A Biocompatible <i>in Vivo</i> Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that d-cysteine and 2-cyanobenzothiazoles can selectively react with each other <i>in vivo</i> to generate a luciferin substrate for firefly luciferase. The success of this ″split luciferin″ ligation reaction has important implications for both <i>in vivo</i> imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a ″caged″ luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free d-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-d-Cys (DEVD-(d-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH₂-CBT) <i>in vivo</i> to form 6-amino-d-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting <i>in vivo</i>

A Biocompatible <i>in Vivo</i> Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that d-cysteine and 2-cyanobenzothiazoles can selectively react with each other <i>in vivo</i> to generate a luciferin substrate for firefly luciferase. The success of this ″split luciferin″ ligation reaction has important implications for both <i>in vivo</i> imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a ″caged″ luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free d-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-d-Cys (DEVD-(d-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH₂-CBT) <i>in vivo</i> to form 6-amino-d-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting <i>in vivo</i>

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival