Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging

Abstract

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Expedient Synthesis and Characterization of π-Extended Luciferins

“…Notably, a similar pattern was observed in prior studies with the former, as well as with certain CouLuc-3/luciferase pairs. 18,20 We hypothesize that this emission pattern might be narrowed through further luciferase engineering. The CouLuc analogues displayed a significant blue shift in emission maximum compared to that of the NapLuc and (Tri)AkaLumine analogues.…”

“…19 The vinyl-extended variant, CouLuc-3-NMe 2 , and its optimized mutant luciferase (Pistachio) display near-infrared (NIR) emission maxima at 720 nm and photon output on par with that of the Fluc/D-luc pair (Figure 1B). 20 efforts provide a generalizable strategy for red-shifting emission maxima while simultaneously creating orthogonal pairs for multiplexed imaging. A critical component in the development of any BLI pair is a concise synthesis of the small molecule luciferin.…”

“…With a single methine linking coumarin and thiazoline ring systems, CouLuc-1-NMe 2 and its corresponding optimized luciferase, Pecan, emit at 620 nm . The vinyl-extended variant, CouLuc-3-NMe 2 , and its optimized mutant luciferase (Pistachio) display near-infrared (NIR) emission maxima at 720 nm and photon output on par with that of the Fluc/ d -luc pair (Figure B) . Overall, these efforts provide a generalizable strategy for red-shifting emission maxima while simultaneously creating orthogonal pairs for multiplexed imaging.…”

“…A critical component in the development of any BLI pair is a concise synthesis of the small molecule luciferin. In the prior work discussed above, the synthesis of the CouLuc-1’s and CouLuc-3’s was enabled by direct condensation of vinyl nitriles with cysteine (Figure C). , By contrast, π-extended luciferins have historically been assembled through a multistep heterocycle assembly strategy. Building on our CouLuc studies, we surmised that other π-extended analogues could easily be accessed through a similar strategy.…”

Firefly luciferin methyl ester illuminates the activity of multiple serine hydrolases

“…39 New tools to identify and validate the selectivity and specificity 40 of inhibitors in live cells are thus valuable. Given the everexpanding scope of luminogenic luciferin analogues, 8,[41][42][43][44] we anticipate that modified luciferin structures as well as the incorporation of different esters can be utilized in combination to further refine the specificity and expand the scope of activitybased bioluminescence probes for enzymes that process lipid substrates, potentially including ABHD6 and ABHD12, among others. Finally, our results indicate that the hydrolytic activity typically ascribed to ''esterases'' can also arise from several serine hydrolases that are involved in specialized aspects of lipid metabolism.…”