Ligands with polyfluorophenyl moieties promote a local structural rearrangement in the Spinach2 and Broccoli aptamers that increases ligand affinities.

The interaction of nucleic acids with their molecular targets often involves structural reorganization that may traverse a complex folding landscape. With the more recent recognition that many RNAs, both coding and noncoding, may regulate cellular activities by interacting with target molecules, it becomes increasingly important to understand how nucleic acids interact with their targets and how drugs might be developed that can influence critical folding transitions. We have extensively investigated the interaction of the Spinach2 and Broccoli aptamers with a library of small molecule ligands modified by various extensions from the imido nitrogen of DFHBI [(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one] that reach out from the Spinach2 ligand binding pocket. Studies of the interaction of these compounds with the aptamers revealed that polyfluorophenyl-modified ligands initiate a slow change in aptamer affinity that takes an extended time (half-life of ∼40 min) to achieve. The change in affinity appears to involve an initial disruption of the entrance to the ligand binding pocket followed by a gradual transition to a more defined structure for which the most likely driving force is an interaction of the gateway adenine with a nearby 2'OH group. These results suggest that polyfluorophenyl modifications might increase the ability of small molecule drugs to disrupt local structure and promote RNA remodeling.

Synthesis, Fabrication, and Characterization of Functionalized Polydiacetylene Containing Cellulose Nanofibrous Composites for Colorimetric Sensing of Organophosphate Compounds.

Organophosphate (OP) compounds, a family of highly hazardous chemical compounds included in nerve agents and pesticides, have been linked to more than 250,000 annual deaths connected to various chronic diseases. However, a solid-state sensing system that is able to be integrated into a clothing system is rare in the literature. This study aims to develop a nanofiber-based solid-state polymeric material as a soft sensor to detect OP compounds present in the environment. Esters of polydiacetylene were synthesized and incorporated into a cellulose acetate nanocomposite fibrous assembly developed with an electrospinning technique, which was then hydrolyzed to generate more hydroxyl groups for OP binding. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), Instron® tensile tester, contact angle analyzer, and UV-Vis spectroscopy were employed for characterizations. Upon hydrolysis, polydiacetylene esters in the cellulosic fiber matrix were found unaffected by hydrolysis treatment, which made the composites suitable for OP sensing. Furthermore, the nanofibrous (NF) composites exhibited tensile properties suitable to be used as a textile material. Finally, the NF composites exhibited colorimetric sensing of OP, which is visible to the naked eye. This research is a landmark study toward the development of OP sensing in a protective clothing system.

Characterization of a Cytosolic Acyl-Activating Enzyme Catalyzing the Formation of 4-Methylvaleryl-CoA for Pogostone Biosynthesis in Pogostemon Cablin.

Pogostone, a compound with various pharmaceutical activities, is a major constituent of the essential oil preparation called Pogostemonis Herba, which is obtained from the plant Pogostemon cablin. The biosynthesis of pogostone has not been elucidated, but 4-methylvaleryl-CoA (4MVCoA) is a likely precursor. We analyzed the distribution of pogostone in P. cablin using gas chromatography-mass spectrometry (GC-MS) and found that pogostone accumulates at high levels in the main stems and leaves of young plants. A search for the acyl-activating enzyme (AAE) that catalyzes the formation of 4MVCoA from 4-methylvaleric acid was launched, using an RNAseq-based approach to identify 31 unigenes encoding putative AAEs including the PcAAE2, the transcript profile of which shows a strong positive correlation with the distribution pattern of pogostone. The protein encoded by PcAAE2 was biochemically characterized in vitro and shown to catalyze the formation of 4MVCoA from 4-methylvaleric acid. Phylogenetic analysis showed that PcAAE2 is closely related to other AAE proteins in P. cablin and other species that are localized to the peroxisomes. However, PcAAE2 lacks a peroxisome targeting sequence 1 (PTS1) and is localized in the cytosol.

Ozonolysis of Alkynes-A Flexible Route to Alpha-Diketones: Synthesis of AI-2.

A mild procedure for the low-temperature conversion of alkynes to diketones has been developed and employed in the synthesis of AI-2.

Direct Synthesis of the Phenanthroviridone Skeleton Using a Highly Regioselective Nitroquinone Diels-Alder Reaction.

A variety of nucleophiles react efficiently with in situ generated nitroquinones. The reaction with substituted resorcinols led to the direct synthesis of the phenanthroviridinone and lagumycin skeleton via a highly regioselective Diels-Alder reaction.

A Synthesis of Dihydro Eurotiumide B via a Tandem Butenolide Annulation/Reductive Thiolation Reaction.

Dihydro eurotiumide B was prepared from a 5-phenylthiobutenolide via a tandem annulation/reductive thiolation reaction.

Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the Spinach RNA Aptamer.

( Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one (DFHBI) and its analogues are fluorogenic molecules that bind the Spinach aptamer (a small RNA molecule), which was selected for imaging RNA. They are extremely weakly fluorescent in liquid solvents. It had been hypothesized that photoisomerization is a very efficient nonradiative process of deactivation. We show, consistent with the results of other studies, that if the isomerization is impeded, the fluorescence signal is enhanced significantly. In addition, we provide a thorough characterization of the photophysical behavior of DFHBI and its derivatives, notably that of ( Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-3-((perfluorophenyl)methyl)-3,5-dihydro-4 H-imidazol-4-one (PFP-DFHBI) in various solvent environments. Solvent-dependent studies were performed with various mixtures of solvents. The results suggest that hydrogen bonding or strong interactions of the solvents with the phenolic-OH group change the absorption band near 420-460 nm and the nature of emission near 430 and 500 nm through various degrees of stabilization and the transformation between the neutral and the anionic species at both ground and excited states. These observations are confirmed by using a methoxy-substituted molecule (( Z)-5-(4-methoxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one), where the 420-460 nm band is absent in the presence of methanol and the spectra are similar to those of PFP-DFHBI in noninteracting solvents, such as acetonitrile and dichloromethane. Thus, in addition to the major role of photoisomerization as a nonradiative process of deactivation of the excited state, the fluorescence of DFHBI-type molecules is very sensitively dependent upon the pH of the medium as well as upon solvent-specific interactions, such as hydrogen-bonding ability and polarity.

Synthesis of (±)-Naphthacemycin A9.

Naphthacemycin A9 was synthesized in nine steps by a sequence featuring a Diels-Alder reaction of a hindered aryl butadiene and a Hauser-Kraus annulation reaction.

Identification and characterization of small molecule inhibitors of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent of PRRS, an economically significant disease of swine worldwide. PRRSV is poorly controlled by the currently available vaccines, and alternative control strategies are needed to help prevent the continual circulation of the virus. Previously, we developed a synthetic route for the natural compound atractylodinol and demonstrated anti-PRRSV activity in vitro. However, the synthetic route was inefficient and the yield was poor. To identify PRRSV inhibitors that could be synthesized easily and cost-effectively, we synthesized a series of atractylodinol analogs and characterized their anti-PRRSV activity in vitro. A furan-substituted bis-enyne subunit was found to be critical for PRRSV inhibition. Six analogs had potent inhibitory activity against PRRSV with 50% inhibition concentration (IC50) of 0.4-1.4 µM and 50% cytotoxic concentration (CC50) of 209-1537 µM in MARC-145 cells. Three of the most promising compounds also demonstrated significant antiviral activity and low cytotoxicity in porcine macrophages. Inhibition of PRRSV in MARC-145 cells occurred primarily at a post-entry step during PRRSV replication, between 4 and 12 h post-entry. These results suggest that atractylodinol analogs are promising antiviral candidates that could augment current PRRSV control strategies.

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl.

The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells.

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of "light-up" aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

Synthetic Anthocyanidins from Natural Benzopyrans.

A hydride abstraction strategy can be used to make anthocyanidins and isoflavylium salts from benzopyrans in good yields.

Phthalide Anions in Organic Synthesis. A Direct Total Synthesis of Furomollugin.

Furomollugin was synthesized in three steps from commercially available starting materials using phthalide annulation chemistry.

Synthetic approach to the psoracorylifols.

Core analogs of the psoracorylifols were generated by a five-step route from 2,2-dimethyl-4-cyanobutanal.

Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

First inverse electron-demand Diels-Alder methodology of 3-chloroindoles and methyl coumalate to carbazoles.

The first successful inverse electron-demand Diels-Alder has been demonstrated with the 2-pyrone methyl coumalate in conjunction with substituted indoles. Utilizing 1-alkyl-3-chloroindoles as the electron-rich dienophile efficiently generates carbazoles without the need for additional metal catalysts. Through a thermal, one-pot Diels-Alder/decarboxylation/elimination domino sequence, access to a class of 3-methylcarbazoles is rapidly generated with exclusive regiocontrol in up to 90% yield.

Synthesis of 3-farnesyl salicylic acid, a novel antimicrobial from Piper multiplinervium.

Both 3-farnesyl salicylic acid and 3-geranyl salicylic acid were synthesized from 2,6-dibromophenol and showed low levels of antimicrobial activity against E. coli strains.

Mechanistic insights into ring-opening and decarboxylation of 2-pyrones in liquid water and tetrahydrofuran.

2-Pyrones, such as triacetic acid lactone, are a promising class of biorenewable platform chemicals that provide access to an array of chemical products and intermediates. We illustrate through the combination of results from experimental studies and first-principle density functional theory calculations that key structural features dictate the mechanisms underlying ring-opening and decarboxylation of 2-pyrones, including the degree of ring saturation, the presence of C═C bonds at the C4═C5 or C5═C6 positions within the ring, as well as the presence of a ß-keto group at the C4 position. Our results demonstrate that 2-pyrones undergo a range of reactions unique to their structure, such as retro-Diels-Alder reactions and nucleophilic addition of water. In addition, the reactivity of 2-pyrones and the final products formed is shown to depend on the solvent used and the acidity of the reaction environment. The mechanistic insights obtained here provide guidance for the selective conversion of 2-pyrones to targeted chemicals.

Total synthesis of paracaseolide A.

The total synthesis of paracaseolide A, a valuable cell-cycle progression inhibitor, was accomplished in 8 steps from known compounds, with 6.6% overall yield. The synthetic strategy creates strong potential for diversification.

Synthesis of uliginosins A and B.

The uliginosins have potentially very useful biological activity, but their availability from natural sources is limited. Moreover, the one synthesis can not be reproduced. Here we report reproducible conditions for the synthesis of the uliginosins.