Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells

Abstract

In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomolecules in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here we report a bioinspired strategy in which molecular shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles-precursors of nitrile imines, we discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddition over the competing nucleophilic addition. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aqueous medium, owing to its unique molecular shape that hinders the approach of a nucleophile as shown by DFT calculations. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.

Light‐Controlled Orthogonal Covalent Bond Formation at Two Different Wavelengths

“…[12] Recently,t he reversibility of the styrylpyrene dimerization was exploited to enable chain ligation and single-chain coupling [13] as well as crosslinking of polymer networks [14] in asequence-independent l-orthogonal fashion.Here,w ep resent ac atalyst-free ligation system forming covalent bonds,w here the color of light and the solvent environment determine the reaction outcome,independent of which wavelength is employed first and despite overlapping absorption spectra of the chromophores.S uch sequenceindependent l-orthogonality may open avenues to the remote control of reaction outcomes in applications ranging from multimaterial 3D direct laser lithography [15] and the synthesis of sequence-defined macromolecules [16] to in vivo ligation. [17] Our previous work [18] led us to assess the potential lorthogonality of diaryltetrazoles and o-methylbenzaldehydes as substrates for reaction with electron-poor dienophiles.The combination of o-methylbenzaldehyde thioether A (R 1 = CH 3 (O(CH 2 ) 2 ) n -, 31 < n < 64;S cheme 1), methoxy-substituted diphenyltetrazole B (R 2 = CH 3 (O(CH 2 ) 2 ) n NHCO-, 31 < n < 64), and N-ethylmaleimide was found to be the most promising system according to the observed absorptivity and reactivity of the chromophores.A sh as been highlighted by Feringa and co-workers, [19] to achieve sequence-independ-…”

Chemical Methods for Selective Labeling of Proteins

“…Later, Davis and co‐workers demonstrated genetic incorporation of S‐allylhomocysteine into proteins for cross‐metathesis reaction with an olefin (Scheme d) . In parallel, Lin's group reported the insertion of a cyclopropene derivative to react with a 1,3‐dipole generated by photoactivation of tetrazoles . Also, Chin and co‐workers synthesized cyclopropene engineered trastuzumab and used it for chemoselective conjugation with tetrazine derivative of MMAE drug .…”

Light‐Induced Tetrazole‐Quinone 1,3‐Dipolar Cycloadditions

“…This "photoclick" chemistry has proven to be highly valuable with a variety of alkenes [13,14] and found applicationi nt he light-ligation and labellingo fb iomolecules. [15,16] Other dipolarophiles, including alkynes [17] and other counterparts, [18] have evidenced the usefulness of this atom-economical transformation.However,t oo ur knowledge,t he use of quinones as dipolarophiles [19] in this processr emainedu nexplored.…”