Expanding the Genetic Code for Photoclick Chemistry in E. coli, Mammalian Cells, and A. thaliana

Abstract

Bioorthogonal chemical reactions together with techniques to expand the genetic code have provided exciting new means for protein labeling and visualization in living systems, as well as for optimizing the efficacy of therapeutic proteins. Toward these goals, amino acids with small bioorthogonal functional groups, such as azide, alkyne, or cyclopropene moieties, as well as larger reactive bioorthogonal groups, such as cyclooctyne, norbornene, trans-cyclooctene, aryltetrazole, or aryltetrazine, have been site-specifically incorporated into proteins, allowing for selective conjugation of biophysical probes through azide–alkyne click chemistry (AAC), tetrazole–alkene photoclick chemistry (TAP), and reverse-electron demand Diels–Alder reactions. The main advantages of the photoclick reaction (Supporting Information, Scheme S1) are: 1) its fast rate (up to 50m 1 s ); 2) that spatiotemporal control is initiated by a photo-induced reaction; 3) that the photoclick reaction is fluorogenic, allowing for high-contrast fluorescence imaging without tedious washing steps. In previous studies, we reported the site-specific incorporation of p-(2-tetrazole)phenylalanine (p-Tpa) and N-e-(1-methylcycloprop-2-enecarboxamido)lysine (CpK) in E. coli and mammalian cells. Subsequent photoirradiation of labeled proteins with UV light facilitates selective conjugation with dimethyl fumarate or diaryltetrazole, respectively. By expanding the genetic code and introducing photoclick chemistry to plants, important problems in plant chemical biology can be addressed, such as photosynthesis and stress response, which can only be studied at the organismal level. Recently, expansion of the genetic code has been used to optimize therapeutic proteins produced in bacteria and mammalian cells. Because plants offer an attractive alternative to microbial fermentation and animal cell cultures for high-yield production of recombinant proteins on an agricultural scale, expanding the genetic code in plants would be useful for producing recombinant therapeutic proteins and enzymes with enhanced properties, better safety, and lower costs. To fully realize the potential of photoclick reaction for tracking fast cellular processes, it is desirable that the unnatural amino acid (UAA) used has a small functional group such that there is minimal perturbation of the target protein, and a very brief exposure to long-wavelength UV light or violet-blue light is used to drive the photoclick reaction to minimize damage to cells and plants. Herein, we addressed these issues by genetically incorporating N-eacryllysine (AcrK, Figure 1A), in response to an amber stop codon (TAG) in bacterial cells, mammalian cells, and plants. This new strategy was then used to efficiently label proteins both in vitro and in vivo. In comparison to lysine, AcrK has only four extra non-hydrogen atoms, which is significantly less than other UAAs. Replacing one lysine residue with AcrK should cause only minimal perturbation to the target protein. In addition, the electron-withdrawing amido group should activate the terminal alkene group to achieve a higher photoclick reaction rate. Indeed, the photoclick reaction between tetrazole and acrylamide was found to proceed nearly one hundred times faster than that of allylphenylether and 1.5 times faster than that of cyclopropene. AcrK was synthesized by reacting N-a-Boc-lysine with acryloyl chloride in a basic ethyl acetate/water solution at 0 8C (Figure 1A), followed by deprotection with HCl gas with an overall yield of 72%, without the need for metal catalysts. AcrK was found to be relatively stable in the presence of glutathione, an abundant biomolecule both amine and thiol groups; greater than 95% of AcrK remained following incubation with 5 mm reduced glutathione in a buffer at pH 7 for 24 hours (Figure S1). At neutral pH, most primary amines are protonated and most thiols are neutral. Therefore, glutathione could react slowly with the acrylamido group through a Michael addition reaction. While AcrK has comparable stability to CpK under physiological conditions, the synthetic route for CpK requires six steps, expensive heavy metal catalysis, with an overall yield of 15%. Because a large amount of the UAA is required for modifying plant proteins on an agricultural scale, it is essential that the UAA is synthesized in an economically, without the use of toxic heavy-metal catalysts. We chose diaryltetrazole 2 (Figure 1B) because it is highly reactive for photoclick reactions and is soluble in water. Because the molar extinction coefficients of 2 at 365 nm and 405 nm are around 100m 1 cm 1 (Figure S2) and the quantum yields for the photolysis of diaryltetrazoles are very high (0.5– 0.9), 2 should be efficiently activated by long-wavelength UV light or violet light. An amine functional group provides a convenient handle for further derivatization. Upon irradi[*] F. H. Li, H. Zhang, Y. Sun, Y. C. Pan, J. Z. Zhou, Prof. Dr. J. Y. Wang Laboratory of Non-coding RNA, Institute of Biophysics Chinese Academy of Sciences 15 Datun Road, Chaoyang District, Beijing, 100101 (China) E-mail: jwang@ibp.ac.cn [] These authors contributed equally to this work.