Probing the Mechanism of Photoaffinity Labeling by Dialkyldiazirines through Bioorthogonal Capture of Diazoalkanes.

Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for biological applications where instantaneous labeling is desirable, as carbene insertions display different chemoselectivity and are much faster than competing mechanisms involving diazo or ylide intermediates. Here, deuterium labeling and diazo compound trapping experiments are employed to demonstrate that both carbene and diazo mechanisms operate in the reactions of a dialkyldiazirine motif that is commonly utilized for biological applications. For the fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling on a model protein under aqueous conditions.

Stereoselective Synthesis of Bicyclo[6.1.0]nonene Precursors of the Bioorthogonal Reagents s-TCO and BCN.

The cyclooctyne BCN and the trans-cyclooctene s-TCO are widely used in bioorthogonal chemistry. A bottleneck for their synthesis had been a poorly selective cyclopropanation with ethyl diazoacetate. Here, we describe that low catalyst loadings (0.27 mol %) of Rh2( S-BHTL)4 provide the BCN precursor with 79:21 syn/ anti selectivity. The synthesis of the s-TCO precursor was best achieved through a sequence of Rh2(OAc)4 (0.33 mol %)-catalyzed cyclopropanation, followed by ester hydrolysis under epimerizing conditions. Both sequences could be carried out on multigram scale.