Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate, and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate “hits” were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multi-component imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

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 analogs. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogs 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.

Cell-cell interactions underlie fundamental biological processes but remain difficult to visualize over long times and large distances in tissues and live organisms. Bioluminescence imaging with luciferase-luciferin pairs is sufficiently sensitive to image cells in vivo but lacks the spatial resolution to identify cellular locations and interactions. To repurpose this technology for visualizing cellular networks, we developed a "caged" luciferin that produces light only when cells are in close contact. This molecule comprises a nitroaromatic core that can be selectively reduced ("uncaged") by one cell type, liberating a luciferin that can be selectively consumed by neighboring, luciferase-expressing cells. When the two cell types are in contact, robust light emission is observed. This imaging strategy will enable the noninvasive visualization of cell-cell interactions relevant to organismal biology.

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.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed using a common synthetic route. All analogs produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed via computation and photophysical measurements. The “brightest” brominated luciferin was further evaluated in cell and animal models. At low doses, the analog outperformed the native substrate in cells. The remaining luciferins, while weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

Supplemental Figure 1: Chemical structures of photoproximity probes used in this study. Supplemental Figure 3: Sub-cellular localization and photocleavage with the PhotoPPI system. A) Fluorescence microscopy images of HeLa cells transiently transfected with SNAP-FLAG-NLS, which exhibit strong nuclear localization of the SNAP protein. B) Fluorescence and brightfield microscopy images of HeLa cells transientlytransfected with SNAP-FLAG-NLS show nuclear-localization of FITC signal in cells treated with the non-cleavableFITC-BnG, or PF-BnG in the absence of UV irradiation (left three columns). Progressive exposure to UVirradiation at 365 nm results in complete loss of FITC signal in less than 10 min. Note that DAPI co-staining of the nucleus could not be performed due to overlap in the wavelength for DAPI excitation and nitroveratryl cleavage. C) Magnified view of cells in (B) above and reproduced from Fig. 1C here for comparison. Scale bars in A, B and C equal 8.3, 50 and 8.3 µm, respectively. Supplemental Figure 4: In vitro photoproximity labeling in the presence of whole cell proteome. Anti-biotin (streptavidin-800) and anti-mouse Western blot analyses of PP1 labeled SNAP-FLAG/ a-FLAG antibody complex with and without UV irradiation prior to analysis. Photolabeling was performed in the presence of whole cell lysate. Labels for individual proteins are included at appropriate molecular weights: LC, light chain; HC, heavy chain; "SNAP" label represents SNAP-Tag protein without the FLAG epitope. Supplemental Figure 5: Schematic depicting C-terminal (KEAP1-SNAP) and N-terminal (SNAP-KEAP1) genetic fusions used in photoproximity profiling of KEAP1 in cells. GxS represents a glycine-serine spacer, with X indicating the number of glycines. General synthetic methodsReagents purchased from commercial suppliers were analytical grade and used without further purification. All reactions were carried out in oven dried flasks using anhydrous solvents (Acros) unless otherwise specified. Reaction progress was monitored by thin-layer chromatography on Macherey-Nagel SIL G-25 UV254 TLC plates, visualized with UV light, ceric ammonium molybdate (CAM), p-anisidine, bromophenol blue, 2,4-dinitrophenyl hydrazine (DNP), or KMnO4 TLC stains. Nuclear magnetic resonance spectra were acquired using either a Bruker AVANCE II+ 500; 11.7 Tesla NMR or Bruker DRX 400; 9.3 Tesla NMR instrument. Accurate mass measurements were obtained using an Agilent 6224 Tof-MS instrument. When necessary, compounds were purified via flash column chromatography using Siliaflash F60 60 Å, 230-400 mesh silica gel (Silicycle) Chemical synthesis of photoproximity profiling (PhotoPPI)-probes 4-[[(2,2,2-trifluoroacetyl)amino]methyl]benzoic acid (2)Solid 4-(aminomethyl)benzoic acid 1 (15.1 g, 100 mmol) was dissolved in TFAA (42 mL) cooled to 0 °C. Once dissolved, the ice bath was removed and the reaction was allowed to stir at rt until starting material was consumed, ~2 hr. Upon completion, the reaction was quenched with H2O (100 mL) and precipitate collected via vacuum...

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

Herein, the synthesis and characterization of an alkyne‐modified luciferin is reported. This bioluminescent probe was accessed using C−H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d‐luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.