Orthogonal Bioluminescent Probes from Disubstituted Luciferins

Abstract

Bioluminescence imaging with luciferase−luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme−substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase−luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging

“…S5 †). Similar trends have been observed with other sterically 26,57 and electronically modied luciferins 33,34 exhibiting comparable emission values (Fig. S6 †).…”

“…Another mutation common to multiple hits was F243M, a residue previously shown to aid in processing bulky luciferin analogs. 29,57 In vitro and in cellulo characterization of lead mutant From the screening hits, we selected the F243M/S347G mutant (dubbed Pecan) for additional characterization with CouLuc-1-NMe 2 . Pecan was particularly attractive for orthogonal probe development as it has been previously used in vivo.…”

Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging

“… 63 A triple-BLI system has also been demonstrated using FLuc/ d -luciferin, GLuc/CTZ, and the codon-optimized luciferase from Vargula hilgendorfii (Vluc) with its substrate vargulin. 64 Prescher’s group has expanded the toolbox of multicomponent BLI by synthesizing novel orthogonal luciferase–luciferin systems based on FLuc/ d -luciferin system, 65 , 66 including a class of π-extended luciferins enabling red-shifted emission. 67 The challenge with these systems, however, remains their low sensitivity in vivo , limiting their detection capability in deep tissues.…”

Synthesis of 2-Cyanobenzothiazoles via Pd-Catalyzed/Cu-Assisted C-H Functionalization/Intramolecular C-S Bond Formation from N-Arylcyanothioformamides

“…Nevertheless, this yield was still lower than the one previously obtained by our group (58%) when compound 2af was subjected to microwave-assisted thermolysis at 150 °C in N -methylpyrrolidinone (NMP) [ 51 ]. Notably, Prescher also observed the same drawback and finally heated 2,5-disubstituted cyanothioformamides at 170 °C in sulfolane to obtain the attempted 4-bromo-7-methyl-benzothiazole-2-carbonitrile derivatives, also in low yields (10–20%) [ 32 ].…”

“…In the last decade, 2-cyanobenzothiazoles substituted at the C6 position by a primary amine or hydroxyl group have become tools of choice in the development of click chemistry methods targeting cysteine residues under physiological and biocompatible conditions. This strategy was applied for the in situ assembly or self-assembly of biomolecules and nanostructures for various applications in drug targeting and delivery, specific labeling of peptides and biorthogonal ligation reactions [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ].…”