Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform

• Published in: Bioconjugate chemistry 2018-06, Vol.29 (6), p.1922-1931
• Main Authors: Nishihara, Ryo, Hoshino, Emi, Kakudate, Yoshiki, Kishigami, Satoshi, Iwasawa, Naoko, Sasaki, Shin-ichi, Nakajima, Takahiro, Sato, Moritoshi, Nishiyama, Shigeru, Citterio, Daniel, Suzuki, Koji, Kim, Sung Bae
• Format: Article
• Language: English
• Subjects: Animals; Azide; Azides - chemical synthesis; Azides - chemistry; Bioassays; Bioluminescence; Catalysis; Cells; Cercopithecus aethiops; Chemical synthesis; Chemiluminescence; COS Cells; Crosstalk; Dyes; Energy transfer; Fluorescence; Fluorescent dyes; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent indicators; Imidazoles - chemical synthesis; Imidazoles - chemistry; Kinetics; Luciferases - chemistry; Luminescent Agents - chemistry; Luminescent Measurements; Lysates; Molecular chains; Molecular Imaging; Molecular structure; Multiplexing; Optical properties; Organic chemistry; Photoproteins; Pyrazines - chemical synthesis; Pyrazines - chemistry; Spectra; Substrate specificity; Substrates
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.8b00188

Native coelenterazine (nCTZ) is a common substrate to most marine luciferases and photoproteins. In this study, nine novel dye- and azide-conjugated CTZ analogues were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of the nCTZ backbone to obtain bulkiness-driven substrate specificity and potential chemiluminescence/bioluminescence resonance energy transfer (C/BRET). The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly biased bioluminescence to ALucs and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living animal cells or lysates. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than nCTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). No or less spectral overlap occurs among [Furimazine–NanoLuc], [6-N3-CTZ–ALuc26], [6-pi-OH-CTZ–RLuc8.6], and [6-N3-CTZ–RLuc8.6] pairs, because of the substrate-driven luciferase specificity and color shifts, providing a crosstalk-free multiplex bioassay platform. The unique bioluminescence system appends a new toolbox to bioassays and multiplex molecular imaging platforms. This study is the first example that systematically synthesized fluorescent dye-conjugated CTZs and applied them for a bioluminescence assay system.