Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Abstract

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.

A methylated lysine is a switch point for conformational communication in the chaperone Hsp90

“…The positive hit will further be dubbed as metK-RS. This aaRS also incorporates several other unnatural amino acids 85,86 .…”

Synthesis of 3-Alkyl-6-Methyl-1,2,4,5-Tetrazines via a Sonogashira-Type Cross-Coupling Reaction

“…Tetrazine-containing amino acids are interesting compounds in chemical biology, and several have been genetically incorporated in proteins in vivo. 17,33,34,39,40 Therefore, following our previous findings, N-Boc and MOM esters were selected as appropriate protecting groups. Thus, N-Boc protected MOM esters of L-propargylglycine (8a) and 4-ethynyl-L-phenylalanine (8b) were synthesised and reacted with 1 under the optimized Sonogashira conditions to afford the methyltetrazinylamino acid derivatives 9a-b.…”

“…Therefore, we envisaged that 3-alkyl-6-methyl-1,2,4,5-tetrazines might strike a balance between stability and reactivity, improving their potential for many applications in chemical biology. 30,31,[33][34][35] The revision of existing literature on cross-coupling reactions at position 3-or 6-of the tetrazine ring seems to confirm that chlorine is more inert than bromine as the halogen, 36 thus requiring electronically enriched tetrazines and strong reaction conditions; in contrast, bromine can react under milder conditions, and a larger number of interesting substrates can potentially be accessed. 30 Consequently, we directed our efforts towards the improvement of the overall synthetic route for 1.…”

Photo-controllable bioorthogonal chemistry for spatiotemporal control of bio-targets in living systems

“…Fast rates of photolysis and the following bioorthogonal ligation process are important for the spatiotemporal resolution of a photo-controlled reaction. 28,29,34,35 It should be noted that this type of photo-controllable bioorthogonal reaction is only a modified version of known bioorthogonal reactions. Cross-talk of the original version and photo-controllable version of the same bioorthogonal reactions is therefore not avoidable.…”