title: Aminoluciferins as functional bioluminogenic substrates of firefly luciferase.
abstract: Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD = the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Takakura
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kojima
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
mesh_terms: D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry; D053148:Caspase 3 / Q000378:metabolism; D049454:Firefly Luciferin / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000378:metabolism; D019265:Spectroscopy, Near-Infrared; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D049454:Firefly Luciferin; D001710:Biotin; D049409:Luciferases, Firefly; D053148:Caspase 3