The first steps in the application of SpyCatcher/SpyTag system for (photo)catalytic processes

Modern chemical industries seek greener solutions, while improving the enantiomeric purity of the product is a great challenge. Enzymes are ideal candidates for green catalysis, since they can promote stereoselective reactions under ambient conditions with high selectivity. By protein redesign new artificial enzymes can be devised even for non-natural enzymatic reactions. This can be reached by the introduction of artificial metal cofactors to a protein. The control of the binding and tailoring the first and second coordination spheres of the metal centre is a challenging task. In this work SPY technology is used to tackle this problem in order to obtain photocatalysts. The SPY tagging system consists of a SpyTag peptide (ST) and a SpyCatcher protein (SC), which bind to each other with a spontaneously formed peptide bond formed between amino acid side chains in a well-defined position [1]. This covalent bond ensures a strong connection, thus this method is widely used in various fields but no examples are reported about SPY-based artificial metalloenzymes. This technology provides a tool, which makes possible to construct a well-defined positioning and secondary coordination sphere in the artificial metalloenzyme. This is crucial for catalytic processes as it is a requirement of products with high enantiomer purity. In this work the first steps of SPY-based artificial metalloenzymes are presented, including the synthesis of Ru(II) and Cu(II) bound STs. Metal complexes were synthesized using Schlenk techniques. Metalated STs were synthesized by solid phase peptide synthesis, and the metalloenzymes will be gained in an aqueous reaction between SC and ST. The computational models of the SPY systems will provide information about the amino acids present in the second coordination sphere. The optimization step will involve the exchange of these amino acids in order to improve their catalytic activity in photostimulated reactions including atom transfer radical addition and isomerization reactions, focusing on stereoselectivity. The resulting library of SPY-based metalloenzymes will provide an insight on the modification of the second coordination sphere and its effect on the product. Acknowledgement: Funded by the European Union. Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Executive Agency (REA). Neither the European Union nor the granting authority can be held responsible for them. Selected references [1] D. Hatlem, T. Trunk, D. Linke, J. C. Leo, Int. J. Mol. Sci., 2019, 20, 2129.