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Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase
Author(s) -
Takakura Hideo,
Kojima Ryosuke,
Urano Yasuteru,
Terai Takuya,
Hanaoka Kenjiro,
Nagano Tetsuo
Publication year - 2011
Publication title -
chemistry – an asian journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.18
H-Index - 106
eISSN - 1861-471X
pISSN - 1861-4728
DOI - 10.1002/asia.201000873
Subject(s) - luciferase , bioluminescence , luciferases , cyanine , substrate (aquarium) , chemistry , biophysics , transfection , fluorescence , luciferin , luminescent measurements , biochemistry , light emission , luminescence , biology , materials science , gene , optoelectronics , ecology , physics , quantum mechanics
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.