Final Report Summary - PERBIOIMAGE (Exploring pericyclic reactions for bioorthogonal imaging of biological processes with molecular precision)
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Sac-tagged proteins in the test tube, but the use of such approach in live cells failed because of selectivity issues (unpublished).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.