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Genetically Encoded Copper-Free Click Chemistry**

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DOI: 10.1002/anie.201008178
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Genetically Encoded CopperFree Click Chemistrynull In Vivo Protein Labeling DOI: 10.1002/anie.201008178 Genetically Encoded Copper-Free Click Chemistry** Tilman Plass, Sigrid Milles, Christine Koehler, Carsten Schultz,* and Edward A. Lemke* The ability to visualize biomolecules within living specimen by engineered fluorescence tags has become a major tool in modern biotechnology and cell biology. Encoding fusion proteins with comparatively large fluorescent proteins (FPs) as originally developed by the Chalfie and Tsien groups is currently the most widely applied technique.[1] As synthetic dyes typically offer better photophysical properties than FPs, alternative strategies have been developed based on genet- ically encoding unique tags such as Halo and SNAP tags, which offer high specificity but are still fairly large.[2] Small tags like multi-histidine[3] or multi-cysteine motifs[4] may be used to recognize smaller fluorophores, but within the cellular environment they frequently suffer from poor specificity as their basic recognition element is built from native amino acid side chains. Such drawbacks may be overcome by utilizing bioorthogonal chemistry that relies on coupling exogenous moieties of non-biological origin under mild physiological conditions. A powerful chemistry that fulfils these require- ments is the Huisgen type (3+2) cycloaddition between azides and alkynes (a form of click chemistry[5]). By utilizing supplementation-based incorporation techniques and click reactions Beatty et al. coupled azide derivatized dyes to Escherichia coli expressing proteins bearing linear alkynes.[6] However, this azide–alkyne cycloaddition required copper(I) as a catalyst (CuAAC), which strongly reduces biocompati- bility (but see Ref. [7]). This limitation has been overcome by Bertozzi and co-workers, who showed that the “click” reaction readily proceeds when utilizing ring-strained alkynes as a substrate[8] and since then this strain-promoted azide– alkyne cycloaddition (SPAAC) has fou

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