- Aminoluciferins as functional bioluminogenic substrates of firefly luciferase
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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.
- Takakura, Hideo,Kojima, Ryosuke,Urano, Yasuteru,Terai, Takuya,Hanaoka, Kenjiro,Nagano, Tetsuo
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p. 1800 - 1810
(2011/12/16)
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- Novel luciferin derivatives
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A compound represented by the following general formula (I) or a salt thereof: [wherein R1 and R2 represent hydrogen atom, a C1-6 alkyl group, or a group represented by the following formula (A): [wherein X1 and X2 represent hydrogen atom, or a group represented as —N(R3)(R4) (R3 and R4 represent hydrogen atom, a C1-6 alkyl group, a C1-6 alkylcarbonyl group, or a C1-6 alkyloxycarbonyl group); and n represents an integer of 1 to 6], provided that R1 and R2 do not simultaneously represent hydrogen atom), which is a novel luciferin derivative that serves as a luciferase substrate.
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Page/Page column 6
(2008/06/13)
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