| Part I BackgroundBioluminescence is a natural luminescence phenomenon that emits visible light produced by a chemical reaction between in vivo chemical substance and specific enzymes within a living organism. The conversion efficiency from chemical energy into light energy is very close to 100%. Bioluminescence can be discovered in various living organisms, including bacteria, insects, and marine living. Currently, firefly luciferase bioluminescence system is the most widely studied.Bio luminescent imaging (BLI) represents an emerging imaging technology that utilizes the invisible fluorescent produced by bioluminescence phenomenon to reveal biological processes. Bioluminescence imaging technology seems to have a lot of advantages, such as simple, intuitive, high specificity, high sensitivity, and less damage to the body. This technology is commonly used in a number of important subjects:medicine, molecular biology, biochemistry, and microbiology. Bio luminescent imaging plays a significant role in monitoring tumor growth and metastasis tracer, drug high-throughput screening, gene therapy, target gene expression detection, stem cell tracking and protein-protein interactions.Part II Design and synthesis of novel firefly luciferase substratesThe firefly luciferase substrates the most widely used are D-luciferin and aminoluciferin. The drawbacks of D-luciferin and aminoluciferin seriously hinder the development of bioluminescence imaging technology:narrow bio luminescent wavelength, short in vivo bioluminescence time and low blood-brain barrier permeability. To address these issues, there are two possible solutions:identifying mutations that redshift the emitted light to wavelengths, and synthesizing luciferin analogues. Over the past few years, a variety of studies focused on improving the sensitivity of in vivo BLI through engineering the genes of firefly luciferase and modulating the properties of luciferin that play important roles on its cellular permeability and pharmacokinetic properties in vivo. Therefore, we prepared the cyclic N-aminoluciferins with higher lipid solubility derived from aminoluciferin. Fluoronitrobenzene and various cyclic amines as a starting material,13 novel firefly luciferase substrates was developed after nucleophilic substitution reaction, DBU desulfurization reaction, cyclization reaction and a reduction reaction.Part III Evaluation of novel firefly luciferase substratesWe have completed bioluminescence emission spectra assay, in vitro bioluminescence assay, cell bioluminescence imaging, nude xenograft tumor model imaging, FVB-Tg mice imaging and brain bioluminescence imaging of nude mice for the novel firefly luciferase substrates.Bioluminescence emission spectra assay:we used fluorescence spectrophotometer to measure bioluminescence emission spectra. Compared to D-luciferin and aminoluciferin. there are significant red-shift for the bioluminescence emission maxima of the novel aminoluciferin analogues, conducive to the applications of in vivo imaging with firefly luciferase. In vitro bioluminescence assay:In vitro, there is the positive correlation relationship between the bioluminescence intensity and concentration for our aminoluciferin analogues. Like natural substrates D-luciferin and aminoluciferin, the bioluminescence intensity of aminoluciferin analogues have significant positive correlation relationship with the increase of ATP concentration, indicating that the novel analogues could be used to monitor the level of ATP in vivo. Cell bioluminescence imaging:we used a Xenogen IVIS Spectrum imaging system to measure dose-response analysis of novel substrates, D-luciferin and aminoluciferin in ES-2-Fluc,4T1-Fluc, A549-Fluc and U87-Fluc. The bioluminescence intensity enhanced with increasing concentrations of the analogues. These results suggest that the synthesized aminoluciferin analogues 1.1,1.2 and 1.3 have excellent cell permeability and biological activity compared to the original substrate and aminoluciferin. Nude xenograft tumor model imaging:we carried on further bioluminescence imaging of well-established mice xenograft tumor models. The nude mice with tumor xenografts were injected i.p. with 1.1,1.2,1.3 and D-luciferin and aminoluciferin. The aminoluciferin analogues all yielded a> 10-fold higher bio luminescent signal than D-luciferin at equivalent doses. FVB-Tg mice imaging:we chose 1.3 that performed more excellent in the nude mice with tumor xenografts assay for further bioluminescence imaging in transgenic mouse ubiquitously expressing firefly luciferase (FVB-Tg mice). As expected, when 100 μL 1.3 (1 mM) injected i.v to FVB-Tg mice, its bioluminescence intensity presented up to 10-fold higher than that of D-luciferin and aminoluciferin. Brain bioluminescence imaging of nude mice:To evaluate the ability of 1.3 to access the blood-brain barrier, we imaged nude mouse that had been inoculating with luciferase-expressing ES-2-Luc cells in brain hippocampus. Significantly,1.3 showed 30-fold higher bioluminescence intensity than D-luciferin.Part IV ConclusionBioluminescence imaging based on the firefly luciferin-luciferase system has been widely used in the biomedical fields owing to its noninvasiveness, high sensitivity which is noninvasive, high sensitivity, visualization and real-time in detection. Here we report the design and synthesis of a series of cyclic N-aminoluciferins (cyLs) that overcome major drawbacks of naturally-occurring firefly luciferase substrates, including weak red light emission, short in vivo bioluminescence time and low blood-brain barrier permeability. These aminoluciferin analogues 1.1,1.2 and 1.3 are competent substrates for luciferase that can yield a>10-fold higher bio luminescent signal than D-luciferin and have nearly 7 hours bioluminescence light emission time. Most notably, novel substrate 1.3 showed>30-fold higher bioluminescence intensity than D-luciferin in brain bioluminescence imaging. These cyclic N-aminoluciferins will expand the imaging toolkit and inspire new applications of bioluminescence technology. |
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