A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes

Molecules

Ágnes Szatmári¹  Tibor Á. Molnár¹  Péter Kele¹  Gergely B. Cserép¹  Krisztina Németh¹  Bianka Söveges¹  Adrienn Biró¹  György Várady²  Edit Szabó² 

[1] Chemical Biology Research Group, Institute of Organic Chemistry, ELKH Research Centre for Natural Sciences, Magyar Tudósok Krt 2, H-1117 Budapest, Hungary;Molecular Cell Biology Research Group, Institute of Enzymology, ELKH Research Centre for Natural Sciences, Magyar Tudósok Krt 2, H-1117 Budapest, Hungary;

关键词: non-canonical amino acid (ncAA);    genetic code expansion (GCE);    orthogonal-bioorthogonal;    dual colour labeling;    self-labeling peptide tag (SLPT);    HaloTag;   

DOI  :  10.3390/molecules26164988

来源: DOAJ

【 摘 要 】

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.

【 授权许可】

Unknown