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Simultaneous “One Pot” Expressed Protein Ligation and CuI‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization

Proteins, enzymes in particular, are becoming more and more important in synthesis and the creation of stereoand regioselectively novel products with high specificity. They also play an important role in the development of diagnostic systems, microarrays and biosensors. The anchoring of proteins to solid supports can facilitate these systems, but to maintain the native properties and homogeneity, site specific immobilization is preferred, which in turn calls for site specific introduction of reactive groups into the protein structure. However, modifications can influence protein stability, activity or electrostatic properties of the protein surface. Alternatively, naturally occurring functionalities, for example, the e-amino group of lysine or the thiol group of cysteine, can be employed. However, frequently more than one such reactive group is exposed on the protein surface leading to formation of several distinct protein species. To circumvent these problems orthogonal ligation strategies are promising alternatives. A commonly used method is expressed protein ligation (EPL). The wide scope of combining C-terminal thioesters with N-terminal cysteine residues of another molecule has been demonstrated in various cases, as recently reviewed. The formation of native peptide bonds, the selectivity and the mild reaction conditions are just some advantages of this technique. Another ligation method is the Cu-catalyzed azide/alkyne cycloaddition (CuAAC), which was independently described by the groups of Meldal and Sharpless. This catalyzed Huisgen [3+2] cycloaddition is accelerated 10-fold, and regioselectively forms the 1,4-disubstituted 1,2,3-triazole through Cu catalysis. The robustness to side reactions, regioand chemoselectivity, and compatibility with an aqueous environment facilitates its application to biomolecules. Accordingly, the reaction can be classified as a “click” reaction. We have combined expressed protein ligation and Cu-catalyzed azide/alkyne cycloaddition in a single simultaneous reaction step to circumvent purification of the ligation product after EPL (Scheme 1). With this combination we have succeeded in immobilizing two model proteins, enhanced green fluo-