PERBIOIMAGE · Exploring pericyclic reactions for bioorthogonal imaging of biological processes with molecular precision

Exploring pericyclic reactions for bioorthogonal imaging of biological processes with molecular precision

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at predetermined sites. These reactions are now used to selectively install particular modifications on recombinant proteins for many biological and therapeutic applications (Figure 1, top). Bioorthogonal site-selective chemistry may also be used to label specific proteins in living cells which allows uptake and intracellular trafficking to be tracked as well as for physiological parameters to be measured optically. Bioorthogonal chemistry is now permitting to site-selectively install modifications on individual proteins in complex biological mixtures, including in living organisms (Nature Chem 2016, 103). In this project, I have explored a conceptually novel bioorthogonal approach that combines the use of small, stable handles that may be labelled upon chemical activation in a temporal controlled manner. The initial approach was focused on a new photo triggered [2+2] cycloaddition bioorthogonal ligation between two alkene containing partners as well as the known inverse electron-demand Diels-Alder cycloaddition (IEDDA) reaction between the minimal alkene-tag and a tetrazine. First, we developed a method for the site-specifically installation of the unstrained S-allyl cysteine (Sac) amino acid into proteins (Angew Chem Int Ed 2016,14683). We found that the photo [2+2] cycloaddition reaction between Sac and an alkene moiety (maleimide) allows controlled labelling of Sac-tagged proteins in the test tube, but the use of such approach in live cells failed because of selectivity issues (unpublished). On the contrary, we have demonstrated that unstrained S-allyl handles precisely installed at predefined cysteine residues within the sequence of a protein are suitable chemical handles for IEDDA reactions with tetrazine dyes. This strategy allowed for selective labelling of proteins in live cells using a pretargeting approach (Angew Chem Int Ed 2016,14683). The easy site-specific installation and the small size of the allyl handle, which is potentially less disruptive compared to non-canonical amino acids bearing bulky strained alkenes, is likely to be of a general benefit for other sensitive protein systems including antibodies used in pre-targeting approaches. As such, we believe that the simple site-specific labelling strategy disclosed here, which enables bioorthogonal live-cell imaging, will find significant use in the biological community, allowing imaging of specific targets with minimal effects on their intrinsic properties.

Data: CORDIS, © European Union