A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes.

Ágnes Szatmári,Bianka Söveges,Krisztina Németh,György Várady,Edit Szabó,Adrienn Biró,Gergely B Cserép,Péter Kele,Tibor Á Molnár

doi:10.3390/molecules26164988

Abstract

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.

Highlights

An ever-growing demand of life science research is to be able to study multiple subcellular structures and events in their natural ambience, in cellulo
An activated isonitrile residue was synthesized in a reaction of 4,4dimethyl-2-oxazoline (1) with p-nitrophenyl-chloroformate, using n-BuLi as a base
The results indicate that Tet-SiR reacted with BCN in either combination

Summary

Introduction

An ever-growing demand of life science research is to be able to study multiple subcellular structures and events in their natural ambience, in cellulo. Fusion of engineered fluorescent proteins (e.g., GFP, mCherry) or tags (e.g., Halo, CLIP, SNAP) to the protein of interest represent routinely accomplished labeling methods. While both of these techniques offer highly selective means of probe installation, the size of these fusion tags often perturbs certain characteristics (mobility, function etc.) of the protein of interest (POI). Point mutations with non-canonical amino acids (ncAAs) carrying, e.g., a bioorthogonal function, are efficiently carried out using genetic code expansion techniques Proteins carrying such a bioorthogonalized amino acid are efficiently and selectively modified with fluorescent or fluorogenic probes bearing the complementary bioorthogonal function. The most established means for the extension of the genetic code to site- incorporate ncAAs is effected by the suppression of the Amber STOP codon (UAG), but the use of other STOP codons in mammalian cells [1], or via quadruplet codons [2,3], has been described as well

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