Rapid and scalable assembly of firefly luciferase substrates

Abstract

Bioluminescence imaging with luciferase-luciferin pairs is a popular method for visualizing biological processes in vivo. Unfortunately, most luciferins are difficult to access and remain prohibitively expensive for some imaging applications. Here we report cost-effective and efficient syntheses of D-luciferin and 6′-aminoluciferin, two widely used bioluminescent substrates. Our approach employs inexpensive anilines and Appel's salt to generate the luciferin cores in a single pot. Additionally, the syntheses are scalable and can provide multi-gram quantities of both substrates. The streamlined production and improved accessibility of luciferin reagents will bolster in vivo imaging efforts.

Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole

“…The synthesis of ACBT 8 was completed by reduction of the nitro group in 13 (Scheme 3). In our hands, the use of iron powder in acetic acid proved more practical than the previously reported procedures with zinc powder and ammonium chloride [7]. It resulted in more consistent yields, an easier work-up, and no persistent contamination of 8 with nitroso intermediates [6].…”

“…Indeed, the treatment of 2-chloro-6-nitrobenzothiazole ( 6 ) with DABCO (15 mol %) and sodium cyanide (NaCN, 1.05 equiv) in DMSO/water 1:1, at room temperature, resulted in full conversion to 2-cyano-6-nitrobenzothiazole ( 13 , Table 1), a known precursor of ACBT 8 [7]. Any unreacted cyanide in the reaction mixture was safely quenched by the addition of an iron(III) chloride solution, and pure ACBT 8 was isolated in 90% yield after (extensive) extractive work-up and flash chromatography.…”

“…The typical preparation of luciferins 3 involves straightforward condensation of CBTs 1 with D-cysteine ( 2 ) (Scheme 1). For BLI applications, luciferins are often generated in vivo from CBTs [4–7], which are easier to modify and handle due to their higher stability, cell permeability, and reactivity than the full luciferin scaffolds [2,5,8]. …”

“…Therefore, this route does not allow straightforward scale-up under laboratory conditions. Prescher and co-workers reported an alternative synthesis of ACBT that avoids the use of cyanide (Scheme 2) [7]. This route makes use of 4,5-dichloro-1,2,3-dithiazolium chloride (Appel’s salt, 12 ), and variations of the procedure have enabled the synthesis of different CBT derivatives, some of which on multi-gram scale [6–7 16–17].…”

“…Prescher and co-workers reported an alternative synthesis of ACBT that avoids the use of cyanide (Scheme 2) [7]. This route makes use of 4,5-dichloro-1,2,3-dithiazolium chloride (Appel’s salt, 12 ), and variations of the procedure have enabled the synthesis of different CBT derivatives, some of which on multi-gram scale [6–7 16–17]. However, Appel’s salt is relatively expensive and available from a limited number of suppliers, and its synthesis requires the use of the highly toxic reagent sulfur monochloride [18].…”