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

Abstract

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.

Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

“…In contrast to the Ar tag and in agreement with previous reports, we have found that all the alkyl diazirine tags tested in this study are prone to intramolecular rearrangements to form unreactive side-products. 24 In the case of the DF-photoaffinity probe the formation of undesirable side-products may occur to such a degree no products of intramolecular reactions are detected in our decomposition experiments (Fig. S11 † ).…”

“…S8–S12 † ). 24 LC-MS peaks corresponding to intermolecular reaction (capture of solvent) were observed for all probes with the exception of DF–F.…”

“…It has previously been reported that a drawback in the use of alkyl diazirines is rearrangement to form metastable diazo species that react with nucleophilic protein side chains rather than undergoing insertion reactions, increasing the radius of labeling. 24 The diazo side product resulting from irradiation of the aromatic diazirines, in contrast, has been considered to be virtually unreactive and is thus expected to produce reduced background labeling. 39 The high degree of background labeling that we have observed here, coupled with kinetics studies showing the clear albeit slow reaction of the diazo side-product resulting from irradiation of the Ar–F ( Fig.…”

“…23 More recent investigations have led to a better understanding of amino acid reactivity preferences, as well as mechanistic insight into diazirine photo-induced reactivity, which includes the formation of a reactive carbene, ylides, and electrophilic diazo intermediates. 24–27 However, the impact of FF tags on the proteome-wide profiles of molecular recognition groups has not been evaluated. Herein, we directly compare and characterize five diazirine-based FF tags containing dialkyl and aryl diazirines as well as a newly reported ‘terminal’ diazirine for their abilities to map small-molecule protein interactions in cells.…”

Structure, Bonding, and Photoaffinity Labeling Applications of Dialkyldiazirines

“…Unlike -aryl and -heteroatom-stabilized diazo compounds, dialkyldiazo compounds are prone to protonation at the diazo substituted carbon; displacement of N 2 by a nucleophile can result in 'pseudo-carbene' photolabeling. 49,52,53 Because the intermediate diazo compound has a lifetime of milliseconds, 50 as compared to the picoseconds lifetime of the excited state carbene, this results in a broader labeling radius that is not diffusion limited.…”