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Insight & Intelligence™ : Jul 30, 2012

New Luciferins

Researchers describe a rapid method to synthesize D-luciferin along with a novel set of electronically modified analogues.
  • Doug Auld, Ph.D.

ASSAY & Drug Development Technologies offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents one article that was analyzed in the "Literature Search and Review" column, a paper published in Journal of the American Chemical Society titled "Expedient synthesis of electronically modified luciferins for bioluminescence imaging." Authors of the paper are McCutcheon DC, Paley MA, Steinhardt RC, and Prescher JA.

Abstract from Journal of the American Chemical Society

Bioluminescence imaging with luciferase enzymes requires access to light-emitting, small-molecule luciferins. Here, we describe a rapid method to synthesize D-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogues. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogues produced with this approach emit light with Fluc in vitro and in live cells. Collectively, our work increases the number of substrates that can be used for bioluminescence imaging and provides a general strategy for synthesizing new collections of luciferins.

Commentary

Luciferase assays are widely used in biochemical and cell-based assays. New luciferases isolated from various bioluminescent species continue to be developed to provide high-sensitivity bioluminescence assays. Firefly luciferase (Fluc) is the most commonly used reporter in cell-based reporter-gene assays (RGAs).

The bioluminescence of Fluc ranges from 500 to 700 nm with a broad emission peak at 550–570 nm. Although red-shifted variants of Fluc have been produced by mutating the D-luciferin binding site, these variant enzymes may show dimmer bioluminescence and many are not widely available. Another approach to developing luciferase assays with different emission maximums involves synthesizing new luciferins. This article describes a facile method to construct new luciferins using Appel's salt (4,5-dichloro-1,2,3-dithiazolium chloride).

The authors were able to synthesize luciferins with benzimidazole and imidazoline rings. Benzimidazoles are known competitive inhibitors of Fluc, and previous work has shown that such heterocycles can be excited to emit light. After obtaining these nitrogenous luciferins, these compounds were used as substrates for Fluc in the presence of ATP and CoASH. No bioluminescence could be detected for analog 3 (see Figure), and only a very weak signal was obtained for analog 4. However, analogs 2 and 15 showed detectable signals, although they were approximately 100-fold weaker than the one obtained with D-luciferin. The benzimidazole analog 2 showed a red-shifted emission maximum of 578 nm.

Interestingly, analog 15 showed a greatly blue-shifted emission peak compared to other luciferins with a λmax = 460 nm. This emission is similar to what is found with coelenterazine if used as a substrate for the luciferase isolated from Renilla reniformis. These analogs were also shown to function in a cell-based Fluc assay using HEK293 cells.

Studies such as these should extend the range of available substrates for Fluc providing researchers new formulations for use in both in vitro and in vivo studies.