Hoechst modification by strain-promoted azide-alkyne cycloaddition for transport of functional molecules into the cell nucleus.

ABSTRACT

The delivery of functional molecules to the cell nucleus enables the visualization of nuclear function and the development of effective medical treatments. In this study, we successfully modified the Hoechst molecule, which is a well-documented nuclear-staining agent, using the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We prepared Hoechst derivatives bearing an azide group (Hoe-N3) and characterized their SPAAC reactions in the presence of corresponding molecules with a dibenzylcyclooctyne unit (DBCO). The SPAAC reaction of Hoe-N3 with alkylamine bearing DBCO, fluorescent TAMRA, or Cy5 molecules bearing DBCO led to the formation of the coupling products Hoe-Amine, Hoe-TAMRA, and Hoe-Cy5, respectively. These Hoechst derivatives retained their DNA-binding properties. In addition, Hoe-TAMRA and Hoe-Cy5 exhibited properties of dual accumulation in the cell nucleus and mitochondria. Initial incubation of these molecules in living cells resulted in its accumulation in mitochondria, while after mitochondrial depolarization, it was smoothly released from mitochondria and translocated into the cell nucleus. Thus, mitochondrial depolarization could be monitored by measuring the emission of Hoe-TAMRA and Hoe-Cy5 at the cell nucleus.