Imidazo[1,5-a]pyridine-Based Fluorescent Probes: A Photophysical Investigation in Liposome Models.

Imidazo[1,5-a]pyridine is a stable scaffold, widely used for the development of emissive compounds in many application fields (e.g., optoelectronics, coordination chemistry, sensors, chemical biology). Their compact shape along with remarkable photophysical properties make them suitable candidates as cell membrane probes. The study of the membrane dynamics, hydration, and fluidity is of importance to monitor the cellular health and to explore crucial biochemical pathways. In this context, five imidazo[1,5-a]pyridine-based fluorophores were synthesized according to a one-pot cyclization between an aromatic ketone and benzaldehyde in the presence of ammonium acetate and acetic acid. The photophysical features of prepared compounds were investigated in several organic solvents and probes 2-4 exhibited the greatest solvatochromic behavior, resulting in a higher suitability as membrane probes. Their interaction with liposomes as artificial membrane model was tested showing a successful intercalation of the probes in the lipid bilayer. Kinetic experiments were carried out and the lipidic phase influence on the photophysical features was evaluated through temperature-dependent experiments. The results herein reported encourage further investigations on the use of imidazo[1,5-a]pyridine scaffold as fluorescent membrane probes.

Dye-Conjugated Spinach RNA by Genetic Alphabet Expansion.

Light-emitting systems using an RNA aptamer-dye pair, such as Spinach RNA, are an attractive method for imaging and tracing RNA expression in vitro and in vivo. We present an alternative Spinach method by genetic alphabet expansion using an unnatural base pair system, in which a dye-conjugated unnatural base substrate is site-specifically incorporated at a specific position in Spinach RNA by transcription involving the third base pair. The incorporation position was predicted by molecular dynamics simulations. This dye-conjugated Spinach RNA increased the thermal stability of the fluorescence, the robustness against ion sensitivity, and the resistance against photobleaching. Furthermore, we applied our method to Baby Spinach, a shorter version of Spinach, for dye conjugation toward the visible detection of transcripts. This is the first demonstration of an alternative RNA imaging method for a detection system using genetic alphabet expansion.

Behavior of Ternary Mixtures of Hydrogen Bond Acceptors and Donors in Terms of Band Gap Energies.

Three ternary mixtures composed by choline chloride (ChCl), ethylene glycol (EG), and a second hydrogen bond donor (HBD) as ethanol (A), 2-propanol (B), and glycerol (C) were studied in terms of composition related to the band gap energy (BGE). A Design of Experiments (DoE) approach, and in particular a Simple Lattice three-components design, was employed for determining the variation of the BGE upon the composition of each system. UV-VIS analysis and subsequent Tauc plot methodology provided the data requested from the DoE, and multivariate statistical analysis revealed a drop of the BGE in correspondence to specific binary compositions for systems A and B. In particular, a BGE of 3.85 eV was registered for the mixtures ChCl/EtOH (1:1) and ChCl/2-propanol (1:1), which represents one of the lowest values ever observed for these systems.

Real-Time Monitoring of Human Guanine Deaminase Activity by an Emissive Guanine Analog.

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (thN) and isothiazole[4,3-d]pyrimidine (tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, thGN, the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, tzXN. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

Optimization of Nazarov Cyclization of 2,4-Dimethyl-1,5-diphenylpenta-1,4-dien-3-one in Deep Eutectic Solvents by a Design of Experiments Approach.

The unprecedented Nazarov cyclization of a model divinyl ketone using phosphonium-based Deep Eutectic Solvents as sustainable non-innocent reaction media is described. A two-level full factorial Design of Experiments was conducted for elucidating the effect of the components of the eutectic mixture and optimizing the reaction conditions in terms of temperature, time, and substrate concentration. In the presence of the Deep Eutectic Solvent (DES) triphenylmethylphosphonium bromide/ethylene glycol, it was possible to convert more than 80% of the 2,4-dimethyl-1,5-diphenylpenta-1,4-dien-3-one, with a specific conversion, into the cyclopentenone Nazarov derivative of 62% (16 h, 60 °C). For the reactions conducted in the DES triphenylmethylphosphonium bromide/acetic acid, quantitative conversions were obtained with percentages of the Nazarov product above 95% even at 25 °C. Surface Responding Analysis of the optimized data furnished a useful tool to determine the best operating conditions leading to quantitative conversion of the starting material, with complete suppression of undesired side-reactions, high yields and selectivity. After optimization, it was possible to convert more than 90% of the model substrate into the desired cyclopentenone with cis percentages up to 77%. Experimental validation of the implemented model confirmed the robustness and the suitability of the procedure, leading to possible further extension to this specific combination of experimental designs to other substrates or even to other synthetic processes of industrial interest.

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.

Enzymatic Syntheses and Applications of Fluorescent Cyclic Dinucleotides.

Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non-emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase-mediated synthesis and phosphodiesterase (PDE)-mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.

Fluorescing Isofunctional Ribonucleosides: Assessing Adenosine Deaminase Activity and Inhibition.

The enzymatic conversion of isothiazolo[4,3-d]pyrimidine-based adenosine (tz A) and 2-aminoadenosine (tz 2-AA) analogues to the corresponding isothiazolo[4,3-d]pyrimidine-based inosine (tz I) and guanosine (tz G) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri-Michaelis-Menten analyses provides the foundation for a high-throughput screening assay, and the efficacy of the assay is showcased by fluorescence-based analysis of tz A conversion to tz I in the presence of known and newly synthesized inhibitors.

Off-line and real-time monitoring of acetaminophen photodegradation by an electrochemical sensor.

The photochemistry of N-acetyl-para-aminophenol (acetaminophen, APAP) is here investigated by using differential pulse voltammetry (DPV) analysis to monitor APAP photodegradation upon steady-state irradiation. The purpose of this work is to assess the applicability of DPV to monitor the photochemical behaviour of xenobiotics, along with the development of an electrochemical set-up for the real-time monitoring of APAP photodegradation. We here investigated the APAP photoreactivity towards the main photogenerated reactive transients species occurring in sunlit surface waters (hydroxyl radical HO, carbonate radical CO3-, excited triplet state of anthraquinone-2-sulfonate used as proxy of the chromophoric DOM, and singlet oxygen 1O2), and determined relevant kinetic parameters. A standard procedure based on UV detection coupled with liquid chromatography (HPLC-UV) was used under identical experimental conditions to compare and verify the DPV-based results. The latter were in agreement with HPLC data, with the exception of the triplet-sensitized processes. In the other cases, DPV could be used as an alternative to the well-tested but more costly and time-consuming HPLC-UV technique. We have also assessed the reaction rate constant between APAP and HO by real-time DPV, which allowed for the monitoring of APAP photodegradation inside the irradiation chamber. Unfortunately, real-time DPV measurements are likely to be affected by temperature variations of the irradiated samples. Overall, DPV appeared as a fast, cheap and reasonably reliable technique when used for the off-line monitoring of APAP photodegradation. When a suitable real-time procedure is developed, it could become a very straightforward method to study the photochemical behaviour of electroactive xenobiotics.

Emissive Synthetic Cofactors: Enzymatic Interconversions of tz A Analogues of ATP, NAD+ , NADH, NADP+ , and NADPH.

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3-d]pyrimidine analogue of adenosine (tz A), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and tz ATP to yield Ntz AD+ , which can be enzymatically phosphorylated by NAD+ kinase and ATP or tz ATP to the corresponding Ntz ADP+ . The latter can be engaged in NADP-specific coupled enzymatic transformations involving conversion to Ntz ADPH by glucose-6-phosphate dehydrogenase and reoxidation to Ntz ADP+ by glutathione reductase. The Ntz ADP+ /Ntz ADPH cycle can be monitored in real time by fluorescence spectroscopy.

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue.

The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NtzAD+ is converted to NtzADH, reflecting a complementary photophysical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding tzADP-ribose. NtzAD+ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD+.

Expanding a fluorescent RNA alphabet: synthesis, photophysics and utility of isothiazole-derived purine nucleoside surrogates.

A series of emissive ribonucleoside purine mimics, all comprised of an isothiazolo[4,3-d]pyrimidine core, was prepared using a divergent pathway involving a key Thorpe-Ziegler cyclization. In addition to an adenosine and a guanosine mimic, analogues of the noncanonical xanthosine, isoguanosine, and 2-aminoadenosine were also synthesized and found to be emissive. Isothiazolo 2-aminoadenosine, an adenosine surrogate, was found to be particularly emissive and effectively deaminated by adenosine deaminase. Competitive studies with adenosine deaminase with each analogue in combination with native adenosine showed preference for the native substrate while still deaminating the isothiazolo analogues.

Polymerase-Mediated Site-Specific Incorporation of a Synthetic Fluorescent Isomorphic G Surrogate into RNA.

An enzyme-mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with th G, an emissive isomorphic G surrogate, is reported. Transcription in the presence of th G and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5'-end modified transcripts that can be mono-phosphorylated and ligated to provide longer site-specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non-canonical purine-containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme-mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy.

Chemical Mutagenesis of an Emissive RNA Alphabet.

An evolved fluorescent ribonucleoside alphabet comprising isomorphic purine ((tz)A, (tz)G) and pyrimidine ((tz)U, (tz)C) analogues, all derived from isothiazolo[4,3-d]pyrimidine as a common heterocyclic core, is described. Structural and biochemical analyses illustrate that the nucleosides, particularly the C-nucleosidic purine analogues, are faithful isomorphic and isofunctional surrogates of their natural counterparts and show improved features when compared to an RNA alphabet derived from thieno[3,4-d]-pyrimidine. The restoration of the nitrogen in a position equivalent to the purines' N7 leads to "isofunctional" behavior, as illustrated by the ability of adenosine deaminase to deaminate (tz)A as effectively as adenosine, the native substrate.

A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Pyrene-based quantitative detection of the 5-formylcytosine loci symmetry in the CpG duplex content during TET-dependent demethylation.

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten-eleven-translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence-based probe not only to target 5-formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET-dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET-dependent 5mC oxidation and novel mechanistic insights into the TET-dependent 5mC oxidation in the mCpG context.

Amphiphilic dynamic NDI and PDI probes: imaging microdomains in giant unilamellar vesicles.

Dynamic amphiphiles provide access to transmembrane ion transport, differential sensing and cellular uptake. In this report, we introduce dynamic amphiphiles with fluorescent tails. Core-substituted naphthalenediimides (cNDIs) and perylenediimides (cPDIs) are tested. Whereas the latter suffer from poor partitioning, dynamic cNDI amphiphiles are found to be purifiable by RP-HPLC, to partition selectively into liquid-disordered (Ld) microdomains of mixed lipid bilayers and to activate DNA as transporters. Importantly, fluorescence properties, partitioning and activity can be modulated by changes in the structure of mixed amphiphiles. These results confirm the potential of dynamic fluorescent amphiphiles to selectively label extra- and intracellular membrane domains and visualize biological function.

Naphthalene- and perylenediimides with hydroquinones, catechols, boronic esters and imines in the core.

The green-fluorescent protein of the jellyfish operates with the most powerful phenolate donors in the push-pull fluorophore. To nevertheless achieve red fluorescence with the same architecture, sea anemone and corals apply oxidative imination, a process that accounts for the chemistry of vision as well. The objective of this study was to apply these lessons from nature to one of the most compact family of panchromatic fluorophores, i.e. core-substituted naphthalenediimides (cNDIs). We report straightforward synthetic access to hydroxylated cNDI and cPDI cores by palladium-catalyzed cleavage of allyloxy substituents. With hydroxylated cNDIs but not cPDIs in water-containing media, excited-state intramolecular proton transfer yields a second bathochromic emission. Deprotonation of hydroquinone, catechol and boronic ester cores provides access to an impressive panchromism up to the NIR frontier at 640 nm. With cNDIs, oxidative imination gives red shifts up to 638 nm, whereas the expanded cPDIs already absorb at 754 nm upon deprotonation of hydroquinone cores. The practical usefulness of hydroquinone cNDIs is exemplified by ratiometric sensing of the purity of DMF with the "naked eye" at a sensitivity far beyond the "naked nose". We conclude that the panchromatic hypersensitivity toward the environment of the new cNDIs is ideal for pattern generation in differential sensing arrays.

Recent progress with functional biosupramolecular systems.

The objective of this account is to summarize our recent progress with functional biosupramolecular systems concisely. The functions covered are artificial photosynthesis, anion transport, and sensing in lipid bilayer membranes. With artificial photosynthesis, the current emphasis is on the construction of ordered and oriented architectures on solid surfaces. Recent examples include the zipper assembly of photosystems with supramolecular n/p-heterojunctions and oriented antiparallel redox gradients. Current transport systems in lipid bilayers reveal new interactions at work. Examples include anion-macrodipole or anion-π interactions. Current attention with membrane-based sensing systems shifts from biosensor approaches with enzymatic signal generation to aptamers (i.e., the DNA version of immunosensing) and differential sensing with dynamic polyion-counterion transporters. The functional diversity accessible with biosupramolecular systems is highlighted, as is the critical importance of cross-fertilization at intertopical convergence zones.