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

Ryo Nishihara, Emi Hoshino, Yoshiki Kakudate, Satoshi Kishigami, Naoko Iwasawa, Shin Ichi Sasaki, Takahiro Nakajima, Moritoshi Sato, Shigeru Nishiyama, Daniel Citterio, Koji Suzuki, Sung Bae Kim

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Abstract

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.

Original languageEnglish
Pages (from-to)1922-1931
Number of pages10
JournalBioconjugate Chemistry
Volume29
Issue number6
DOIs
Publication statusPublished - 2018 Jun 20

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ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Nishihara, R., Hoshino, E., Kakudate, Y., Kishigami, S., Iwasawa, N., Sasaki, S. I., Nakajima, T., Sato, M., Nishiyama, S., Citterio, D., Suzuki, K., & Kim, S. B. (2018). Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform. Bioconjugate Chemistry, 29(6), 1922-1931. https://doi.org/10.1021/acs.bioconjchem.8b00188