Development of Molecular Imaging Tools to Investigate Protein Functions by Chemical Probe Design

Abstract

Molecular imaging technologies, which enable the visualization of the behaviors or functions of biomolecules in living systems, have received considerable attention from life scientists. Novel imaging technologies that overcome the limitations of current imaging techniques are desired. In this review, two independent technologies that were recently developed by the authors are described. The first technology is for smart 19 F magnetic resonance imaging (MRI) probes that were developed for in vivo applications. These probes were developed by exploiting paramagnetic relaxation enhancement in order to detect hydrolase activity. With respect to cellular applications, gene expression in cells was visualized using one of the 19 F MRI probes. It was confirmed that this probe design principle is effective for various hydrolases, and broad applications are expected. The second technology is for practical protein labeling. This labeling method is based on a mutant b b -lactamase and its specific labeling probes. Since the probe is fluorescence resonance energy transfer (FRET)-based, this labeling method achieves both specific and fluorogenic labeling of target proteins. In addition, derivatization of the probe enabled the labeling of intracellular proteins and the modification of various functional molecules.

Small Molecule Active Site Directed Tools for Studying Human Caspases

“…408 19 F MRI probe 506 consists of three main parts: (1) caspase-3 cleavable linker sequence DEVD, (2) Gd 3+ complex and (3) 19 F-radiolabeled moiety (Gd-DOTA-DEVD-Tfb presented in Figure 46). The Gd 3+ complex has strong paramagnetic relaxation enhancement (PRE) because of the seven unpaired electrons in the 4f orbital of Gd 3+ 408,409 . Proximity of 19 F to Gd 3+ in the probe weakened the MRI signal which significantly increases after hydrolysis with decrease in the PRE signal.…”

New frontiers and developing applications in 19F NMR

“…The simplest may be adding additional equivalent fluorine atoms [111, 112]. A more subtle approach recently exploited paramagnetic relaxation enhancement (PRE) based on spatial proximity to a relaxing paramagnetic lanthanide center [113]: furthermore, enzyme induced molecular cleavage or pH dependant conformational changes may alter the proximity and signal characteristics [109]. Alternatively transverse relaxation may be enhanced by formation of densely packed molecular agglomerations, potentially liquid crystal type structures with restricted motion causing severe 19 F line broadening [112, 114].…”

“…Recent reports have shown evolution of both color pigment or fluorescence and 19 F NMR signal [109, 184, 185]. New β-gal (β-galactosidase) reporters simultaneously show 19 F chemical shift and 1 H contrast effects [186, 187].…”

“…In most cases the chemical shift response is larger when the reporter moiety is aromatic, as opposed to aliphatic, and fluoronitrophenols have been popular. Proximity of the signaling fluorine to the site of cleavage is also critical, although in some cases a self-immolative cascade allows greater spacing [109, 166, 187]. …”

“…Recent reports have demonstrated a PRE approach to detecting enzyme activity or ions [109, 163–166, 187, 326]. Substrates include a 19 F moiety in close proximity to paramagnetic center exerting strong T 2 * relaxation and rendering it “invisible”.…”