A Comparative Study of Bioorthogonal Reactions with Azides

Abstract

Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chemical reporter. Once introduced into target biomolecules, either metabolically or through chemical modification, the azide can be tagged with probes using one of three highly selective reactions: the Staudinger ligation, the Cu(I)-catalyzed azide-alkyne cycloaddition, or the strain-promoted [3 + 2] cycloaddition. Here, we compared these chemistries in the context of various biological applications, including labeling of biomolecules in complex lysates and on live cell surfaces. The Cu(I)-catalyzed reaction was found to be most efficient for detecting azides in protein samples but was not compatible with live cells due to the toxicity of the reagents. Both the Staudinger ligation and the strain-promoted [3 + 2] cycloaddition using optimized cyclooctynes were effective for tagging azides on live cells. The best reagent for this application was dependent upon the specific structure of the azide. These results provide a guide for biologists in choosing a suitable ligation chemistry.

N-Myristoyl transferase-mediated protein labelling in vivo

“…38, ‡ Interestingly, the efficiency of 30 capture by [3 + 2] cycloaddition (or 'click' chemistry) appears to be superior to that of capture with the Staudinger-Bertozzi reagent under the conditions used, an observation that corroborates a previous comparative study of bioorthogonal ligation approaches. 5 [3 + 2] capture of azide tagged protein 35 with 14 is also superior to that of alkyne tagged protein with 13 under identical conditions, an observation noted previously by Cravatt and co-workers. 39 This is likely due to the enhancement in rate in the presence of a large excess of activated alkyne in the case of capture using reagent 14; in the 40 case of capture with 13, the rate is presumably limited by the relatively lower concentration of activated alkynyl-protein.…”

Copper-free click chemistry for dynamic in vivo imaging

“…We previously explored the use of ring strain, demonstrating that cyclooctynes, the smallest of the stable cycloalkynes, can achieve bioorthogonal labeling of azides (14). Although the sensitivity of this strain-promoted cycloaddition was no better than that of the Staudinger ligation, the cyclooctyne probes demonstrated no cellular toxicity and offered a platform for further reaction development using the principles of physical organic chemistry (15).…”

Selective chemical protein modification

“…In its original format, the SPAAC reaction displayed similar, relatively slow, kinetics to the Staudinger ligation (Fig. 4a) 67 . To improve the rate, both difluorinated cyclooctynes 92 (DIFO) and dibenzocylooctynes 93 were independently reported allowing the visualisation of dynamic processes.…”

“…More recently, Serwa et al 66 reported a phosphite-Staudinger reaction with a stalled intermediate phosphoramidate. In addition to negating the problem of possible phosphine oxidation 67 , this 'traceless' reaction valuably allows installation of phosphate mimics 66 .…”

“…Despite its obvious strengths, the use of the Staudinger ligation has diminished recently due to slower associated kinetics, the retained triarylphosphine oxide appendage in 'non-traceless' variants and problems related to phosphine oxidation and possible side reactions of phosphines in 'traceless' variations 65,67 .…”