Chromophore Analogues Research Articles

A novel fluorescent protein chromophore analogue to simultaneously probe lysosome viscosity and β-amyloid fibrils

Alzheimer's disease (AD) is an incurable neurodegenerative disease. Better diagnosis of AD is extremely important for therapeutic interventions. The amyloid cascade hypothesis and its revised version identify insoluble β-amyloid deposition as a good diagnostic biomarker for AD. We hypothesize that lysosomal viscosity may also act as an auxiliary biomarker for AD based on literature research. Herein, we have successfully developed a FP chromophore analogue C25 as a dual-functional fluorescent probe which can respond effectively to the Aβ fibrils and enhanced lysosomal viscosity with distinguishable turn-on fluorescence signals around 570 nm and 600 nm, respectively. The probe C25 can also monitor the Aβ degradation in the lysosomes. The probe C25 can serve as a promising tool for developing early diagnosis and innovative therapeutics of AD.

A turn-on fluorescent GFP chromophore analog for highly selective and efficient detection of H2S in aqueous and in living cells

Hydrogen sulphide is a gaseous neurotransmitter responsible for neuronal function and controls vast range of physiological functions. Herein, we report the synthesis and evaluation of novel Green Fluorescent Protein (GFP) chromophore analog, acryloyl-4-(p-hydroxybenzylidene)-5-imidazolidinone (AHBI) for turn-on fluorescent detection of H2S over wide range of anions and various biologically important competitive thiols. AHBI probe exhibited high selectivity and sensitivity, high fluorescence stability, large stokes shift and lower detection limit (15.85 ppb) for H2S in complete water medium. Cell imaging studies in human colon cancer cells (HCT116) and normal human dermal fibroblasts (HDF) confirmed the compatibility and versatility of AHBI probe at micromolar level. Overall, we believe the AHBI, as an optical probe will be useful to investigate the role of H2S in various physiological processes, regulation of cancer cell growth, and in pathogenic events.

Crystal structure and spectral studies of green fluorescent protein (GFP) chromophore analogue ethyl 2-[(4Z)-(6-hydroxy naphthalen-2-yl) methylene)-2-methyl-5-oxo-4,5-di hydro-1H-imidazol-1-yl] acetate

Synthetically modified green fluorescent protein chromophore derivative was prepared, its crystal structure and spectral properties were studied. Crystal data for C19H18N2O4: triclinic, space group P-1 (no. 2), a = 8.2506(17) Å, b = 11.934(2) Å, c = 17.461(4) Å, α = 102.89(3)°, β = 94.62(3)°, γ = 96.68(3)°, V = 1654.5(6) Å3, Z = 4, T = 173(2) K, μ(MoKα) = 0.096 mm-1, Dcalc = 1.358 g/cm3, 7227 reflections measured (4.722° ≤ 2Θ ≤ 53.996°), 7227 unique (Rint = 0.0453, Rsigma = 0.0662) which were used in all calculations. The final R1 was 0.0561 (I > 2σ(I)) and wR2 was 0.1658 (all data). The single crystal structure showed, the benzylidine moiety adopts Z-conformation in solid state and the molecules were associated by various O−H···O and C−H···O non-covalent interactions. The UV absorption-emission spectral analysis indicated that a significant red shift of emission observed at alkaline pH indicating its utility for live cell imaging applications.

S1/S0 Potential Energy Surfaces Experience Different Types of Restricted Rotation: Restricted Z/ E Photoisomerization and E/ Z Thermoisomerization by an Out-of-Plane Benzyl Group or In-Plane m-Pyridinium Group?

Any method that can enhance the fluorescence of fluorophores is highly desirable. Fluorescence enhancement accomplished by restricted Z/ E photoisomerization through intramolecular steric hindrance or relatively high bond order of a C═C double bond in a S1 excited state has rarely been studied. In this article, we used green fluorescent protein (GFP) chromophore analogues as a model to get new physical insights into the restricted Z/ E photoisomerization and E/ Z thermoisomerization phenomena. We found that the S1 and S0 potential energy surfaces (PESs) of the GFP chromophore analogues experience two dramatically different types of restricted rotation, and 2b can be a representative example. In its S1 PES, it is not the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group but the relatively high bond order of the I-bond in the S1 excited state of 2b that makes it have a higher barrier for the Z/ E photoisomerization, a smaller Z/ E photoisomerization quantum yield, and a higher fluorescence quantum yield. In its S0 PES, it is not the reduced bond order of the I-bond in the S0 ground state of 2b but the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group that makes it have an extra higher barrier for E/ Z thermoisomerization and a much smaller E/ Z thermoisomerization rate constant.

Synthesis of the New Green Fluorescent Protein Chromophore Analogue Starting from a Cinnamic Aldehyde Derivative

A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphenyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was prepared by the synthetic approach based on the use of carboxyimidate. Comparison of optical properties of the obtained compound and of the classical GFP chromophore analogue (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one showed the shift of absorption and emission maxima to the long-wavelength region induced by the increase of the conjugated π-system in the benzylidene fragment.

Synthesis and spectroscopic characterization of novel GFP chromophore analogues based on aminoimidazolone derivatives.

In order to improve the fluorescence properties of the green fluorescent protein chromophore, p‑HOBDI ((5‑(4‑hydroxybenzylidene)‑2,3‑dimethyl‑3,5‑dihydro‑4H‑imidazol‑4‑one), sixteen dihydroimidazolone derivates were synthesized from thiohydantoin and arylaldehydes. The synthesis developed is an efficient, novel, one-pot procedure. The study provides a detailed description of the spectroscopic characteristics of the newly synthesized compounds, using p‑HOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than p‑HOBDI, up to 28 times higher quantum yields. An experimental and theoretical investigation of the relationship of the fluorescence properties with the molecular structure was also carried out. A good correlation was found between the emission wavenumber and the Hammett constant of the functional group, which suggests the intermolecular charge transfer (ICT) mechanism between the aromatic groups.

Synthesis of Green Fluorescent Protein Chromophore Analogues for Interdisciplinary Learning for High School Students

Interdisciplinary outreach activities with easy-to-implement experiments have been developed to enhance high school students’ interest in chemistry and their inquisitive minds with active learning strategies. The synthesis of a model chromophore of the green fluorescent protein was employed as a key motif. Students simply enjoyed observing changes in the substances as well as witnessing a variety of fluorescent emissions. They gradually realized the importance of molecular design in the understanding and development of chemistry, biology, physics, and medicine.

Synthesis and environment-dependent fluorescence behavior of a biaryl-conjugated (diphenylmethylene)imidazolinone.

The newly designed green fluorescent protein (GFP) chromophore analog, bar-DAIN, which has a 2-biaryl-conjugated 5-(diphenylmethylene)imidazolinone structure, was effectively synthesized using the Suzuki coupling reaction. Bar-DAIN showed environment-dependent fluorescence behavior; for example, the thienyl analog emitted yellow-green fluorescence in viscous solution (λem: 535 nm), yellow-orange fluorescence in suspension (λem: 551 nm), and cyan fluorescence in a powder state (λem: 497 nm) although it showed almost no emission in common solvents such as dichloromethane. The dynamic discoloration of the fluorescence was observed by changing environmental conditions from suspension to viscous.

Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins.

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

Electronic State Mixing Controls the Photoreactivity of a Rhodopsin with all- trans Chromophore Analogues.

Rhodopsins hosting synthetic retinal protonated Schiff base analogues are important for developing tools for optogenetics and high-resolution imaging. The ideal spectroscopic properties of such analogues include long-wavelength absorption/emission and fast/hindered photoisomerization. While the former may be achieved, for instance, by elongating the chromophore π-system, the latter requires a detailed understanding of the substituent effects (i.e., steric or electronic) on the chromophore light-induced dynamics. In the present letter we compare the results of quantum mechanics/molecular mechanics excited-state trajectories of native and analogue-hosting microbial rhodopsins from the eubacterium Anabaena. The results uncover a relationship between the nature of the substituent on the analogue (i.e., electron-donating (a Me group) or electron-withdrawing (a CF3 group)) and rhodopsin excited-state lifetime. Most importantly, we show that electron-donating or -withdrawing substituents cause a decrease or an increase in the electronic mixing of the first two excited states which, in turn, controls the photoisomerization speed.

Protective Effect of a Locked Retinal Chromophore Analog against Light-Induced Retinal Degeneration

Continuous regeneration of the 11-cis-retinal visual chromophore from all-trans-retinal is critical for vision. Insufficiency of 11-cis-retinal arising from the dysfunction of key proteins involved in its regeneration can impair retinal health, ultimately leading to loss of human sight. Delayed recovery of visual sensitivity and night blindness caused by inadequate regeneration of the visual pigment rhodopsin are typical early signs of this condition. Excessive concentrations of unliganded, constitutively active opsin and increased levels of all-trans-retinal and its byproducts in photoreceptors also accelerate retinal degeneration after light exposure. Exogenous 9-cis-retinal iso-chromophore can reduce the toxicity of ligand-free opsin but fails to prevent the buildup of retinoid photoproducts when their clearance is defective in human retinopathies, such as Stargardt disease or age-related macular degeneration. Here we evaluated the effect of a locked chromophore analog, 11-cis-6-membered ring-retinal against bright light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Using in vivo imaging techniques, optical coherence tomography, scanning laser ophthalmoscopy, and two-photon microscopy, along with in vitro histologic analysis of retinal morphology, we found that treatment with 11-cis-6-membered ring-retinal before light stimulation prevented rod and cone photoreceptor degradation and preserved functional acuity in these mice. Moreover, additive accumulation of 11-cis-6-membered ring-retinal measured in the eyes of these mice by quantitative liquid chromatography-mass spectrometry indicated stable binding of this retinoid to opsin. Together, these results suggest that eliminating excess of unliganded opsin can prevent light-induced retinal degeneration in Abca4-/-Rdh8-/- mice.

The effect of the chromophoric group modification on the optical properties of retinal proteins

The effects of chromophoric group structures on the functional properties of bacteriorhodopsin (BR) and proteorhodopsin from E. sibiricum (ESRh) were compared. ESRh retinal binding site was found as preserving the similar stereo- and spatial restrictions on the chromophore structure during the retinal protein reconstitution process (except for C25-analog AR8). It was revealed that the structure peculiarities of the chromophore analog molecules affect the optical parameters of ESRh and BR pigment families in similar ways.

Enhancing the Anti‐Solvatochromic Two‐Photon Fluorescence for Cirrhosis Imaging by Forming a Hydrogen‐Bond Network

Two-photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor-acceptor (D-A) type molecules are the most widely employed two-photon scaffolds. However, current D-A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D-A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen-bond network in water to improve the two-photon efficiency. Our design results in two-photon chalcone (TPC) dyes with 0.80 quantum yield and large two-photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two-photon materials with anti-solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H2 S generation and scavenging in the cirrhotic rat liver for the first time.

Enhancing the Anti‐Solvatochromic Two‐Photon Fluorescence for Cirrhosis Imaging by Forming a Hydrogen‐Bond Network

AbstractTwo‐photon imaging is an emerging tool for biomedical research and clinical diagnostics. Electron donor–acceptor (D–A) type molecules are the most widely employed two‐photon scaffolds. However, current D–A type fluorophores suffer from solvatochromic quenching in aqueous biological samples. To address this issue, we devised a novel class of D–A type green fluorescent protein (GFP) chromophore analogues that form a hydrogen‐bond network in water to improve the two‐photon efficiency. Our design results in two‐photon chalcone (TPC) dyes with 0.80 quantum yield and large two‐photon action cross section (210 GM) in water. This strategy to form hydrogen bonds can be generalized to design two‐photon materials with anti‐solvatochromic fluorescence. To demonstrate the improved in vivo imaging, we designed a sulfide probe based on TPC dyes and monitored endogenous H2S generation and scavenging in the cirrhotic rat liver for the first time.

Simultaneous Detection of Local Polarizability and Viscosity by a Single Fluorescent Probe in Cells

Many intracellular reactions are dependent on the dielectric (“polarity”) and viscosity properties of their milieu. Fluorescence imaging offers a convenient strategy to report on such environmental properties. Yet, concomitant and independent monitoring of polarity and viscosity in cells at submicron scale is currently hampered by the lack of fluorescence probes characterized by unmixed responses to both parameters. Here, the peculiar photophysics of a green fluorescent protein chromophore analog is exploited for quantifying and imaging polarity and viscosity independently in living cells. We show that the polarity and viscosity profile around a novel hybrid drug-delivery peptide changes dramatically upon cell internalization via endosomes, shedding light on the spatiotemporal features of the release mechanism. Accordingly, our fluorescent probe opens the way to monitor the environmental effects on several processes relevant to cell biochemistry and nanomedicine.

Synthetic green fluorescent protein (GFP) chromophore analog for rapid, selective and sensitive detection of cyanide in water and in living cells

Here, we report Green Fluorescent Protein (GFP) chromophore analog as a turn-on fluorescent chemodosimeter (THBI) for selective detection of cyanide in water, on solid state and in living cells. The detection limit was found to be 0.17 μM (4.5 ppb). The time dependent study revealed that there is a rapid enhancement in fluorescence intensity (in less than 5s) and was constant over the period of 1 h. Cell imaging data exhibited that THBI was successfully crossed cell membrane and visualized fluorescence response in live HCT cells.

Protonation of the imino nitrogen deactivates the excited state of imidazolin-5-one in the solid state

The imino nitrogen of p-methoxybenzylideneimidazolinone (pMBDI) was protonated using aqueous $$\hbox {HClO}_{4}$$ and HCl acid and crystallized in monoclinic crystal system with $$\textit{P2}_{\textit{1}}/n$$ space group. Interestingly, the protonated compounds are fluorescent in polar solvents but non-fluorescent in non-polar solvent as well as in the solid state. These results point to the existence of a non-radiative pathway involving the imidazole nitrogen in the quenching of excited states in these compounds. SYNOPSIS The imino nitrogen of p-methoxybenzylideneimidazolinone (pMBDI) was protonated and crystallized in monoclinic crystal system with $$\textit{P2}_{\textit{1}}/n$$ space group. Interestingly, the protonation at imino nitrogen of imidazolinone ring results in the loss of the fluorescence property of gfp chromophore analogue (p-methoxybenzylideneimidazolinone).

Colorimetric Determination of Sulfate via an Enzyme Cascade for High-Throughput Detection of Sulfatase Activity.

High-throughput screening (HTS) methods have become decisive for the discovery and development of new biocatalysts and their application in numerous fields. Sulfatases, a broad class of biocatalysts that hydrolyze sulfate esters, are involved in diverse relevant cellular functions (e.g., signaling and hormonal regulation) and are therefore gaining importance, particularly in the medical field. Additionally, various technical applications have been recently devised. One of the major challenges in the field of enzyme development is the sensitive and high-throughput detection of the actual product of the biocatalyst of interest without the need for chromophore analogues. Addressing this issue, a colorimetric assay for sulfatases was developed and validated for detecting sulfate through a two-step enzymatic cascade, with a linear detection range of 3.3 (limit of detection) up to 250 μM. The procedure is compatible with relevant compounds employed in sulfatase reactions, including cosolvents, cations, and buffers. The assay was optimized and performed as part of a 96-well screening workflow that included bacterial growth, heterologous sulfatase expression, cell lysis, sulfate ester hydrolysis, inactivation of cell lysate, and colorimetric sulfate determination. With this procedure, the activity of an aryl and an alkyl sulfatase could be confirmed and validated. Overall, this assay provides a simple and fast alternative for screening and engineering sulfatases from DNA libraries (e.g., using metagenomics) with medical or synthetic relevance.

Unveiling Structural Motions of a Highly Fluorescent Superphotoacid by Locking and Fluorinating the GFP Chromophore in Solution.

Superphotoacidity involves ultrafast proton motions implicated in numerous chemical and biological processes. We used conformational locking and strategic addition of electron-withdrawing substituents to synthesize a new GFP chromophore analogue: p-HO-3,5-diF-BDI:BF2 (diF). It is highly fluorescent and exhibits excited-state proton transfer (ESPT) in various solvents, placing it among the strongest photoacids. Tunable femtosecond stimulated Raman spectroscopy with unique resonance conditions and transient absorption are complementarily employed to elucidate the structural basis for superphotoacidity. We reveal a multistep ESPT reaction from diF to methanol with an initial proton dissociation on the ∼600 fs time scale that forms a charge-separated state, stabilized by solvation, and followed by a diffusion-controlled proton transfer on the ∼350 ps time scale. A ∼1580 cm-1 phenolic ring motion is uncovered to accompany ESPT before 1 ps. This study provides a vivid movie of the photoinduced proton dissociation of a superphotoacid with bright fluorescence, effectively bridging fundamental mechanistic insights to precise control of macroscopic functions.

Synthetic green fluorescent protein chromophore analogues with a positive charge at the phenyl-like group.

Synthetic green fluorescent protein (GFP) chromophore analogues with a positive charge at the phenyl-like group have the highly electrophilic amidine carbon, smaller LUMO-HOMO energy gap, red-shifted electronic absorptions and fluorescent emissions, and accelerated E-Z thermoisomerization rates. They are water-labile and their hydrolysis results in ring-opening of the imidazolinone moiety with a half life around 25-37h in D2O at 25°C.