Regioselectivity of the 1,3-Dipolar Cycloaddition of Organic Azides to 7-Heteronorbornadienes. Synthesis of ß-Substituted Furans/Pyrroles.

An efficient procedure for the preparation of ß-substituted furans/pyrroles is presented. The methodology is based on the use of 7-oxa/azanorbornadienes as dipolarophiles in 1,3-dipolar cycloaddition with benzyl azide. The triazoline cycloadduct thus formed spontaneously decomposes via a retro-Diels-Alder (rDA) reaction to afford a ß-substituted furan/pyrrole derivative and a stable triazole. The scope of this tandem 1,3-dipolar cycloaddition/rDA reaction was studied with thirteen 7-heteronorbornadienes. This study allowed a deep knowledge of the regioselectivity of the reaction, which can be tuned through the substituents of the heteronorbornadienic systems.

Stable Pyrrole-Linked Bioconjugates through Tetrazine-Triggered Azanorbornadiene Fragmentation.

An azanorbornadiene bromovinyl sulfone reagent for cysteine-selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole-linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene-tagged protein through inverse electron demand Diels-Alder cycloaddition with subsequent double retro-Diels-Alder reactions to form a stable pyrrole linkage. The sequence of site-selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.

Azabicyclic vinyl sulfones for residue-specific dual protein labelling.

We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels-Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1-5 equiv. of reagent in NaPi pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels-Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.