Expedient Synthesis of Electronically Modified Luciferins for Bioluminescence Imaging

Abstract

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.

How Does the Local Electrostatic Field Influence Emitted Wavelengths and Bioluminescent Intensities of Modified Heteroaromatic Luciferins?

“…In order to check whether the above results are reliable, the emitted excited energies, wavelengths, and relative fluorescent intensities for the OxyLH 2 and its analogs under the electric fields induced by the PpyLuc and the mutant R218Q enzyme are evaluated and listed in Table 2. In the microenvironment of the PpyLuc enzyme, the first excited energy differences between the computational and experimental values are 0.05 eV (OxyLH 2 ), −0.02 eV (OxyBiLH 2 ), 0.01 eV (OxyBoLH 2 ), and 0.06 eV (OxyBtLH 2 ) respectively, 16,17 which indicates that computational and experimental results match well and the above LEF simulation is reliable. In the microenvironment of the mutant R218Q luciferase, the difference between the computational and experimental excitation energy for the OxyLH 2 is 0.04 eV.…”

“…The benzimidazole ring in the OxyBiLH 2 undergoes rapid N−H isomerization (between X 3 = NH, X 4 = N and X 3 = N, X 4 = NH), which results in broadened 1 H and 13 C NMR signals. 16,17 Our calculation shows that the emission spectrum of the isomer of the OxyBiLH 2 (X 3 = N, X 4 = NH) is out of the visible light range, so it is inactive in visible light production. The rapid tautomerization suppresses the fluorescent quantum yield of the OxyBiLH 2 .…”

“…The experimental data show that the fluorescent intensities for the OxyLH 2 , OxyBoLH 2 , and OxyBtLH 2 are at the same order but that of the OxyBiLH 2 is about 28-fold lower than that of the OxyLH 2 . 16,17 The big difference between the computational and experimental fluorescent intensity of the OxyBiLH 2 can be explained by the tautomerization. The rapid tautomerization suppresses the fluorescent quantum yield of the OxyBiLH 2 .…”

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

“…Furthermore, although we did not observe bioluminescence from the mammalian ACSLs in CHO cells, which have lower homology to firefly luciferase, mammalian ACS enzymes are known to adenylate xenobiotics such as ibuprofen (32,33). We therefore expect that probing the intersection between the luminogenic chemistry of small-molecule luciferin analogs (16,(34)(35)(36)(37)(38)(39)(40) and the activation chemistry of existing adenylating enzymes (33) will reveal that latent luciferase activity is more common than previously thought.…”

The Spectral–Structural Relationship of a Series of Oxyluciferin Derivatives

“…[22] To date, one of the most important applications of the multicolored bioluminescence of fireflies is bioluminescence imaging (BL), which has been a focus of research in cancer biology to effectively monitor gene expression, gene delivery, tumor growth, enzyme activity, responses to experimental drug therapies, and protein-protein interactions. [22][23][24][25] Interactions between luciferase and luciferin produces a multicolored emission, however due to the lack of a suitable luciferase, the wavelength of bioluminescence is usually lower than 600 nm and decreases with tissue depth, thus restricting the application of BL to small animals and superficial depths. Compared with mutating the luciferase, changing the structure of d-luciferin is a relatively easy way to modulate the emission wavelength.…”