Diels-Alder reaction–triggered bioorthogonal protein decaging in living cells

Abstract

Small molecules that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small molecule-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chemically caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.

Playing with the Molecules of Life

“…A recent study incorporated a cyclooctene lysine derivative into a reporter luciferase protein in mammalian cells. 260 Incubation with a tetrazine induced a Diels-Alder reaction that led to the removal of the cyclooctene cage, restoring wild-type lysine and consequently fluorescence. Palladium-catalyzed allene decaging 261 and phosphine-mediated decaging of azides via a Staudinger reduction have also been employed to chemically modulate protein function in vivo.…”

“…Proof-of-concept experiments were performed via caging luciferase, quenching luminescence until the tetrazine reagent is added. 260 …”

Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation

“…5–12 Recently, bioorthogonal chemistry has been utilized in payload release strategies, with the aim of triggering diverse events including drug delivery, gene expression, and modulating materials properties. 13–15 There has also been a growing interest in using external stimuli to induce bioorthogonal reactivity. In particular, photoinducible reactions have emerged as a method for turning on bioorthogonal reactions with temporal and spatial control.…”

Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells

“…Once the 4-phenoxy-4,5-dihydropyridazine (int2)isobtained, it readily tautomerizes into 4-phenoxy-1,4-dihydropyridazine (int4), which can swiftly decage through an elimination reaction. Of note,o ther dihydropyridazine tautomers previously reported to undergo decaging (int3), [6,8] are unreactive in this reaction. To support the theoretical data, we mixed 1a The initial cycloaddition is the rate-limitingstep.…”

“…[1c,2] Fore xample,p hotodeprotection of agenetically encoded caged cysteinecould be used to reveal the active native protein in live cells. [3] Similarly,p alladium-mediated depropargylation, [4] phosphine-mediated Staudinger reduction, [5] and tetrazine-triggered inverse electron-demand Diels-Alder (IEDDA) elimination reactions [6] were successfully employed to restore the activity of proteins bearing acaged lysine residue in the active site.B ond-cleavage reactions are also attractive for drugdelivery applications.P alladium-catalyzed deprotection of a5 -fluoroacil prodrug was shown as am ethod for controlled drug release in vivo. [7] TheI EDDAr eaction between at etrazine and ac aged doxorubicin derivative efficiently releases the cytotoxic drug.…”