PROJECT SUMMARY/ABSTRACT Bioorthogonal reactions are powerful tools capable of altering and visualizing biological processes. Accordingly, the ability to develop organic reactions into bioorthogonal reactions has the potential to advance biomedicine. Although a number of organic reactions have been applied in a bioorthogonal manner, most are limited by the poor synthetic accessibility, aqueous solubility, or chemical stability of reagents, or by slow reaction rates. A computationally guided rational design approach will be used to improve the utility and accessibility of cyclopentadiene-based bioorthogonal reactions, ultimately making them a highly efficient tool for applications in biological systems. The goal of the proposed research is to develop cyclopentadiene-based bioorthogonal reactions based upon an in silico screening procedure that evaluates reactivity and stability. First, state-of-the-art computational methods that provide accurate activation energies will be used to evaluate the reactivity of cyclopentadienes with known bioorthogonal 2π cycloaddends. Second, the cyclopentadienes calculated to have suitable reaction kinetics (k > 1 M–1s–1 at room temperature in water) by computational screening will be synthesized and evaluated experimentally as reactants. Third, the stability of these cyclopentadienes will be assessed under physiological conditions. Finally, the cyclopentadienes that emerge from the first three aims will be used in an in cellulo experiment to detect the nascent biosynthesis of DNA within living human cells. This work will be carried out in the Raines group at MIT. The Raines group has solved problems in chemistry and biology for three decades and has all of the facilities necessary to carry out the computational, chemical, and biological aspects of the proposed research.