Recent advances in Si-rhodamine-based fluorescent probes for live-cell imaging.

Fluorescence imaging is a powerful technique for visualizing biological events in living samples with high temporal and spatial resolution. Fluorescent probes emitting far-red to near infrared (NIR) fluorescence are particularly advantageous for in vivo imaging due to their high tissue permeability and low autofluorescence, as well as their suitability for multicolor imaging. Among the far-red to NIR fluorophores, Si-rhodamine is one of the most practical fluorophores for the development of tailor-made NIR fluorescent probes because of the relative ease of synthesis of various derivatives, the unique intramolecular spirocyclization behavior, and the relatively high water solubility and high photostability of the probes. This review summarizes these features of Si-rhodamines and presents recent advances in the synthesis and applications of far-red to NIR fluorescent probes based on Si-rhodamines, focusing on live-cell imaging applications such as fluorogenic probes, super-resolution imaging and dye-protein hybrid-based indicators.

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.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5'-phosphate (PLP) via Schiff base formation.

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

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.

Amide nitrogen pyramidalization changes lactam amide spinning.

Although cis-trans lactam amide rotation is fundamentally important, it has been little studied, except for a report on peptide-based lactams. Here, we find a consistent relationship between the lactam amide cis/trans ratios and the rotation rates between the trans and cis lactam amides upon the lactam chain length of the stapling side-chain of two 7-azabicyclo[2.2.1]heptane bicyclic units, linked through a non-planar amide bond. That is, as the chain length increased, the rotational rate of trans to cis lactam amide was decreased, and consequently the trans ratio was increased. This chain length-dependency of the lactam amide isomerization and our simulation studies support the idea that the present lactam amides can spin through 360 degrees as in open-chain amides, due to the occurrence of nitrogen pyramidalization. The tilting direction of the pyramidal amide nitrogen atom of the bicyclic systems is synchronized with the direction of the semicircle-rotation of the amide.