A bioluminogenic substrate for in vivo imaging of beta-lactamase activity.

b-Lactamases are an important family of bacterial enzymes for research in bacterial infection and biotechnology. Bacterial resistance to antibiotics is becoming a growing global health problem, and bacteria expressing b-lactamases are resistant to b-lactam antibiotics because b-lactamases efficiently hydrolyze b-lactam antibiotics such as penicillins and cephalosporins. Rapid and sensitive detection of b-lactamase activity in vitro and in vivo is thus clinically important for the identification of bacterial pathogens that are resistant to b-lactam antibiotics. A number of colorometric and fluorometric substrates have been developed for the detection of b-lactamase in vitro and in living cells, but a substrate that can detect b-lactamase in living animals has not yet been reported. Highly sensitive imaging of bacteria will find many health and environmental applications. In contrast, b-lactamases have been developed as a powerful tool with a number of biotechnology applications. For example, TEM-1 b-lactamase (Bla), a small (29 kDa) and monomeric enzyme, is a sensitive reporter system for detecting and imaging biological processes and interactions in single living mammalian tissue culture cells. It has been applied for examining promoter/regulatory elements activity, imaging RNA splicing, monitoring viral infection, and detecting protein–protein interactions. The b-lactamase enzymatic activity has recently also been applied to control formation of supramolecular hydrogels. To extend the reporter utility of Bla to whole living animals is highly desirable but has been impeded by lack of a suitable substrate that can image the Bla activity in vivo. Herein, we report the first bioluminogenic substrate for Bla and demonstrate its application for in vivo imaging of the Bla activity. Our detection strategy utilizes the bioluminescent enzyme firefly luciferase (fLuc) to image the Bla activity in vivo, because luciferase-based bioluminescence imaging offers great sensitivity for probing biology in small living animals (Scheme 1). Firefly luciferase catalyzes the oxidation of its substrate d-luciferin in the presence of O2, ATP, andMg 2+ and