Structural Information about the trans-to-cis Isomerization Mechanism of the Photoswitchable Fluorescent Protein rsEGFP2 Revealed by Multiscale Infrared Transient Absorption.

RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.

Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy.

RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.

Biodegradable Polymersomes with Structure Inherent Fluorescence and Targeting Capacity for Enhanced Photo-Dynamic Therapy.

Biodegradable nanostructures displaying aggregation-induced emission (AIE) are desirable from a biomedical point of view, due to the advantageous features of loading capacity, emission brightness, and fluorescence stability. Herein, biodegradable polymers comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-P(CLgTMC)), with tetraphenylethylene pyridinium-TMC (PAIE) side chains have been developed, which self-assembled into well-defined polymersomes. The resultant AIEgenic polymersomes are intrinsically fluorescent delivery vehicles. The presence of the pyridinium moiety endows the polymersomes with mitochondrial targeting ability, which improves the efficiency of co-encapsulated photosensitizers and improves therapeutic index against cancer cells both in vitro and in vivo. This contribution showcases the ability to engineer AIEgenic polymersomes with structure inherent fluorescence and targeting capacity for enhanced photodynamic therapy.

E-Z Isomerization Mechanism of the Green Fluorescent Protein Chromophore: Remote Regulation by Proton Dissociation of the Phenol Group.

Dronpa, a GFP (green fluorescent protein)-like fluorescent protein, allows its fluorescent and nonfluorescent states to be switched to each other reversibly by light or heat through E-Z isomerization of the GFP chromophore. In this article, a GFP chromophore (p-HBDI) in water is used as a model to explore this E-Z isomerization mechanism. Based on the experimental solvent isotope effect (kH2O/kD2O = 2.30), the E-Z isomerization of p-HBDI in water is suggested to go through the remote-proton-dissociation-regulated direct mechanism with a proton transfer in the rate-determining step. The fractionation factor (ϕ) of the water-associated phenol proton of p-HBDI in the transition state is found to be 0.43, which is exactly in the range of 0.1-0.6 for the fractionation factor (ϕ) of the transferring proton in the transition state of R2O···H···O+H2 in water. This means that the phenol proton of E-p-HBDI in the transition state is on the way to the associated water oxygen during the E-Z isomerization. The proton dissociation from the phenol group of p-HBDI remotely regulates its E-Z isomerization. Less proton dissociation from the phenol group (pKa = 8.0) at pH = 1-4 results in a modest reduction in the E-Z isomerization rate of p-HBDI, while complete proton dissociation from the phenol group at pH = 11-12 also reduces its E-Z isomerization rate by one order of magnitude because of the larger charge separation in the transition state of the p-HBDI anion. All of these results are consistent with the remote-proton-dissociation-regulated direct mechanism but against the water-assisted addition/elimination mechanism.

Tetraphenylethene-Based Supramolecular Coordination Frameworks with Aggregation-Induced Emission for an Artificial Light-Harvesting System.

Supramolecular coordination is an efficient strategy to construct supramolecular coordination frameworks with predesigned structures, assembled shapes, and specific function. In this work, we report the synthesis, structural characterization, and photophysical property of two tetraphenylethene-based supramolecular coordination frameworks 1a and 1b formed from 1,1,2,2-tetrakis(4-(pyridin-4-yl)phenyl)ethene (2a) or 1,1,2,2-tetrakis(4-((E)-2-(pyridin-4-yl)vinyl)phenyl)ethene (2b) and a linear difunctional platinum(II) ligand (3a) via coordination-driven self-assembly. Controlled by the specific angularity and geometry of tetraphenylethene (with 60° and 120°) and difunctional Pt(II) linker (with 180°), these supramolecular coordination frameworks possess a well-defined and two-dimensional (2D) rhombic network-type topology with good periodicity and porosity. Given the aggregation-induced emission (AIE) property of tetraphenylethene units and the porosity of frameworks, 1a and 1b have been successfully used as fluorescent platforms and energy donors to fabricate efficient artificial light-harvesting materials with two fluorescent acceptors (Nile Red and Sulforhodamine 101) via noncovalent interactions in aqueous solution. Furthermore, these light-harvesting materials have been applied for promoting cancer cell imaging with a full shift of imaging channels from blue/green channels to the red channel. Thus, this study provides an effective approach to fabricate functional frameworks as fluorescent platforms for developing more fluorescent materials.

A nitroxyl-responsive near-infrared fluorescent chemosensor for visualizing H2S/NO crosstalk in biological systems.

We present a near-infrared (NIR) fluorescent probe, NR-HNO, which was successfully applied to visualizing H2S/NO "crosstalk" by the fluorescence detection of nitroxyl with a fast response time (5 min) and a large Stokes shift (131 nm) in living cells and tissue; it was also used to image nitroxyl in live mice.

Specific Targeting, Imaging, and Ablation of Tumor-Associated Macrophages by Theranostic Mannose-AIEgen Conjugates.

Tumor-associated macrophages (TAMs) that exist in tumor microenvironment promote tumor progression and have been suggested as a promising therapeutic target for cancer therapy in preclinical studies. Development of theranostic systems capable of specific targeting, imaging, and ablation of TAMs will offer clinical benefits. Here we constructed a theranostic probe, namely, TPE-Man, by attaching mannose moieties to a red-emissive and AIE (aggregation-induced emission)-active photosensitizer. TPE-Man can specifically recognize a mannose receptor that is overexpressed on TAMs by the sugar-receptor interaction and enables fluorescent visualization of the mannose-receptor-positive TAMs in high contrast. The histologic study of mouse tumor sections further verifies TPE-Man's excellent targeting specificity being comparable with the commercial mannose-receptor antibody. TAMs can be effectively eradicated upon exposure to white light irradiation via a photodynamic therapy effect. To our knowledge, this is the first small molecular theranostic probe for TAMs that revealed combined advantages of low cost, high targeting specificity, fluorescent light-up imaging, and efficient photodynamic ablation.

Screening of two-photon activated photodynamic therapy sensitizers using a 3D osteosarcoma model.

Photodynamic therapy (PDT) involves a photosensitizing agent activated with light to induce cell death. Two-photon excited PDT (TPE-PDT) offers numerous benefits compared to traditional one-photon induced PDT, including an increased penetration depth and precision. However, the in vitro profiling and comparison of two-photon photosensitizers (PS) are still troublesome. Herein, we report the development of an in vitro screening platform of TPE-PS using a 3D osteosarcoma cell culture. The platform was tested using three different two-photon (2P) active compounds - a 2P sensitizer P2CK, a fluorescent dye Eosin Y, and a porphyrin derivative (TPP). Their 2P absorption cross-sections (σ2PA) were characterised using a fully automated z-scan setup. TPP exhibited a remarkably high σ2PA at 720 nm (8865 GM) and P2CK presented a high absorption at 850 nm (405 GM), while Eosin Y had the lowest 2P absorption at the studied wavelengths (<100 GM). The cellular uptake of PS visualized using confocal laser scanning microscopy showed that both TPP and P2CK were internalized by the cells, while Eosin Y stayed mainly in the surrounding media. The efficiency of the former two TPE-PS was quantified using the PrestoBlue metabolic assay, showing a significant reduction in cell viability after two-photon irradiation. The possibility of damage localization was demonstrated using a co-culture of adipose derived stem cells together with osteosarcoma spheroids showing no signs of damage to the surrounding healthy cells after TPE-PDT.

A novel colorimetric and ratiometric fluorescent probe for sensing SO2 derivatives and their bio-imaging in living cells.

We report a novel fluorescent probe HBN-TCF for the detection of SO2 derivatives. This probe exhibited near-infrared fluorescence emission with an excitation wavelength of 620 nm. After reacting with SO32-, the emission channel at 664 nm decreased, while the new strong emission channel at 482 nm increased (λex = 400 nm), with a large emission distance (Δλ = 182 nm) observed. This probe exhibited the rapid and selective detection of SO2 derivatives compared with other sulfur-containing species and featured a low detection limit (82 nM). This colorimetric and ratiometric fluorescent probe showed high selectivity and sensitivity for detecting SO2 derivatives. The probe was also successfully exploited for the fluorescence imaging of intracellular and exogenous SO2 derivatives in BEL-7402 cells.

Synthesis and properties of geometrical 4-diarylmethylene analogs of the green fluorescent protein chromophore.

We have developed a novel analog of the GFP chromophore: geo-DAIN. Since geo-DAIN is equipped with an E/Z-photoisomerizable geometrical diarylmethylene moiety instead of benzylidene of the GFP chromophore, different-colored reversible emissions are expected. We synthesized geo-DAIN by a condensation with methyl imidate and N-(diarylmethylene)glycinate. We found the emission from geo-DAIN to be different from that of benzylidene-type analogs; in the powder state, the E- and Z-isomers of geo-DAIN emitted different fluorescence colors.

Apparent viscosity of p-methoxybenzylidene-p'-n-butylaniline in the presence of electrohydrodynamic convection.

The apparent shear viscosity of p-methoxybenzylidene-p'-n-butylaniline in the presence of electrohydrodynamic convection (EHC) is investigated experimentally. In the absence of an electric field, directors are almost aligned along the flow direction such that the viscosity is close to the minimum of the Miesowicz viscosities. Since EHC disturbs the flow-aligned director configuration, the viscosity increases as the applied voltage is increased in the low-voltage regime. In the high-voltage regime, however, further increasing the voltage leads to a decrease in viscosity. Microscope observations using a rheometer reveal that the decrease in viscosity occurs in the dynamic scattering mode 2 (DSM2) state, whose spatial director distribution is anisotropic due to the shear flow. By adopting the Ericksen-Leslie theory for the shear flow under the electric field, we find that the viscosity decrease can be attributed to the negative contribution of the electric stress caused by the anisotropic director distribution of the DSM2 state.

Reductive ring opening of o-nitrobenzylidene acetals of monosaccharides: synthesis and photolysis of some photolabile sugars.

[figure: see text] A 6-O-o-nitrobenzyl methylglucoside and methylmannoside were synthesized by reacting 4,6-O-o-nitrobenzylidene acetals with triethylsilane and boron trifluoride etherate. A 2,6-di-O-o-nitrobenzyl and a 3,6-di-O-o-nitrobenzyl methylmannoside were obtained from a 2,3:4,6-di-O-o-nitrobenzylidene methylmannoside by the same method. The photolabile sugars obtained were deprotected by irradiation at 350 nm to afford methylglycosides.

Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy.

Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.