Acceptor Fluorophores Research Articles

Storehouse of Peculiar Supramolecular Architecture: Probing the Charge-Transfer Phenomenon and Effect of Altering the pH in a Meta-Oriented Single Donor-Double Acceptor Fluorophore.

The investigation of excited-state intramolecular charge transfer (ESICT) has been a fascinating area of research. Although the ESICT events have been studied mostly for para-disubstituted donor-acceptor type molecules, the meta-oriented donor-acceptor type molecules have also shown tremendous potential as ESICT active molecules. In the current work, a small fluorescent probe diethyl 5-amino isophthalate (DE-5A-IPA) was investigated as a potential model to investigate ESICT events in the solution as well as solid phase. DE-5A-IPA was synthesized easily starting from commercially available 5-amino isophthalic acid in excellent yield. In the solid state, differing extents of CH···π-type binding led to formation of fully planar and puckered "cyclobutane" mimic supramolecular architecture, as evidenced from the crystal structure analysis. In addition, the single crystal structure of DE-5A-IPA shows that the donor (-NH2) and acceptor (-CO2Et) are situated in the same plane, thereby assuring the prerequisites of a through-space charge transfer. DE-5A-IPA undergoes noticeable Stokes shift (in cm-1) when the polarity of the medium was changed (4820 cm-1 in hexane; 9375 cm-1 in water). The excited-state dipole moment (μe) was calculated to be 4.0 units higher than the ground-state dipole moment (μg). DE-5A-IPA had an appreciable quantum yield (0.10 ± 0.03 to 0.27 ± 0.04). The ESICT phenomenon was also investigated by ground- and excited-state structural calculations using Gaussian 16 software. The excited-state lifetime measured by the time correlated single photon counting technique was found to vary with the polarity of the solvent, thereby providing further support to the ESICT phenomenon being operative in DE-5A-IPA. Taking advantage of the -NH2 group (which is susceptible to protonation) in DE-5A-IPA, steady state and time-resolved photodynamics were investigated in solutions of varying pH values. Interestingly, the emission quantum yields as well as the emission lifetime increase with an increase in pH value, thereby establishing DE-5A-IPA as a pH-sensitive probe. The current findings shall boost the understanding of meta-oriented ESICT enabled compounds in terms of their excited-state photophysics.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

DNA tweezer-assembled, regenerated nanosensor for one-step and sustainable detection of viral RNA and Hg2+

Transcription-polymerase chain reaction (RT-qPCR) is the golden standard to detect viral RNA. However, it suffers from several inherent defects such as high false-negative rates, time-consuming and expensive. To exploit better analytical methods for future global pandemics, it is vital to develop rapid, sustainable, and environmentally-friendly sensing methods. Here, a one-step, regenerated nanosensor assembled by DNA tweezers is developed for rapid and sustainable detection of viral RNA. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is selected as model analyte. The DNA tweezer is designed with a central strand and two arm strands containing overhangs complementary to SARS-CoV-2 RNA. The SARS-CoV-2 RNA hybridizes with the two arm overhangs and strains the DNA tweezer to closed conformation, resulting in the close proximity of the donor and acceptor fluorophores. With the addition of an anti-strand complementary to the SARS-CoV-2 RNA, the closed DNA tweezer is relaxed to open conformation through a strand displacement process, leading to the separation of the dual fluorophores. Thus, the DNA tweezer is revived for fresh SARS-CoV-2 RNA. The proposed nanosensor can be regenerated for three cycles with the same performance. Moreover, the viral RNA can be detected within 30 min with a detection limit of 0.04 nM. By altering the sequences of the DNA tweezer, rapid, sensitive, and sustainable detection of Hg2+ is achieved. The proposed nanosensor provides a novel approach for rapid, flexible, and sustainable detection of viral RNA and Hg2+, exhibiting great potential for routine preliminary coronavirus disease tests, epidemic control, and environmental monitoring.

Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation

Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade. Specialized FRET probes placed on Ub and Ub-like proteins have been developed to monitor Ub removal by deubiquitinating enzymes (DUBs) that result in a loss of a FRET signal upon cleavage of the FRET probes. FRET has also been used to understand conformational changes in large complexes such as multimeric E3 ligases and the proteasome, frequently using sophisticated single molecule methods. Overall, FRET is a powerful tool to help unravel the intricacies of the complex ubiquitination system.

POLARISED views and FRETting about probe modulation assays: Learning from High Throughput Screening

Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.

Fused heteroaromatic donor–acceptor fluorophores: enhanced brightness and variable emission characteristics

Abstract Octacyclic imidazophenanthridine derivatives have been synthesized by the intramolecular C–H arylation of phenanthroimidazolylbenzothiadiazoles. A significant increase in the molar extinction coefficient (ε) compared with their non-fused counterparts has been achieved, while retaining the high fluorescence quantum yield (ΦF). Their luminescence properties vary in various solvents, as well as in crystalline and mechanically ground states.

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

Aggregation induced emission (AIE) based donor–π–acceptor fluorophores: an approach to fabricate acidochromic sensors and white light emitting diodes

This work reports the design and synthesis of two AIE active donor–π–acceptor fluorophores (TPASCNPy and TPESCNPy) and evaluation of their potential in the fabrication of acidochromic sensors and white light emitting diodes.

PH Clock Guided Dynamic Broad Spectrum Multi‐Color Fluorescence Modulation in Size‐Oscillatory Vesicles

AbstractAcid–base equilibria of aqueous cellular fluids play a vital role in constituting various signaling and feedback mechanisms in biological systems. To mimic the complex pH‐directed signaling mechanism in laboratory settings, a pH‐responsive vesicular assembly is presented that shows dynamic multicolor fluorescence and size alterations under the influence of a chemically driven pH cycle. The vesicular architecture is a versatile platform for establishing Förster resonance energy transfer (FRET) interactions between multiple donors and acceptor fluorophores. Spontaneously fluctuating interfacial charge of the vesicles during the pH cycle alters the donor fluorophore's emission intensity, affecting the efficiency of FRET interactions among the donor–acceptor pairs. This enables multicolor as well as transient pure white light fluorescence of the vesicles during the pH cycle. The vesicles also display significant size changes in response to varying pH conditions. Furthermore, the vesicles demonstrate excellent adaptability in terms of the lifespan of the oscillatory cycles and the tunability of the fluorescence color transitions.

REGA-SIGN: Development of a Novel Set of NanoBRET-Based G Protein Biosensors.

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gαi/o, GαSs/L, Gα12/13 and Gαq/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor-acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for GαSs/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity.

Time-resolved burst variance analysis

Quantifying biomolecular dynamics has become a major task of single-molecule fluorescence spectroscopy methods. In single-molecule Förster resonance energy transfer (smFRET), kinetic information is extracted from the stream of photons emitted by attached donor and acceptor fluorophores. Here, we describe a time-resolved version of burst variance analysis that can quantify kinetic rates at microsecond to millisecond timescales in smFRET experiments of diffusing molecules. Bursts are partitioned into segments with a fixed number of photons. The FRET variance is computed from these segments and compared with the variance expected from shot noise. By systematically varying the segment size, dynamics at different timescales can be captured. We provide a theoretical framework to extract kinetic rates from the decay of the FRET variance with increasing segment size. Compared to other methods such as filtered fluorescence correlation spectroscopy, recurrence analysis of single particles, and two-dimensional lifetime correlation spectroscopy, fewer photons are needed to obtain reliable timescale estimates, which reduces the required measurement time.

Photodissociative Modules that Control Dual‐Emission Properties in Donor–π–Acceptor Organoborane Fluorophores

AbstractDonor–π–acceptor fluorophores that consist of an electron‐donating amino group and an electron‐accepting triarylborane moiety generally exhibit substantial solvatochromism in their fluorescence while retaining high fluorescence quantum yields even in polar media. Herein, we report a new family of this compound class, which bears ortho‐P(=X)R2‐substituted phenyl groups (X=O or S) as a photodissociative module. The P=X moiety that intramolecularly coordinates to the boron atom undergoes dissociation in the excited state, giving rise to dual emission from the corresponding tetra‐ and tricoordinate boron species. The susceptibility of the systems to photodissociation depends on the coordination ability of the P=O and P=S moieties, whereby the latter facilitates dissociation. The intensity ratios of the dual emission bands are sensitive to environmental parameters, including temperature, solution polarity, and the viscosity of the medium. Moreover, precise tuning of the P(=X)R2 group and the electron‐donating amino moiety led to single‐molecule white emission in solution.

FRET Based Biosensor: Principle Applications Recent Advances and Challenges.

Förster resonance energy transfer (FRET)-based biosensors are being fabricated for specific detection of biomolecules or changes in the microenvironment. FRET is a non-radiative transfer of energy from an excited donor fluorophore molecule to a nearby acceptor fluorophore molecule. In a FRET-based biosensor, the donor and acceptor molecules are typically fluorescent proteins or fluorescent nanomaterials such as quantum dots (QDs) or small molecules that are engineered to be in close proximity to each other. When the biomolecule of interest is present, it can cause a change in the distance between the donor and acceptor, leading to a change in the efficiency of FRET and a corresponding change in the fluorescence intensity of the acceptor. This change in fluorescence can be used to detect and quantify the biomolecule of interest. FRET-based biosensors have a wide range of applications, including in the fields of biochemistry, cell biology, and drug discovery. This review article provides a substantial approach on the FRET-based biosensor, principle, applications such as point-of-need diagnosis, wearable, single molecular FRET (smFRET), hard water, ions, pH, tissue-based sensors, immunosensors, and aptasensor. Recent advances such as artificial intelligence (AI) and Internet of Things (IoT) are used for this type of sensor and challenges.

Impact of Electron-Donating Groups on Attaining Dual-Emitting Imidazole-Based Donor-Acceptor Materials.

Imidazole-based donor-acceptor materials are well known for their polarity-controlled trade-off phenomenon between the localized excitation-based short-wavelength (SW) emission in nonpolar solvents and charge transfer dominated long-wavelength (LW) emission in polar solvents. To attain concurrent SW- and LW-based dual-emission characteristics, a series of imidazole-based donor-acceptor fluorophores (CBImDCN, TPImDCN, PZImDCN) possessing different electron-donating groups such as carbazole, triphenylamine, and phenothiazine linked via the N-position of the imidazole core unit were synthesized and verified by NMR and mass spectroscopic techniques. As a result, the strong donating TPImDCN and PZImDCN exhibited dual emission in different solvents of varying polarity, covering the blue (SW) and green/orange (LW) regions. On the other hand, in contrast, only an SW emission band is observed with the weak donating CBImDCN. Moreover, PZImDCN shows panchromatic emission under 365 nm illumination, while only orange color emission is observed under visible light excitation, revealing two different origins of SW and LW emissions, as also evidenced from DFT calculations. Overall, this work reveals a new approach for attaining concurrent SW and LW emission characteristics from imidazole-based D-A materials and sheds light on the design and development of novel panchromatic emitters with intriguing properties for lighting and display applications.

Design and synthesis of a small molecular NIR-II chemiluminescence probe for in vivo-activated H2S imaging

Chemiluminescence (CL) with the elimination of excitation light and minimal autofluorescence interference has been wieldy applied in biosensing and bioimaging. However, the traditional emission of CL probes was mainly in the range of 400 to 650 nm, leading to undesired resolution and penetration in a biological object. Therefore, it was urgent to develop CL molecules in the near-infrared window [NIR, including NIR-I (650 to 900 nm) and near-infrared-II (900 to 1,700 nm)], coupled with unique advantages of long-time imaging, sensitive response, and high resolution at depths of millimeters. However, no NIR-II CL unimolecular probe has been reported until now. Herein, we developed an H2S-activated NIR-II CL probe [chemiluminiscence donor 950, (CD-950)] by covalently connecting two Schaap's dioxetane donors with high chemical energy to a NIR-II fluorophore acceptor candidate via intramolecular CL resonance energy transfer strategy, thereby achieving high efficiency of 95%. CD-950 exhibited superior capacity including long-duration imaging (~60 min), deeper tissue penetration (~10 mm), and specific H2S response under physiological conditions. More importantly, CD-950 showed detection capability for metformin-induced hepatotoxicity with 2.5-fold higher signal-to-background ratios than that of NIR-II fluorescence mode. The unimolecular NIR-II CL probe holds great potential for the evaluation of drug-induced side effects by tracking its metabolites invivo, further facilitating the rational design of novel NIR-II CL-based detection platforms.

Fluorescence lifetime analysis of smFRET with contribution of PIFE on donor and acceptor.

Single-molecule fluorescence resonance energy transfer (FRET) is a powerful technique based on dipole-dipole interaction between donor and acceptor fluorophores to observe inter- and intra-molecular dynamics in real time with sensitivity to macro-molecular distances (∼2.5-10 nm). That said, some fluorophores have an inherent characteristic known as protein induced fluorescence enhancement (PIFE). PIFE is a photo-physical feature of dyes undergoing cis-trans transitions and occurs for protein-dye interactions closer than 3 nm where FRET is less sensitive. Here, the challenge is uncoupling the PIFE effect in the FRET data. Ignoring the PIFE effect in the analysis of the FRET data may lead to misinterpretation of the system under investigation. As a solution to this problem, we develop a computational framework based on Bayesian statistics to analyze the fluorescence lifetime signals of the donor and acceptor channels which allows us to uncouple the PIFE effects from the FRET. Our framework can extract any changes in the FRET efficiency simultaneously with any changes in the fluorescence lifetimes of the donor and acceptor due to the PIFE effect. In addition, our framework can provide other parameters, such as the donor and acceptor excitation rates, background photon rates, and detectors’ cross-talk ratios. Our framework extracts all these parameters by analyzing a single photon arrival time trace with only a few thousand photons.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Designs and Applications of Multi-stimuli Responsive FRET Processes in AIEgen-Functionalized and Bi-fluorophoric Supramolecular Materials

Materials capable of displaying strong ratiometric fluorescence with Förster resonance energy transfer (FRET) processes have attracted much research interest because of various chemosensor and biomedical applications. This review highlights several popular strategies in designing FRET-OFF/ON mechanisms of ratiometric fluorescence systems. In particular, the developments of organic and polymeric FRET materials featuring aggregation-induced emission-based luminogens (AIEgens), supramolecular assemblies, photochromic molecular switches and surfactant-induced AIE/FRET mechanisms are presented. AIEgens have been frequently employed as FRET donor and/or acceptor fluorophores to obtain enhanced ratiometric fluorescences in solution and solid states. Since AIE effects and FRET processes rely on controllable distances between fluorophores, many interesting fluorescent properties can be designed by regulating aggregation states in polymers and supramolecular systems. Photo-switchable fluorophores, such as spiropyran and diarylethene, provide drastic changes in fluorescence spectra upon photo-induced isomerizations, leading to photo-switching mechanisms to activate/deactivate FRET processes. Supramolecular assemblies offer versatile platforms to regulate responsive FRET processes effectively. In rotaxane structures, the donor-acceptor distance and FRET efficiency can be tuned by acid/base-controlled shuttling of the macrocycle component. The tunable supramolecular interactions are strongly influenced by external factors (such as pH values, temperatures, analytes, surfactants, UV-visible lights, etc.), which induce the assembly and disassembly of host-guest systems and thus their FRET-ON/FRET-OFF behavior. In addition, the changes in donor or acceptor fluorescence profiles upon detections of analytes can also sufficiently alter the FRET behavior and result in different ratiometric fluorescence outputs. The strategies and examples provided in this review offer the insights and toolkits for future FRET-based material developments.

Synthesis of polyfunctionalized dihydro-2-oxypyrroles catalyzed by 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) as a novel donor-acceptor fluorophore

We developed a green radical synthesis method for polyfunctionalized dihydro-2-oxypyrroles based on the Michael–Mannich cyclocondensation of amines, dialkyl acetylenedicarboxylates, and formaldehyde. To generate a renewable energy sources from visible light, a PCET (proton-coupled electron transfer) photocatalyst was used in an ethanol solution in an air environment and at room temperature. In this study, we aim to develop an inexpensive and easily accessible novel donor–acceptor (D–A) fluorophore. Besides its speed-saving features and ease of use, the carbazole-based photocatalyst (4CzIPN) also shows high yields, energy-efficient, and is environmentally friendly. In this way, it is possible to monitor changes in chemical and environmental variables over time. The variety of yields is pretty uniform (84–97%, average 92.3%), and the kind of response times be very speedy (15–25 min, average 17.6 min), and the element noted within the dialogue is that the system tolerates a variety of donating and withdrawing functional groups, at the same time as nevertheless giving very fast rate and tremendous yields. A study of polyfunctionalized dihydro-2-oxypyrroles was conducted to calculate the turnover number (TON) and turnover frequency (TOF). Gram-scale cyclization proves that it can be applied to industry in a practical manner.