A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT).

Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.

General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.

Design and Synthesis of an Activatable Photoacoustic Probe for Hypochlorous Acid.

Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.

Development of a series of near-infrared dark quenchers based on Si-rhodamines and their application to fluorescent probes.

Near-infrared (NIR) fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers, which are nonfluorescent dyes, as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). Here, we describe Si-rhodamine-based NIR dark quenchers (SiNQs), which show broad absorption covering this region. They are nonfluorescent independently of solvent polarity and pH, probably due to free rotation of the bond between the N atom and the xanthene moiety. SiNQs can easily be structurally modified to tune their water-solubility and absorption spectra, enabling flexible design of appropriate FRET pair for various NIR fluorescent dyes. To demonstrate the usefulness of SiNQs, we designed and synthesized a NIR fluorescent probe for matrix metalloproteinase (MMP) activity using SiNQ780. This probe 1 could detect MMP activity in vitro, in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized, by NIR fluorescence. We believe SiNQs will be useful for the development of a wide range of practical NIR fluorescent probes.

A design strategy for small molecule-based targeted MRI contrast agents: their application for detection of atherosclerotic plaques.

Gadolinium(III) ion (Gd(3+)) complexes are widely used as contrast agents in magnetic resonance imaging (MRI), and many attempts have been made to couple them to sensor moieties in order to visualize biological phenomena of interest inside the body. However, the low sensitivity of MRI has made it difficult to develop practical MRI contrast agents for in vivo imaging. We hypothesized that practical MRI contrast agents could be designed by targeting a specific biological environment, rather than a specific protein such as a receptor. To test this idea, we designed and synthesized a Gd(3+)-based MRI contrast agent, 2BDP3Gd, for visualizing atherosclerotic plaques by linking the Gd(3+)-complex to the lipophilic fluorophore BODIPY to stain lipid-rich environments. We found that 2BDP3Gd was selectively accumulated into lipid droplets of adipocytes at the cellular level. Atherosclerotic plaques in the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits were clearly visualized in T1-weighted MR images after intravenous injection of 2BDP3Gd in vivo.

Development of hypoxia-sensitive Gd3+-based MRI contrast agents.

Hypoxia occurs in various diseases, including cancer, ischemia, and acute and chronic vascular diseases. Here we describe the design and synthesis of the first hypoxia-sensitive MRI contrast agents, SAGds. SAGds showed a pH-dependent r(1) relaxivity change associated with intramolecular chelation of the nitrogen atom of the sulfonamide moiety to the Gd(3+) center. There was a correlation between the pK(a) of the r(1) relaxivity change and the sum of the Hammett σ constants of substituents on the aromatic ring. Among the synthesized compounds, 4NO(2)2MeOSAGd was selectively reduced to the amine by rat liver microsomes under hypoxic conditions, resulting in a 1.8-fold increment of the r(1) relaxivity owing to the change in pK(a) of the arylsulfonamide moiety. This enhancement of the r(1) relaxivity could be clearly detected in T(1)-weighted MR images. Thus, 4NO(2)2MeOSAGd is a 'smart' MRI contrast agent for the detection of hypoxia under physiological conditions.