Design

Upon examining the crystal structure of firefly luciferase and luciferin, we identified a large binding pocket surrounding the hydroxyl group of luciferin, thereby allowing for the introduction of a cyclic amino group in that region to augment the affinity with luciferase.

Simultaneously, the incorporation of this group may enhance the lipid-water partition coefficient and blood-brain barrier permeability, as well as prolong the bioluminescence wavelength and duration in vivo.

Considering the above information, we designed cyclic N-aminoluciferins (cyL) as firefly luciferase substrates in bioluminescence imaging.

Built

Following rational design, we undertook the organic synthesis of cyL molecules. We adopted cycloaminobenzothiazole-2-nitriles (the courtesy gift from Nobel Drugs) as the starting material and dissolved it in a mixture of methanol, dichloromethane, and water. Subsequently, we gradually introduced D-cysteine, anhydrous potassium carbonate, and hydrochloric acid to facilitate the cyclization reaction. Upon completion of the reaction, which occurred 20 minutes later under nitrogen gas protection, we obtained cyL compounds including 5-cyL, 6-cyL, 7-cyL, and 8-cyL.

Test

Subsequent to the synthesis of compounds, we conducted a variety of biological evaluations. We assessed their bioluminescent characteristics, enzymatic imaging, cellular imaging, imaging of the xenograft tumor model in nude mice, imaging of transgenic mice, and imaging of the brain tumor model in nude mice.

We employed a spectrophotometer to evaluate the bioluminescent wavelengths of cyL compounds. In comparison to the substrates D-luciferin and N-aminoluciferin, the bioluminescent wavelengths of all cyL compounds exhibited red-shifted enhancement, hence facilitating imaging application in deep tissues. Meanwhile, the lower kinetic constants Km and V_max of 5-cyL, 6-cyL, 7-cyL and 8-cyL suggest that the reaction rate and affinity have obviously potential, compared to D-luciferin and aminoluciferin. These results suggest that these compounds may possess high sensitivity as novel substrates for firefly luciferase.

Learn

In the evaluation of the dependence of enzymatic bioluminescence intensity on ATP and substrate concentrations, we found that while compounds 5-cyL, 6-cyL, and 7-cyL could not surpass the substrates aminoluciferin and D-luciferin, an increase in ATP concentration resulted in a corresponding enhancement of bioluminescent intensity for all substrates, demonstrating a linear correlation. Nonetheless, compound 8-cyL exhibited unreasonable performance; hence, we exclusively implemented compounds 5-cyL, 6-cyL, and 7-cyL in the following experiments.

To evaluate cell-based activity, we initially developed a firefly luciferase-labeled human glioblastoma cell line (U87-Luc) and conducted experiments to determine the relationship between cellular concentrations and the bioluminescent intensity of substrates. Under equivalent substrate concentration conditions, the bioluminescent intensity was directly correlated with the cellular concentrations of U87-Luc brain glioma cells. In comparison to the substrates aminoluciferin and D-luciferin, the compounds 5-cyL, 6-cyL, and 7-cyL exhibited much higher sensitivity, advantageous cellular permeability, and bioluminescent feasibility.

We administered identical doses of the three compounds, D-luciferin and N-aminoluciferin, at varying concentrations to nude mice bearing the U87-Luc subcutaneous xenograft tumor. Following bioluminescent imaging evaluations via the real-time imaging system in animals, we observed that the bioluminescent intensities of 5-cyL, 6-cyL, and 7-cyL at identical concentration levels were much greater than those of the substrates D-luciferin and aminoluciferin. The highest bioluminescence intensity and longest duration was 7-cyL, exhibiting a bioluminescent intensity 25 times greater than that of D-luciferin, with an imaging duration of 7 hours.

Built

Following the imaging results from the nude mice xenograft tumor model, we selected 7-cyL to do the imaging experiment on the FVB-Tg transgenic mice model. Post intravenous administration, we observed that the bioluminescent intensity of 7-cyL exceeded that of the substrates D-luciferin and N-aminoluciferin by more than tenfold, exhibiting a comparatively prolonged in vivo luminescence lifetime of three hours. Upon calculating the ratio of cerebral bioluminescent intensity in FVB-Tg mice to their own bioluminescent intensity, in comparison to D-luciferin and N-aminoluciferin, compound 7-cyL exhibited a comparatively high cerebral imaging ratio and superior blood-brain barrier permeability, indicating its potential significant application value in brain tumor imaging.

Test

Given the elevated cerebral bioluminescent ratio of compound 7-cyL in FVB-Tg mice bioluminescent imaging, we developed a brain tumor model to examine the in vivo imaging of 7-cyL. Compound 7-cyL demonstrated a bioluminescence intensity over 30 times that of D-luciferin, with a steady bioluminescent duration of up to 4 hours, indicating that compound 7-cyL exhibited good permeability across the blood-brain barrier. Consequently, this advantage would enhance bioluminescent brain tumor imaging capabilities.

Learn

In this effort, we developed four cyclic N-aminoluciferins (cyL), with compound 7-cyL exhibiting a bioluminescence intensity exceeding tenfold that of an equivalent dose of D-luciferin, and a bioluminescent duration of seven hours. The brain tumor imaging of 7-cyL demonstrated a bioluminescent intensity exceeding that of the natural substrate D-luciferin by over 30 times. We believe that such cyL substrates would expand the capabilities of existing bioluminescence technologies and stimulate new uses of bioluminescence imaging in brain tumor research.