Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression |
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| Authors: | Eric Lindberg Dr Shin Mizukami Dr Keiji Ibata Dr Atsushi Miyawaki Prof Kazuya Kikuchi |
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| Institution: | 1. Graduate School of Engineering, Osaka University, Osaka, 565‐0871 (Japan), Fax: (+81)?6‐6879‐7925;2. Immunology Frontier Research Center, Osaka University, Osaka, 565‐0871 (Japan);3. Department of Physiology, School of Medicine, Keio University, Tokyo 160‐8582 (Japan);4. RIKEN Brain Science Institute, Saitama, 351‐0198 (Japan);5. Life, Function and Dynamics, Exploratory Research for Advanced Technology (Japan), Science and Technology Agency, Saitama, 351‐0198 (Japan) |
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| Abstract: | Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations. |
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| Keywords: | biosensors cleavage reactions enzymes galactosidase luminescence |
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