Copper-free click chemistry for dynamic in vivo imaging

Abstract

Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 ؉ 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.azide ͉ bioorthogonal reaction ͉ cyclooctyne glycan trafficking ͉ molecular imaging

A Click Chemistry‐Based Proteomic Approach Reveals that 1,2,4‐Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

“…Replacement of an alkyne with an azide group in P2 ( 7 a ) had no detrimental effect on the antimalarial potency of the trioxolane azide probe as indicated in Figure S2, Figure 3 and Table S1. However, the labeling profile intensity with P2 ( 7 a ) was much higher compared to P1 ( 6 a ) (Figure 3 c,d) suggesting greater efficiency of the copper‐free click reaction 8b. Reduction of P2 ( 7 a ) concentrations to 100 n m (LC90) had no impact on the labeling pattern observed (Figure 3 f).…”

Live‐Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions

“…[17] By monitoring the disappearance of the characteristic tetrazine absorption band at 520 nm, we measured a second-order rate constant of 0.137 ±0.004M −1 s −1 at 37°C in a solution of water/ DMSO (12% DMSO by volume; Figure 2c). Although this rate constant is comparable to previous bioorthogonal labeling strategies, [8] it is much slower than the reaction of other strained alkenes with tetrazine, such as trans-cyclooctene and even norbornene. [1] Faster kinetics would improve coupling yields, particularly for applications where it is not possible to flood the target with a large excess of reactant, for example in live-cell intracellular labeling or in vivo.…”

“…[1,6] This situation is in contrast to Staudinger ligations or strain-promoted azide-cycloalkyne cycloadditions that utilize a small azide functional group. [7,8] This requirement has limited the use of tetrazine reactions in methods that require tags with minimal steric impact or nominal effect on the partition ratio. [2] The development of smaller dienophile partners capable of reacting rapidly with tetrazines would therefore represent a major advance.…”

Fast, copper-free click chemistry: a convenient solid-phase approach to oligonucleotide conjugation

“…And third, the required strained cyclic alkynes are available only following long synthetic sequences. 8 Thus, a void exists in the field of chemical biology for a fast, reliable, copper-free, solid-phase oligonucleotide conjugation strategy which will proceed under physiological conditions.…”