Accelerated Strain‐Promoted and Oxidation‐Controlled Cyclooctyne‐Quinone Cycloaddition for Cell Labeling

Abstract

We developed an accelerated strain‐promoted oxidation‐controlled cyclooctyne‐1,2‐quinone cycloaddition (SPOCQ) for protein modification. Here, dopaquinone was generated using potassium nitrosodisulfonate (PTN) in a reaction that is six orders of magnitude faster than the enzymatic one. The dopaquinone generated then reacts with bicyclononyne (BCN) derivatives in the bioconjugation. The PTN yields a single product with defined stereochemistry and temporally controlled conjugation with BCN. The feasibility of this bioconjugation was demonstrated with different proteins. Cell labeling was explored by conjugating annexin‐dibenzocyclooctyne‐PEG4‐fluor545 through the SPOCQ approach for the detection of apoptosis in HeLa cells.

Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling

“…Initially, a small molecule DOPA was selected to demonstrate the feasibility of enzymemediated quinone reaction. To evaluate the tyrosinase-mediated modi cation in DOPA incorporated proteins [Annexin A5 (anX) and Green Fluorescent Protein (GFP)], we selected highly reactive strained cyclic ring containing alkynes namely BCN and DBCO, which was already used in life and material science applications via Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) and Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition (SPOCQ) [7,24,25,26]. Initially, the reaction was optimized with BCN to understand the one-step SPOCQ.…”

Strain‐Promoted Cycloaddition of Cyclopropenes with o‐Quinones: A Rapid Click Reaction

“…The strain‐promoted oxidation‐controlled cyclooctyne‐1,2‐quinone cycloaddition (SPOCQ) shown in Scheme , is another example of such a click strategy. The fast kinetics of this reaction in solution ( k 2 =496±70 m −1 s −1 ) makes it amenable for example, accelerated site‐specific protein conjugation, cell labelling and hydrogel preparation . An additional feature of this reaction is that quinone formation can be triggered by enzymatic or electrochemical oxidation, thus providing an inducible click handle.…”

Genetically Encoded Dihydroxyphenylalanine Coupled with Tyrosinase for Strain Promoted Labelling

“…Residue speci c incorporation of DOPA was carried out in the E. coli JW2581 containing pQE80L-anX and GFP vector as described by George et al [26]. The cells harboring pQE80L-anX and GFP plasmids for residue-speci c incorporation was grown in LB broth containing ampicillin and were then incubated overnight at 37 ºC.…”

“…Earlier FREMY'S SALT was tested for the direct conversion of tyrosine to Dopaquinone without the formation of any intermediate. The chemical oxidant-mediated conversion of tyrosine to dopaquinone requires a longer incubation time (20 h) and a high concentration of FREMY'S salt (10M excess) for polymerization, Intra cross-linking of proteins and peptides and conjugation of uorophore [26][27][28][29][30][31][32][33][34][35][36][37] . Secondly, FREMY'S SALT is a strong oxidant, hence it could polymerize, aggregate and precipitate the protein.…”

“…However, DOPA is also a suitable substrate similar to tyrosine for enzyme recognition. To evaluate the tyrosinase-mediated modi cation in DOPA incorporated proteins [Annexin A5 (anX) and green uorescent protein (GFP)], we selected highly reactive strained cyclic ring containing alkynes namely bicyclonon[6.1.0]-4-yne (BCN) and dibenzocyclooctyne-PEG-Fluor 545 (DBCO), which was already used in life and material science applications via Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) and Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition (SPOCQ) [24][25][26][27] . Initially, the reaction was optimized with BCN to understand the one-step SPOCQ.…”