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

Published on 2018-05-16T13:58:27Z (GMT) by
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-N<sub>3</sub>-CTZ–ALuc26], [6-pi-OH-CTZ–RLuc8.6], and [6-N<sub>3</sub>-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.

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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 (2018): Azide- and Dye-Conjugated Coelenterazine Analogues

for a Multiplex Molecular Imaging Platform. ACS Publications. Collection.