control collective behaviors. We probe this strategy using Staphylococcus aureus. Pro- and anti-quorum-sensing molecules can be covalently attached to surfaces using click chemistry, where they retain their abilities to influence bacterial behaviors. We investigate key features of the compounds, linkers, and surfaces necessary to appropriately position molecules to interact with cognate receptors, and the ability of modified surfaces to resist long-term storage, repeated infections, host plasma components, and flow-generated stresses. Our studies highlight how this surface approach can be used to make colonization-resistant materials against S. aureus and other pathogens and how the approach can be adapted to promote beneficial behaviors of bacteria on surfaces.

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Section: Resultsmentioning

confidence: 99%

“…We calculate the coverage density of the clicked azide to be 2.1 × 10 4 μm −2 (±0.11). We assume that the alkyne dye and the Alkyne-AIP-I have identical reactivity in the click reaction 33 , thus rendering the same surface coverage density, which is sufficient to stimulate the S. aureus Agr quorum-sensing response (Fig. S6e).…”

Section: Resultsmentioning

confidence: 99%

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

et al. 2017

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“…A method, described in ref. 45, was tried to overcome the observed site reactions. In order to prevent the copper-induced precipitation and protein degradation through formation of reactive oxygen species, excess ligand, aminoguanidine and NiCl 2 were added to the reaction.…”

Section: Resultsmentioning

confidence: 99%

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

et al. 2015

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The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/ connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nanoobject assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

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“…Thus, cells were not depleted of Met, and Anl was added to standard growth medium (Materials and Methods). After 4 h, Anllabeled proteins were detected by Cu (I)-catalyzed azide-alkyne ligation (24) to Alexa Fluor 488-alkyne in fixed (permeabilized) cells. Imaging of cells by fluorescence microscopy revealed prominent labeling of proteins in HEK293-8D3 cells (Fig.…”

Section: Expression Of Nll-ecmetrs Enables Incorporation Of Anl Into mentioning

confidence: 99%

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Ngo

Schuman

Tirrell

2013

Proc. Natl. Acad. Sci. U.S.A.

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Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications.

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Analysis and Optimization of Copper‐Catalyzed Azide–Alkyne Cycloaddition for Bioconjugation

Cited by 325 publications

References 52 publications

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

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