Protein Modification by Strain-Promoted Alkyne–Nitrone Cycloaddition**

The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins. In this approach, an abiotic chemical functionality (reporter) is incorporated into a target biomolecule and can then react with a complementary bioorthogonal functional group linked to one of a diverse set of probes. The azide functional group, which is the most commonly employed reporter, can react in a Staudinger ligation with modified phosphines, in a copper(I)-catalyzed cycloaddition with terminal alkynes (CuAAC), or in a strain-promoted alkyne–azide cycloaddition (SPAAC). The last type of reaction is attractive because it does not require a cytotoxic metal catalyst and therefore provides unique opportunities for the labeling of cell-surface glycans and proteins of living cells, the decoration of polymeric nanostructures, the labeling of lipids, proteomics, and tissue reengineering. The first generation of cyclooctynes suffered from relatively slow reaction rates; however, it has been found that the rate of strain-promoted cycloaddition can be increased by appending electron-withdrawing groups adjacent to the triple bond. For example, reactions of difluorinated cyclooctynes, such as 1 (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives. We have found that derivatives of the 4-dibenzocyclooctynol 2 react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells. Attractive features of dibenzocyclooctynols include easy synthetic access, nontoxicity, and the straightforward attachment of a variety of probes. Recently, we introduced the more polar azacyclooctyne 3, which exhibits a higher rate of reaction. Despite these advances, there is an urgent need for new and faster bioorthogonal reactions for labeling at low concentration. We report herein a novel bioorthogonal reaction pair based on strain-promoted alkyne–nitrone cycloaddition (SPANC) to give N-alkylated isoxazolines with exceptionally fast reaction kinetics. The new methodology was used in a Figure 1. Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides.