Fluorescent Nucleoside Research Articles

Chemical Capture and Sensing of Bovine Serum Albumin with Phenothiazinylcarbaldehyde-Labeled 2′-Deoxyuridine

AbstractChemical-capture-mediated sensing has had a great impact on proteomic research. Toward this end, we demonstrate the chemical trapping of BSA by the reactive formyl functionality of a newly developed fluorescent nucleoside probe, formylphenothiazine-labeled-2′-deoxyuridine. The probe is capable of trapping BSA via Schiff base formation leading to fluorescence ‘switch-on’ sensing with a large hypsochromic shift of ca. 100 nm. The α-amylase does not show any significant change in fluorescence response, demonstrating the efficiency of the probe in selective sensing of BSA. Docking studies suggest the preferential interaction of the phenothiazinylcarbaldehyde-labeled dU with the residual amino acids in site I of the BSA protein as compared to site II.

Synthesis of base-modified fluorescent furo[3,2-c]coumarin nucleosides and their photophysical studies

A small library of fluorescent furo[3,2-c]coumarin nucleoside analogues has been successfully synthesized via a one-pot condensation reaction of 3′,5′-di-O-acetyl-5-formyl-2′-deoxyuridine, alkyl isocyanides, and differently substituted 4-hydroxycoumarins in acetonitrile at 80 °C. Further, the synthesized compounds have been characterized by IR, 1H NMR, 13C NMR, 1H–1H COSY, 1H–13C HETCOR, 2D NOESY NMR experiments, HRMS measurements and single crystal X-ray structure analysis of compound 5-(2′'-N-(cyclohexyl)-amino-4′'H-furo[3,2-c]chromen-4-one-3′'-yl)-2′-deoxyuridine. The electronic structure of these modified nucleosides has been examined by Density Functional Theory (DFT). The synthesized nucleoside analogues exhibited a prominent emission spectrum, featuring a band around 500 nm (with excitation at 380 nm). Photophysical investigations revealed a noteworthy fluorescence intensity, along with excellent Stokes shift values (54–194 nm) and higher quantum yields (0.010–0.515) in comparison to other pyrimidine-based fluorescent nucleosides. The solvatochromic studies of 5-(2′'-N-(cyclohexyl)-amino-8′'‑chloro-4′'H-furo[3,2-c]chromen-4-one-3′'-yl)-2′-deoxyuridine determined the quantum yield (ΦF) to be 0.515 in DMSO (λabs = 388 nm). This emphasizes the potential utility of these modified nucleosides in probing the local structure and dynamics of nucleic acids. Moreover, the scalability of the synthesis protocol was effectively demonstrated by producing one of the compounds on a gram scale, exhibiting its practical viability for large-scale production.

Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues.

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e.it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.

Chemo-Enzymatic Generation of Highly Fluorescent Nucleoside Analogs Using Purine-Nucleoside Phosphorylase

Chemo-enzymatic syntheses of strongly fluorescent nucleoside analogs, potentially applicable in analytical biochemistry and cell biology are reviewed. The syntheses and properties of fluorescent ribofuranosides of several purine, 8-azapurine, and etheno-purine derivatives, obtained using various types of purine nucleoside phosphorylase (PNP) as catalysts, as well as α-ribose-1-phosphate (r1P) as a second substrate, are described. In several instances, the ribosylation sites are different to the canonical purine N9. Some of the obtained ribosides show fluorescence yields close to 100%. Possible applications of the new analogs include assays of PNP, nucleoside hydrolases, and other enzyme activities both in vitro and within living cells using fluorescence microscopy.

1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.

BN-Phenanthrene- and BN-Pyrene-Based Fluorescent Uridine Analogues.

Two unprecedented fluorescent nucleosides that feature BN-doped polycyclic aromatic hydrocarbons are presented. One of them, having a BN-modified phenanthrene moiety incorporated, shows blue fluorescence but suffers from poor stability under aqueous conditions. The other nucleoside comprises an internally BN-doped pyrene as the chromophore. It shows green fluorescence in various solvents and is stable under aqueous and alkaline conditions.

Synthesis, characterization and photophysical studies of dual-emissive base-modified fluorescent nucleosides.

A straightforward and efficient methodology has been employed for the synthesis of a diverse set of base-modified fluorescent nucleoside conjugates via Cu(I)-catalysed cycloaddition reaction of 5-ethynyl-2',3',5'-tri-O-acetyluridine/3',5'-di-O-acetyl-2'-deoxyuridine with 4-(azidomethyl)-N9-(4'-aryl)-9,10-dihydro-2H,8H-chromeno[8,7-e][1,3]oxazin-2-ones in tBuOH to afford the desired 1,2,3-triazoles in 92-95% yields. Treatment with NaOMe/MeOH resulted in the final deprotected nucleoside analogues. The synthesized 1,2,3-triazoles demonstrated a significant emission spectrum, featuring two robust bands in the region from 350-500 nm (with excitation at 300 nm) in fluorescence studies. Photophysical investigations revealed a dual-emissive band with high fluorescence intensity, excellent Stokes shift (140-164 nm) and superior quantum yields (0.068-0.350). Furthermore, the electronic structures of the synthesized triazoles have been further verified by DFT studies. Structural characterization of all synthesized compounds was carried out using various analytical techniques, including IR, 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C HETCOR experiments, and HRMS measurements. The dual-emissive nature of these nucleosides would be a significant contribution to nucleoside chemistry as there are limited literature reports on the same.

Fluorescent 2′-Deoxyuridine (dU) Analogue: Tropolonyl triazolyl-dU (tt-dU) Exhibits Solvatochromism/HeLa Cell Internalization and Its Triphosphate (tt-dUTP) Is Incorporated into DNA Enzymatically

AbstractThis era has witnessed the development and extensive application of modified nucleosides, including fluorescent nucleosides that clinically served humankind. Most fluorescent nucleoside analogues are derived from benzenoid aromatic scaffolds. However, the non-benzenoid aromatic moiety, tropolone, which exhibits unique hydrogen bonding and metal chelating properties, also occurs in nature. Recently, we introduced the tropolone unit at deoxyuridine through an ethyne linker and prepared its DNA analogues, which are fluorescent. This report describes the synthesis of a new troponyl triazolyl-dU (tt-dU) analogue, possessing a triazolyl linker, through click chemistry. tt-dU exhibits fluorescence with solvatochromism and enters into Hela cells without any cytotoxicity. Its triphosphate (tt-dUTP) was also synthesized and incorporated enzymatically into DNA, as shown in primer extension experiments. The unique photophysical properties and metal-chelating ability of the tropolone group make tt-dU a promising modified nucleoside.

4-Isocyanoindole-2ʹ-deoxyribonucleoside (4ICIN): An isomorphic indole nucleoside suitable for inverse electron demand Diels-Alder reactions

Isomorphic nucleosides are powerful tool compounds for interrogating a variety of biological processes involving nucleosides and nucleic acids. We previously reported a fluorescent isomorphic indole nucleoside called 4CIN. A distinguishing molecular feature of 4CIN is the presence of a 4-cyano moiety on the indole that functions as the nucleobase. Given the known chemical reactivity of isonitriles with tetrazines through [4+1]-cycloaddition chemistry, we investigated whether conversion of 4CIN to the corresponding isonitrile would confer a useful chemical probe. Here we report the synthesis of 4-isocyanoindole-2ʹ-deoxyribonucleoside (4ICIN) and the propensity of 4ICIN to undergo inverse electron demand Diels-Alder cycloaddition with a model tetrazine.

Photophysical characterization of isothiazologuanosine, a unique isomorphic and isofunctional fluorescent analogue of guanosine

Application of fluorescence techniques to investigate molecular interactions with nucleic acids is complicated by their poor emission, making the substitution of natural nucleobases by fluorescent nucleoside analogues (FNAs) a useful strategy. A breakthrough in fluorescent nucleoside analogues has been the development of thienoguanosine (thG) and isothiazologuanosine (tzG), two isosteric mimics of guanosine (G). Due to its N7 atom needed in Hoogsteen base pairs and enzyme recognition, tzG is also an isofunctional G surrogate. Herein, we integrated fluorescence spectroscopy measurements with quantum mechanical (QM) calculations to characterize the mechanisms underlying tzG photophysics in different solvents. In dioxane and ethyl acetate, tzG existed primarily as a H1 keto-amino tautomer with short fluorescence lifetime (τ ∼ 2 ns) and low quantum yield (ϕ ∼ 0.10). In buffer, the H1 tautomer (ϕ = 0.36, τ = 8.84 ns) coexisted with a weakly emissive H3 keto-amino tautomer. The two tautomers were also observed in methanol, but with a 30% decrease in ϕ and τ values for the major H1 tautomer. QM calculations suggested that the main non-radiative pathway of tzG-H1 involves NS bond loosening and is responsible for the more solvent-sensitive ϕ and τ values compared to thG. This pathway is much more efficient for tzG-H3, for which an additional pathway to a dark nπ* state and a large coupling with triplet states further explain its very low emission. This study lays the ground for rationally using tzG as a sensitive FNA.

Acyclic 2-Ethynylnaphthalene-Modified 8-Aza-3,7-dideaza-2′-deoxyadenosine Allows Thymine Discrimination by Probing the DNA Minor Groove Environment

AbstractTwo novel acyclic environment-sensitive fluorescent nucleosides named ac37zA and an37zA are synthesized and their photophysical properties are investigated. Both ac37zA and an37zA exhibit dual fluorescence emission depending upon molecular coplanarity. Oligodeoxynucleotide probes containing ac37zA clearly discriminate perfectly matched thymine in target DNA strands by strong charge transfer (CT) emission in the longer wavelength region and by strong quenching of emission from the twisted conformation in the mismatched case.

Rationalizing the environment-dependent photophysical behavior of a DNA luminescent probe by classical and non-adiabatic molecular dynamics simulations.

Environment-sensitive fluorescent nucleoside analogs are of utmost importance to investigate the structure of nucleic acids, their intrinsic flexibility, and sequence-specific DNA- and RNA-binding proteins. The latter play indeed a key role in transcription, translation as well as in the regulation of RNA stability, localization and turnover, and many other cellular processes. The sensitivity of the embedded fluorophore to polarity, hydration, and base stacking is clearly dependent on the specific excited-state relaxation mechanism and can be rationalized combining experimental and computational techniques. In this work, we elucidate the mechanisms leading to the population of the triplet state manifold for a versatile nucleobase surrogate, namely the 2-thienyl-3-hydroxychromone in gas phase, owing to non-adiabatic molecular dynamics simulations. Furthermore, we analyze its behavior in the B-DNA environment via classical molecular dynamics simulations, which evidence a rapid extrusion of the adenine facing the 2-thienyl-3-hydroxychromone nucleobase surrogate. Our simulations provide new insights into the dynamics of this family of chromophores, which could give rise to an integrated view and a fine tuning of their photochemistry, and namely the role of excited-state intramolecular proton transfer for the rational design of the next generation of fluorescent nucleoside analogs.

Thiophene-Extended Fluorescent Nucleosides as Molecular Rotor-Type Fluorogenic Sensors for Biomolecular Interactions.

Fluorescent molecular rotors are versatile tools for the investigation of biomolecular interactions and the monitoring of microenvironmental changes in biological systems. They can transform invisible information into a fluorescence signal as a straightforward response. Their utility is synergistically amplified when they are merged with biomolecules. Despite the tremendous significance and superior programmability of nucleic acids, there are very few reports on the development of molecular rotor-type isomorphic nucleosides. Here, we report the synthesis and characterization of a highly emissive molecular rotor-containing thymine nucleoside (ThexT) and its 2'-O-methyluridine analogue (2'-OMe-ThexU) as fluorogenic microenvironment-sensitive sensors that emit vivid fluorescence via an interaction with the target proteins. ThexT and 2'-OMe-ThexU may potentially serve as robust probes for a broad range of applications, such as fluorescence mapping, to monitor viscosity changes and specific protein-binding interactions in biological systems.

Tropolone-Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization.

Tropolone is a non-benzenoid aromatic scaffold with unique photophysical and metal-chelating properties. Recently, it has been conjugated with DNA, and the photophysical properties of this conjugate have been explored. Tropolonyl-deoxyuridine (tr-dU) is a synthetic fluorescent DNA nucleoside analogue that exhibits pH-dependent emissions. However, its solvent-dependent fluorescence properties are unexplored owing to its poor solubility in most organic solvents. It would be interesting to incorporate it into DNA primer enzymatically. This report describes the solvent-dependent fluorescence properties of the silyl-derivative, and enzymatic incorporation of its triphosphate analogue. For practical use, its cell-internalization and cytotoxicity are also explored. tr-dU nucleoside was found to be a potential analogue to design DNA probes and can be explored for various therapeutic applications in the future.

Synthesis and photophysical characterization of fluorescent indole nucleoside analogues.

Fluorescent nucleosides are useful chemical tools for biochemical research and are frequently incorporated into nucleic acids for a variety of applications. The most widely utilized fluorescent nucleoside is 2-aminopurine-2'-deoxyribonucleoside (2APN). However, 2APN is limited by a moderate Stokes shift, molar extinction coefficient, and quantum yield. We recently reported 4-cyanoindole-2'-deoxyribonucleoside (4CIN), which offers superior photophysical characteristics in comparison to 2APN. To further improve upon 4CIN, a focused library of additional analogues combining the structural features of 2APN and 4CIN were synthesized and their photophysical properties were quantified. Nucleosides 2-6 were found to possess diverse photophysical properties with some features superior to 4CIN. In addition, the structure-function relationship data gained from 1-6 can inform the design of next-generation fluorescent indole nucleosides.

A tolane-modified 5-ethynyluridine as a universal and fluorogenic photochemical DNA crosslinker.

We report the fluorescent nucleoside ToldU and its application as a photoresponsive crosslinker in three different DNA architectures with enhanced fluorescence emission of the crosslinked products. The fluorogenic ToldU crosslinking reaction enables the assembly of DNA polymers in a hybridization chain reaction for the concentration-dependent detection of a specific DNA sequence.

Fluorescent inosine analogues: Synthesis, cytotoxicity activity and self-assembly nanoparticle for live cell image

The fluorescence is of importance for the investigation of pharmacokinetic of nanomedicines. The fluorescent DOI nanoparticles was obtained by self-assembly of amphiphilic 2′,5′-dioleoyl 8-(4-(p-ethoxyphenylethynyl) phenyl)-inosine 5 (DOI) which was synthesized by coupling two oleic acid chains to the sugar moiety of fluorescent inosine analogue synthesized by Suzuki cross-coupling reaction. IC50 value on A549 cells of DOI nanoparticles was 10.99 μg/mL which was much lower than that of free nucleoside 4e (31.16 μg/mL) owing to improving in vitro cell uptake, which was examined via autofluorescence of nucleosides 4e and DOI nanoparticle in live cells. This indicates that the bright fluorescent nucleosides can be used for live cell image and has the potential for investigating pharmacokinetic characteristics of nucleoside nanomedicines.

Probing of Fluorogenic RNA Aptamers via Supramolecular Förster Resonance Energy Transfer with a Universal Fluorescent Nucleobase Analog.

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI+-binding RNA aptamer Chili. This method yields structural restraints that bridge the gap between traditional low-resolution secondary structure probing methods and more elaborate high-resolution methods such as X-ray crystallography and NMR spectroscopy.

Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems; however, approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Furthermore, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

Environmentally sensitive fluorescent nucleoside analogues as probes for nucleic acid – protein interactions: molecular design and biosensing applications

Fluorescent nucleoside analogues (FNAs) are indispensable in studying the interactions of nucleic acids with nucleic acid-binding proteins. By replacing one of the poorly emissive natural nucleosides, FNAs enable real-time optical monitoring of the binding interactions in solutions, under physiologically relevant conditions, with high sensitivity. Besides that, FNAs are widely used to probe conformational dynamics of biomolecular complexes using time-resolved fluorescence methods. Because of that, FNAs are tools of high utility for fundamental biological research, with potential applications in molecular diagnostics and drug discovery. Here I review the structural and physical factors that can be used for the conversion of the molecular binding events into a detectable fluorescence output. Typical environmentally sensitive FNAs, their properties and applications, and future challenges in the field are discussed.