Optical Control of Translation with a Puromycin Photoswitch

Abstract

Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed puroswitch, features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. Puroswitch shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that puroswitch inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, puroswitch reacts with standard puromycin antibodies, which allows for tracking de novo protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, puroswitch can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision puroswitch as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.

Twelve-membered ring photoswitches with excellentZ→Econversion under ambient light

“…The ability of photoswitchable molecules to address two distinct isomers of the same molecule by the non-invasive stimulus "light" opens numerous areas of potential application, including, but not limited to, chemical sensing, 1,2 smart materials, [3][4][5] data storage, 6 logic gates, 7 photopharmacology, [8][9][10][11] and life-like motion. 12 With the broad scope of potential applications arises the need for a wide variety of chemically stable photoswitches with distinct propertiessuch as excitation wavelength, the photostationary state (PSS) under irradiation and under ambient conditions, and thermal half-life τ1 2 .…”

“…[25][26][27] These cyclic azobenzenes represent a small but fast-growing class of molecular switches. The eight-membered ring diazocines, whose photochemical properties have been investigated as early as 2009, 25 have recently been successfully applied in photopharmacology 10,11 and smart materials. 28 Comparison of the ethylene-bridged diazocine 25 with the oxamethylene-bridged diazocine 26 shows increased thermal half-life and photoconversion for the ethylene-bridged analogue.…”

Triplet sensitization enables bidirectional isomerization of diazocine with 130 nm redshift in excitation wavelengths

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] As a result, diazocines have found use in emerging elds, such as stimuli-responsive systems, [14][15][16][17][18][19] photoactuation, 20 and most notably, photopharmacology. [21][22][23][24][25][26][27][28] Unlike conventional azobenzene, whose triplet state properties have been investigated since the 1960s, 29 no studies on the triplet states of diazocine molecules have been published. Knowledge of the triplet state properties has been exploited to achieve sensitized photoswitching of conventional azobenzenes 30 (Scheme 1) in addition to other classes of molecules, such as diarylethenes, 31,32 overcrowded alkenes, 33 stilbenes, 34,35 indigos 36 and norbornadienes.…”

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids

“…Precise and temporal control of protein functions in living cells is essential to dissect dynamic and complex biological processes. − Light-triggered bioorthogonal transformations of small molecule ligands − or protein side chains − have emerged as an efficient approach for rapid gain-of-function manipulation of proteins of interest (POIs) under living conditions. Over the past decade, unnatural amino acids (UAAs) with photoremovable cages have been developed and genetically incorporated into diverse POIs, ,− including photocaged lysine, , tyrosine, − cystine, , and serine, as well as the recently developed photocaged glutamic acid .…”