Bioorthogonal Reaction of β-Chloroacroleins with meta-Aminothiophenol to Develop Near-Infrared Fluorogenic Probes for Simultaneous Two-color Imaging

Abstract

Highly fluorogenic probe based bioorthogonal chemistry has become a promising tool in biomedical applications. However, the majority of fluorogenic probes are designed by introducing a bioorthogonal partner as a fluorescence quencher into classical fluorophores, and these probes exhibit a deteriorating fluorogenicity as the emission wavelength shifts toward the near-infrared (NIR) region, greatly limiting their applications in vivo. Herein, we report a novel fluorogenic bioorthogonal reaction involving β-chloroacroleins (β-CAs) and meta-aminothiophenol (m-AT₁), whose fluorescence increases more than 500-fold upon in situ generating fluorophores. β-CAs are stable under physiological conditions and react rapidly (β-CA₉, k₂ = 2.2 × 10² M^–1 s^–1, in H₂O) and chemoselectively with m-AT₁ in the presence of biological nucleophiles, and delightfully, the reaction proceeds swiftly even under solvent-free conditions. Furthermore, manipulating the conjugate length of β-CAs enables the emission wavelength of the probes to be fine-tuned from 627 to 778 nm. These probes allow the simultaneous labeling of multiple cellular organelles without washing steps, and two-color tumor visualization is achieved in living mice. We believe this study not only provides new insights for the development of NIR fluorogenic probes with superior turn-on behaviors but also presents a promising fluorogenic bioorthogonal reaction CA-AT with widespread potential applications in biomedical research.