Small-molecule Fluorophores Research Articles

Fluorescent Flippers: Small-Molecule Probes to Image Membrane Tension in Living Systems.

Flipper probes have been introduced as small molecule fluorophores to image physical forces, that is, membrane tension in living systems. Their emergence over one decade is described, from evolution in design and synthesis to spectroscopic properties. Responsiveness to physical compression in equilibrium at the ground state is identified as the ideal origin of mechanosensitivity to image membrane tension in living cells. A rich collection of flippers is described to deliver and release in any subcellular membrane of interest in a leaflet-specific manner. Chalcogen-bonding cascade switching and dynamic covalent flippers are developed for super-resolution imaging and dual-sensing of membrane compression and hydration. Availability and broad use in the community validate flipper probes as a fine example of the power of translational supramolecular chemistry, moving from fundamental principles to success on the market.

Nerve Tissue Targeted Fluorophore Synthesis: From Scheme to Administration

Nerve tissue damage as a result of surgical injury is a common negative outcome during many surgical interventions. Depending on the site of the injury and the amount of cellular damage, nerve tissue injury may potentially have a life altering effect on the surgical patient. Post-surgical nerve pain is often a chronic problem which persists long after injuries have healed, and can severely infringe upon quality of life for individuals. Surgery that is fluorescence guided will aid in the visualization of nerve structure patterns, and as a result may significantly improve patient outcomes. Small molecule fluorophores may potentially cross the blood brain barrier and blood nerve barrier, allowing unparalleled future application in surgical interventions involving the central and peripheral nervous systems. In this research, work was done to synthesize a small set of fluorescent contrast agents which may have intraoperative application to highlight nerve tissue in real time, with great sensitivity and in high contrast against background tissues. Though work is ongoing, absorption and fluorescence spectra will be observed as a way to evaluate each synthesized agent. Systemic administration of the novel agents in rodents will help define nerve tissue uptake specificity. In order to evaluate nerve signal to background ratios, images of surgical sites will be collected and quantified. The knowledge garnered from this study will greatly help inform nerve tissue specific, clinical grade, contrast agent development.

Design of a Coumarin-Based Fluorescent Probe for Efficient In Vivo Imaging of Amyloid-β Plaques.

Amyloid-β (Aβ) is the core constituent protein of senile plaques, which is one of the key pathological hallmarks of Alzheimer's disease (AD). Here we describe the design, synthesis, and evaluation of coumarin-derived small molecule fluorophores for Aβ imaging. By embedding the aromatic coumarin framework into π bridge of a push-pull chromophore, a novel fluorescence probe XCYC-3 applicable to efficient Aβ recognition was discovered. XCYC-3 displays higher fluorescent enhancement for aggregated Aβ than monomeric Aβ, and possesses good blood-brain barrier permeability. In vitro staining and in vivo imaging studies demonstrated that XCYC-3 could efficiently recognize Aβ plaques in the brain of AD transgenic mice. These results suggest that XCYC-3 is a promising fluorescence imaging agent for Aβ, which might provide important clues for the future development of potent NIR fluorescent probes for Aβ diagnosis.

Modular and genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular target detection.

Genetically encodable bioluminescent sensors have been broadly used for highly sensitive, low-background, low-phototoxicity bioanalysis and imaging in living biological systems. To achieve real-time quantitative measurement and high-throughput screening, powerful bioluminescence resonance energy transfer (BRET) sensors have been designed, especially based on interactions between a luciferase/luciferin donor and a fluorescent protein acceptor. While fluorescent protein-based BRET sensors have been widely applied in cellular and in vivo studies, more modular and easy-to-engineer RNA-based BRET sensors have never been reported. In this project, we report, for the first time, genetically encoded fluorogenic RNA-based BRET sensors for cellular imaging and quantification of different target analytes and RNA-protein interactions. Fluorogenic RNAs are single-stranded RNA aptamers that can bind specific small-molecule fluorophores and activate the fluorescence signals. By coupling a NanoLuc luciferase donor with different types of fluorogenic RNA acceptor, without reliance on external light, sensitive BRET signals are generated in response to target metabolite, signaling molecule, or RNA levels inside living cells. We envision that this novel genetically encoded RNA-based BRET system can be widely used in bioanalysis and nanomedicine.

Direct optical resolution in the intra-molecular distance range.

Estimations of molecular distances <10 nm have long been the almost exclusive domain of Förster resonance energy transfer (FRET) and related spectroscopic techniques. By site-specifically labeling molecular subunits with (at least) two fluorophores, single-molecule FRET has also allowed for the monitoring of distances within macromolecules. In this presentation, we report on the direct determination of such intra-molecular distances based on the MINFLUX localization concept. We measured intra-macromolecular spacings of photoactivatable small-molecule fluorophores in well-characterized uni- or few-molecular systems, with simple polypeptides and proteins serving as initial examples. Our experiments indicate that the widely employed STORM-type dark-state photoblinking of cyanines reaches an impasse for dye separations below ∼6-10 nm, as the dyes couple and no longer behave independently. The complex photophysics of such dye-dye communication impedes the reliable, isolated detection of their signals. As we show however, sequential photoactivation - providing the required off-on-off transitions in a more orthogonal way - is a viable strategy to read out dye coordinates even for separations as low as few nm, beyond any perceived remaining barriers of resolution. Our results, with direct resolution of intra-molecular distances and conformations, herald the possibility of fluorescence-based analyses of structural biology at room or physiological temperature relying on direct positional measurements rather than on indirect FRET readouts.

Genetic code expansion to enable site-specific bioorthogonal labeling of functional G protein-coupled receptors in live cells.

For use in site-specific bioorthogonal labeling of expressed G protein-coupled receptors (GPCRs) in live cells, we developed a luciferase-based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side-by-side the efficiency of incorporation of three different noncanonical amino acids [4-azido-l-phenylalanine (azF), cyclopropene-l-lysine (CpK), and trans-cyclooct-2-en-l-lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope-tagged C-C chemokine receptor 5 (CCR5)-RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell-based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small-molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine-containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine-containing compounds using the inverse electron demand Diels-Alder ligation was overall slightly more efficient than other reactions tested.

Smart probes for optical imaging of T cells and screening of anti-cancer immunotherapies.

T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes for T cells and anti-cancer immunotherapies.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small-molecule Fluorophores.

Messenger RNA (mRNA) is the fundamental information transfer system in the cell. Tracking single mRNA from transcription to degradation with fluorescent probes provides spatiotemporal information in cells about how the genetic information is transferred from DNA to proteins. The traditional single mRNA imaging approach utilizes RNA hairpins (e.g. MS2) and tethered fluorescent protein as probes. As an exciting alternative, RNA aptamers: small-molecule fluorophores (SFs) systems have emerged as novel single mRNA imaging probes since 2019, exhibiting several advantages including fluorogenic ability and minimal perturbation. This review summarizes all five reported RNA aptamers: SFs systems for single mRNA imaging in living cells so far. It also discusses the challenges and provides prospects for single mRNA imaging applications. This review is expected to inspire researchers to develop RNA aptamers: SFs systems for studying gene expression at single-molecule resolution in cells.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

AbstractMessenger RNA (mRNA) is the fundamental information transfer system in the cell. Tracking single mRNA from transcription to degradation with fluorescent probes provides spatiotemporal information in cells about how the genetic information is transferred from DNA to proteins. The traditional single mRNA imaging approach utilizes RNA hairpins (e.g. MS2) and tethered fluorescent protein as probes. As an exciting alternative, RNA aptamers: small‐molecule fluorophores (SFs) systems have emerged as novel single mRNA imaging probes since 2019, exhibiting several advantages including fluorogenic ability and minimal perturbation. This review summarizes all five reported RNA aptamers: SFs systems for single mRNA imaging in living cells so far. It also discusses the challenges and provides prospects for single mRNA imaging applications. This review is expected to inspire researchers to develop RNA aptamers: SFs systems for studying gene expression at single‐molecule resolution in cells.

Synthesis Processes, Photoluminescence Mechanism, and the Toxicity of Amorphous or Polymeric Carbon Dots.

Fluorescence is the emission of light following photon absorption. This optical phenomenon has many applications in daily life, such as in LED lamps, forensics, and bioimaging. Traditionally, small-molecule fluorophores were most common, but the types of molecules and particles with compelling fluorescence properties have expanded. For example, green fluorescent protein (GFP) was isolated from jellyfish and won the Nobel prize in 2008 due to its significant utility as a fluorescent biomarker. Using the intrinsic fluorescence of GFP, many previously invisible biological processes and substances can now be observed and studied. Other fluorescent materials have also been developed, greatly expanding the potential applications. Semiconductor quantum dots (QDs), which have bright fluorescence and a narrow bandwidth, are a popular choice for display technologies. However, QDs are made of heavy metal elements such as Cd and Se, which pose potential safety concerns to the environment and human health. Thus, new fluorescent organic materials are being developed to mitigate the toxicological concerns while maintaining the QD advantages.One type of new material attracting great attention as an environmentally friendly substitute for semiconductor QDs is carbon dots (CDs). CDs have been developed with strong fluorescence, good photostability, and low toxicity using a variety of precursors, and some synthesis processes have good potential for scale-up. However, since they are made of a variety of materials and through different methods, the structure and properties of CDs can differ from preparation to preparation. There are three major types of CDs: graphene quantum dots (GQDs), carbon quantum dots (CQDs), and amorphous or polymeric carbon dots (PCDs). This Account focuses on PCDs and their unique properties by comparing it with other types of CDs. The synthesis processes, fluorescence properties, fluorescence mechanisms, and toxicity are discussed below with an emphasis on the distinct attributes of PCDs.PCDs can be synthesized from small molecules or polymers. They have an amorphous or cross-linked polymer structure with bright fluorescence. This fluorescence is possibly due to cross-link-enhanced emission or clusteroluminescence that arises from the through-space interactions of heteroatomic-rich functional groups. Other fluorescence mechanisms of CDs, including distinct contributions from the carbon core and surface states, may also contribute. The toxicological profiles of CDs are influenced by the chemical composition, surface functionalization, and light illumination. CDs are generally thought to be of low toxicity, and this can be further improved by removing toxic byproducts, functionalizing the surface, and reducing light exposure to minimize the generation of reactive oxygen species.

Bright V-Shaped bis-Imidazo[1,2-a]pyridine Fluorophores with Near-UV to Deep-Blue Emission.

Ten novel small-molecule fluorophores containing two electron-accepting imidazo[1,2-a]pyridine (ImPy) units are presented. Each ImPy core is functionalized at its C6 position with groups featuring either electron accepting (A) or donating (D) properties, thus providing emitters with general structure X-ImPy-Y-ImPy-X (X=either A or D; Y=phenyl or pyridine). The molecules bear either a phenyl (series 4) or a pyridine (series 5) π bridge that connects the two ImPys via meta (phenyl) or 2,6- (pyridine) positions, yielding an overall V-shaped architecture. The final compounds are synthetized straightforwardly by condensation between substituted 2-aminopyridines and α-halocarbonyl derivatives. All the compounds display intense photoluminescence with quantum yield (PLQY) in the range of 0.17-0.51. Remarkably, substituent effect enables tuning the emission from near-UV to (deep-)blue region while keeping Commission Internationale de l'Éclairage (CIE) y coordinate ≤0.07. The emitting excited state is characterized by a few nanoseconds lifetime and high radiative rate constant, and its nature is modulated from pure π-π* to intramolecular charge transfer (ICT) by the electronic properties of the peripheral X substituent. This is further corroborated by the nature of the frontier orbitals and vertical electronic excitations computed at (time-dependent) density functional level of theory (TD-)DFT. Finally, this study enlarges the palette of bright deep-blue emitters based on the interesting ImPy scaffolds in view of their potential application as photo-functional materials in optoelectronics.

Clostridium botulinum C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages.

The protein toxin C3bot from Clostridium botulinum is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q for covalent linkage of cargos via thiol–maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.

Directed Evolution of Fluorescent Proteins in Bacteria.

Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy.

NIR-II bioimaging of small molecule fluorophores: From basic research to clinical applications

Due to the low autofluorescence and deep-photo penetration, the second near-infrared region fluorescence imaging technology (NIR-II, 1000–2000 nm) has been widely utilized in basic scientific research and preclinical practice throughout the past decade. The most attractive candidates for clinical translation are organic NIR-II fluorophores with a small-molecule framework, owing to their low toxicity, high synthetic repeatability, and simplicity of chemical modification. In order to enhance the translation of small molecule applications in NIR-II bioimaging, NIR-II fluorescence imaging technology has evolved from its usage in cells to the diagnosis of diseases in large animals and even humans. Although several examples of NIR-II fluorescence imaging have been used in preclinical studies, there are still many challenges that need to be addressed before they can finally be used in clinical settings. In this paper, we reviewed the evolution of the chemical structures and photophysical properties of small-molecule fluorophores, with an emphasis on their biomedical applications ranging from small animals to humans. We also explored the potential of small-molecule fluorophores.

J-aggregates albumin-based NIR-II fluorescent dye nanoparticles for cancer phototheranostics.

Phototheranostics, relying on energy conversions of fluorophores upon excitation, integrating diagnostic fluorescence imaging and photo-driven therapy, represents a promising strategy for cancer precision medicine. Compared with the first near-infrared biological window (NIR-I), fluorophores imaged in the second window (NIR-II, 1000–1700 ​nm) exhibit a higher temporal and spatial resolution and tissue penetration depth. Polymethine cyanine-based dye IR1061 is a typical NIR-II small-molecule organic fluorophore, but its low water solubility and short circulation time limiting its biological applications. Therefore, human serum albumin (HSA) nanoparticles with great biocompatibility and biosafety were employed to fabricate hydrophobic IR1061, which exhibited red-shifted absorption band as typical for J-aggregates. Moreover, IR1061@HSA nanoparticles can be successfully used for NIR-II imaging to noninvasively visualize the tumor vascular networks, as well as real-time intraoperative image-guided tumor resection. Interestingly, benefiting from the high photothermal conversion efficiency brought by J-aggregates, IR1061@HSA nanoparticles were also explored for photothermal therapy (PTT) and cause efficient thermal ablation of tumors. Overall, IR1061@HSA, as a novel J-aggregates albumin-based NIR II dye nanoparticle with high biocompatibility, provides an integrated versatile platform for cancer phototheranostics with promising clinical translation prospects.

One-Pot Reductive Methylation of Nitro- and Amino-Substituted (Hetero)Aromatics with DMSO/HCOOH: Concise Synthesis of Fluorescent Dimethylamino-Functionalized Bibenzothiazole Ligands with Tunable Emission Color upon Complexation.

One-pot reductive N,N-dimethylation of suitable nitro- and amino-substituted (hetero)arenes can be achieved using a DMSO/HCOOH/Et3N system acting as a low-cost but efficient reducing and methylating agent. The transformation of heteroaryl-amines can be accelerated by using dimethyl sulfoxide/oxalyl chloride or chloromethyl methyl sulfide as the source of active CH3SCH2+ species, while the exclusion of HCOOH in the initial stage of the reaction allows avoiding N-formamides as resting intermediates. The developed procedures are applicable in multigram-scale synthesis, and because of the lower electrophilicity of CH3SCH2+, they also work in pathological cases, where common methylating agents provide N,N-dimethylated products in no yield or inferior yields due to concomitant side reactions. The method is particularly useful in one-pot reductive transformation of 2-H-nitrobenzazoles to corresponding N,N-dimethylamino-substituted heteroarenes. These, upon Cu(II)-catalyzed oxidative homocoupling, afford 2,2'-bibenzazoles substituted with dimethylamino groups as charge-transfer N^N ligands with intensive absorption/emission in the visible region. The fluorescence of NMe2-functionalized bibenzothiazoles remains intensive even upon complexation with ZnCl2, while emission maxima are bathochromically shifted from the green/yellow to orange/red spectral region, making these small-molecule fluorophores, exhibiting large emission quantum yields and Stokes shifts, an attractive platform for the construction of various functional dyes and light-harvesting materials with tunable emission color upon complexation.

Photostability investigation of a near-infrared-II heptamethine cyanine dye

The development of small-molecule organic fluorophores in the second near-infrared region (NIR-II, 1000–1700 nm) has recently received immense attention due to deep tissue light penetration and reduced autofluorescence. Polymethine cyanine dyes as important building blocks are frequently used to design NIR-II light-activated fluorophores. However, applications of these fluorophores were restricted by their poor photostability. Herein, we synthesized a NIR-II heptamethine cyanine dye 4 as the model compound to investigate its photostability and identify its photoproducts. Compound 4 shows improved photostability compared to ICG, and remains relatively stable even under sunlight irradiation. The cyanine scaffold’s good photostability promoted its stable photothermal properties. The effective photothermal activity of cyanine dye 4 and the low toxicity of its one major photoproduct demonstrated its potential for further photo-therapeutic applications. This work would pave the way for the development of NIR-II cyanines with photostability through delicate structure design.

Synthesis, spectral analysis, and theoretical investigations of diphenyl fumarate

Diphenyl fumarate is synthesized from maleic anhydride and phenol under solvent-free conditions. The structure of the product is characterized by FTIR, 1H NMR, and 13C NMR. The molecular structure is optimized using the density functional theory approach at the B3LYP/6-311G(d,p) level and the structural parameters are analyzed. Theoretical vibrational frequencies are investigated at the same level; the 1H NMR and 13C NMR are simulated using the default gauge-independent atomic orbital method at the B3LYP/6-31G(d) level and the solvation model density implicit continuum solvation model for chloroform; chemical shifts are scaled using scaling factors available on the cheshirenmr.info website (accessed 12 September 2021). In addition, the UV-Vis absorption and fluorescence emission are simulated using the time-dependent method at the PBE38/6-311G(d,p) level. All the calculated spectral values are compared with the experimental data. A generally good agreement between the experimental and theoretical results is found. The results reveal diphenyl fumarate as a candidate substrate for small-molecule organic fluorophores with a large Stokes shift. This work provides a detailed experimental and theoretical study on the structural and spectral properties of diphenyl fumarate, and thus provides proof for the novel applications of diphenyl fumarate and its derivatives.

Prediction of Maximum Absorption Wavelength Using Deep Neural Networks.

Fluorescent molecules are important tools in biological detection, and numerous efforts have been made to develop compounds to meet the desired photophysical properties. For example, tuning the wavelength allows an appropriate penetration depth with minimal interference from the autofluorescence/scattering for a better signal-to-noise contrast. However, there are limited guidelines to rationally design or computationally predict the optical properties from first principles, and factors like the solvent effects will make it more complicated. Herein, we established a database (SMFluo1) of 1181 solvated small-molecule fluorophores covering the ultraviolet-visible-near-infrared absorption window and developed new machine learning models based on deep neural networks for accurately predicting photophysical parameters. The optimal system was applied to 120 out-of-sample compounds, and it exhibited remarkable accuracy with a mean relative error of 1.52%. In this new paradigm, a deep learning algorithm is promising to complement conventional theoretical and experimental studies of fluorophores and to greatly accelerate the discovery of new dyes. Due to its simplicity and efficiency, data from newly developed fluorophores can be easily supplemented to this system to further improve the accuracy across various dye families.

Epidermal growth factor-targeted fluorescence is unaffected by standard neoadjuvant therapies in human sarcomas.

Curative surgery for other many cancers requires that the tumor be removed with a zone of normal tissue surrounding the tumor with 'negative' margins. Sarcomas, cancers of the bones, muscles, and fat, require WLE for cure. Unfortunately, 'positive' margins occur in 20-25% of sarcoma surgeries, associated with cancer recurrence and reduced survival. Our group successfully tested a small-molecule fluorophore (ABY-029) in sarcomas that targets the epidermal growth factor receptor. We sought to evaluate human sarcoma xenografts for epidermal growth factor receptor expression and binding of ABY-029 with and without exposure to standard presurgical chemotherapy and radiation. We inoculated groups of 24 NSG mice with five cell lines (120 mice total). Eight mice from each cell line received: 1) radiation alone; 2) chemotherapy alone; or 3) chemotherapy and radiation. We administered ABY-029 2-4 hours before surgery. Tumor and biopsy portions of background tissues were removed. All tissues were imaged on a LI-COR Odyssey and processed in pathology. There were no significant reductions in epidermal growth factor receptor expression or in ABY-029-mediated fluorescence in tumors exposed to chemotherapy, radiation, or both. fluorescence-guided surgery demonstrates strong promise to improve curative surgical cancer care, particularly for sarcomas where the positive margin rate is substantial. Fluorophore performance must be evaluated under circumstances that duplicate accurately the biological milieu relevant to a particular cancer. This work shows that human sarcoma xenografts subjected to standard therapies do not demonstrate a change in epidermal growth factor receptor expression or in epidermal growth factor receptor-targeted fluorescence, thereby indicating that epidermal growth factor receptor-targeted fluorescence-guided surgery should be feasible under normal therapeutic conditions in the clinic.