Bioorthogonal chemistry: recent progress and future directions

Abstract

The ability to use covalent chemistry to label biomolecules selectively in their native habitats has greatly enhanced our understanding of biomolecular dynamics and function beyond what is possible with genetic tools alone. To attain the exquisite selectivity that is essential in this covalent approach a "bottom-up" two-step strategy has achieved many successes recently. In this approach, a bioorthogonal chemical functionality is built into life's basic building blocks-amino acids, nucleosides, lipids, and sugars-as well as coenzymes; after the incorporation, an array of biophysical probes are selectively appended to the tagged biomolecules via a suitable bioorthogonal reaction. While much has been accomplished in the expansion of non-natural building blocks carrying unique chemical moieties, the dearth of robust bioorthogonal reactions has limited both the scope and utility of this promising approach. Here, we summarize the recent progress in the development of bioorthogonal reactions and their applications in various biological systems. A major emphasis has been placed on the mechanistic and kinetic studies of these reactions with the hope that continuous improvements can be made with each reaction in the future. In view of the gap between the capabilities of the current repertoire of bioorthogonal reactions and the unmet needs of outstanding biological problems, we also strive to project the future directions of this rapidly developing field.

Figure 1

Schematic representation of bioorthogonal chemistry approach for labeling of a targeted biomolecule with a small-molecule probe: Blue rectangle: biomolecular target; yellow circle, bioorthogonal chemical reporter; red rectangle, cognate reaction partner of the reporter; green star, small-molecule probe.

Figure 2

(a) Bioorthogonal chemistry and (b) antibody-based approaches toward biomolecular labeling with a small-molecule probe.

Figure 3

Photoinduced lipidation of EGFP carrying a tetrazole motif at its C-terminus: (a) Scheme for a photoinduced lipidation by a lipid dipolarophile. (b) Fluorescence imaging (top panel, λ_ex = 365 nm) and Coomassie Blue staining (bottom panel) of EGFP-Tet and EGFP upon photoirradiation in the presence or absence of the lipid dipolarophile. Duration of 1-min 302-nm UV irradiation was applied to the samples.

Figure 4

Selective labeling of alkene-Z by tetrazole in E. coli cells: (1) reaction scheme; (2) CFP channel (top row) and DIC (Differential Interference Contrast) channel (bottom row) images of bacteria expressing either alkene-Z (left) or wt-Z (right) proteins after treatment of 100 µM tetrazole 1.

Figure 5

Observing a bent nitrile imine structure in the solid state: (a) scheme for the photocrystallography; (b) photodifference map based on the Fo,(after)-Fo (before). Blue, 2.0; light blue, 1.0; orange, −1.0; red, −2.0 e/A³. Only one half of the map is shown because of the 2-fold symmetry; (c) ORTEP representation of the geometry-refined nitrile imine structure.

Scheme 1

Acid-catalyzed (a) and aniline-catalyzed (b) condensations of aldehyde/ketone with reactive amine nucleophiles.

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8