Bis(Benzofuran-1,3-N,N-heterocycle)s as Symmetric and Synthetic Drug Leads against Yellow Fever Virus.

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran−1,3-imidazolidin-4-one)s and bis(benzofuran−1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC50 of <3.54 µM and SI of >15.3. The results are valuable for the development of novel antiviral drug leads against emerging diseases.

Diversity-Oriented Synthesis of a Molecular Library of Immunomodulatory α-Galactosylceramides with Fluorous-Tag-Assisted Purification and Evaluation of Their Bioactivities in Regard to IL-2 Secretion.

Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia-Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF3-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.

Computer-Aided Design and Synthesis of (Functionalized quinazoline)-(α-substituted coumarin)-arylsulfonate Conjugates against Chikungunya Virus.

Chikungunya virus (CHIKV) has repeatedly spread via the bite of an infected mosquito and affected more than 100 countries. The disease poses threats to public health and the economy in the infected locations. Many efforts have been devoted to identifying compounds that could inhibit CHIKV. Unfortunately, successful clinical candidates have not been found yet. Computations through the simulating recognition process were performed on complexation of the nsP3 protein of CHIKV with the structures of triply conjugated drug lead candidates. The outcomes provided the aid on rational design of functionalized quinazoline-(α-substituted coumarin)-arylsulfonate compounds to inhibit CHIKV in Vero cells. The molecular docking studies showed a void space around the ß carbon atom of coumarin when a substituent was attached at the α position. The formed vacancy offered a good chance for a Michael addition to take place owing to steric and electronic effects. The best conjugate containing a quinazolinone moiety exhibited potency with EC50 = 6.46 µM, low toxicity with CC50 = 59.7 µM, and the selective index (SI) = 9.24. Furthermore, the corresponding 4-anilinoquinazoline derivative improved the anti-CHIKV potency to EC50 = 3.84 µM, CC50 = 72.3 µM, and SI = 18.8. The conjugate with 4-anilinoquinazoline exhibited stronger binding affinity towards the macro domain than that with quinazolinone via hydrophobic and hydrogen bond interactions.

Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.

CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.

A Modular Chemoenzymatic Synthesis of Disialosyl Globopentaosylceramide (DSGb5Cer) Glycan.

The total synthesis of the oligosaccharide moiety of disialosyl globopentaosylceramide (DSGb5 Cer), a dominant ganglioside isolated from malignant renal cell carcinoma tissues, is reported. The synthetic strategy relies on a chemical α(2,6)-sialylation at the internal GalNAc unit of a Gb5 pentasaccharide backbone that furnishes a Neu5Acα(2,6)GalNAc-linked hexasaccharide, suitable for an enzymatic α(2,3)-sialylation of the terminal Gal residue to construct a heptasaccharide glycan. Convergent access to this key α(2,6)-sialylated hexasaccharide was also achieved through a [3+3] glycosylation building upon a Galß(1,3)[Neu5Acα(2,6)]GalNAc-based trisaccharide donor and a Gb3 acceptor. The synthetic DSGb5 glycan bearing a 6-azidohexyl aglycon at the reducing end could undergo further regioselective functionalization. This approach represents a viable chemoenzymatic method for accessing complex ganglioside glycans and should be useful for the synthesis and biological investigation of DSGb5 derivatives.

Domino Reaction for the Synthesis of Polysubstituted Pyrroles and Lamellarin R.

A three-component annulation reaction was developed for the synthesis of pyrroles, a class of compounds with various properties valuable to biomedical and polymer industries. Treatment of α-silylaryl triflates, Schiff bases, and alkynes generated polysubstituted pyrroles in good yields (61-86%) with regioselectivity. This domino reaction involved completion of five sequential steps in a single flask, which comprised aryne formation through 1,2-elimination, their alkylation by Schiff bases through 1,2-addition, 1,4-intramolecular proton transfer, Hüisgen 1,3-dipolar cycloaddition, and dehydrogenative aromatization. It was then successfully applied as the key step in the synthesis of the natural product lamellarin R. This new reaction represents an efficient, sustainable process for the production of chemical materials.

Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells.

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.

Enterovirus Inhibition by Hinged Aromatic Compounds with Polynuclei.

The modern world has no available drugs for the treatment of enteroviruses (EV), which affect millions of people worldwide each year. The EV71 is a major causative disease for hand, foot, and mouth disease; sometimes it is associated with severe central nervous system diseases. Treatment for enteroviral infection is mainly supportive; treatment for aseptic meningitis caused by enteroviruses is also generally symptomatic. Upon the urgent request of new anti-enterovirus drugs, a series of hinged aromatic compounds with polynulei were synthesized through two different chemical pathways. Among these morpholine-furan/thiophene/pyrrole-benzene-pyrazole conjugates, three new agents exhibited inhibitory activity with EC50 = 2.29-6.16 µM toward EV71 strain BrCr in RD cells. Their selectivity index values were reached as high as 33.4. Their structure-activity relationship was deduced that a thiophene derivative with morpholine and trifluorobenzene rings showed the greatest antiviral activity, with EC50 = 2.29 µM.

Synthesis of an S-Linked α(2→8) GD3 Antigen and Evaluation of the Immunogenicity of Its Glycoconjugate.

Replacing the interglycosidic oxygen atom of oligosaccharides with a nonhydrolyzable sulfur atom has attracted significant interest because it provides opportunities for developing new glycoconjugate vaccines. Herein, a stereocontrolled and highly convergent method to synthesize a non-reducing-end inter-S-glycosidic variant of the GD3 antigen (S-linked α(2→8) GD3) that is resistant to enzymatic hydrolysis is reported. The key steps in the synthesis are a regio- and stereoselective α(2→3) sialylation of a lactoside acceptor with a C8-iodide-derivatized sialyl donor and an anomeric S-alkylation, which enable stereoselective construction of a terminal S-linked α(2→8) disialyl residue. The sulfhydryl-reactive maleimide group was used as the linker for the well-defined conjugation of these antigens to the immunogenic protein keyhole limpet hemocyanin (KLH). Groups of mice were immunized with the GD3-KLH and S-linked GD3-KLH glycoconjugates in the presence of complete Freund's adjuvant. Microarray analysis of the sera showed the promise of the S-linked GD3-KLH vaccine: it stimulated a high immunoglobulin G response against S-linked GD3 and cross-reactivity with the O-linked GD3 antigen was low. The activity of the S-linked GD3-KLH vaccine was comparable to that of the O-linked GD3-KLH vaccine, which highlighted the effectiveness of generating glycoconjugate vaccines and immunotherapies by relatively simple means.

Syntheses of Chroman-2-ones and α-Amino Acids through a Diastereoselective Domino Reaction.

Many 3-aminochroman-2-ones and ß,ß-diarylalanines exhibit significant biological activities. A new method was thus developed for the syntheses of these compounds with high efficiency and diastereoselectivity. First, treatment of various phenols with Erlenmeyer-Plochl (Z)-azlactones and AlCl3 in toluene produced the desired cis-3-aminochroman-2-ones in 65-90% yields under kinetic control. This coupling reaction involved a domino process of Friedel-Crafts alkylation, 1,4-AlCl3 shift, transesterification, and protodealumination in a "single-flask." The corresponding products, however, were not generated by replacement of AlCl3 with a protonic acid. Second, hydrolysis of the resultant 3-amino-4-arylchroman-2-ones by NaHCO3 in a mixture of THF and water gave α-(N-benzoyl)amino acids. Further deprotection of these isolated compounds by use of hydrochloric acid (12 N) in methanol afforded the desired free amino acids in 80-88% yields. Under these optimized conditions, epimerization did not occur at the α carbons of α-(N-benzoyl)- and free α-amino acids. These new findings provide a convenient way to generate 3,4-disubtituted chroman-2-ones and ß,ß-diarylalanine derivatives with very high stereoselectivity.

Syntheses of Platinum-Sulindac Complexes and Their Nanoparticles as Targeted Anticancer Drugs.

Platinum(II)-sulindac complexes [{η2 -C5 H4 SN(O)}Pt(DMSO){O(C=O)Sulindac}], [{η2 -C5 H4 SN(O)}PtCl{(S=O)Sulindac}], [{η2 -C5 H4 SN(O)}PtCl{(S=O)Sulindac-succinimide}], and [{η2 -C5 H4 SN(O)}PtCl{(S=O)Sulindac-thymidine}] were synthesized that exhibited IC50 values of 2.9-4.8 µm against human oral cancer cells OECM1. The poly(lactic-co-glycolic acid) (PLGA) encapsulated [{η2 -C5 H4 SN(O)}PtCl{(S=O)Sulindac}] also showed cytotoxic activity although less potent than the pristine species.

Cytotoxicity of Postmodified Zeolitic Imidazolate Framework-90 (ZIF-90) Nanocrystals: Correlation between Functionality and Toxicity.

Using a simple method, the aldehyde groups of zeolitic imidazolate framework-90 (ZIF-90) nanocrystals were converted into carboxyl, amino, and thiol groups, without affecting the integrity of the framework. Notably, for the first time, correlations between functionality and cytotoxicity are also demonstrated via in vitro cytotoxicity assays. The positive charged aminated-ZIF-90 presumably results in either perturbation of cell membrane, more efficient cell uptake, or both. Therefore, the half-maximal effective (EC50 ) concentration of aminated-ZIF-90 has a higher cytotoxicity of about 30 µg mL(-1) .

Synthesis and Structure-Activity Relationships of Imidazole-Coumarin Conjugates against Hepatitis C Virus.

A series of new conjugated compounds with a -SCH2- linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 µM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents.

Aryne-Induced Novel Tandem 1,2-Addition/(3+2) Cycloaddition to Generate Imidazolidines and Pyrrolidines.

A new "single-flask" method was developed for the synthesis of imidazolidines and pyrrolidines with high stereoselectivity. First, a Schiff base was arylated with an aryne. Second, an intramolecular proton transfer took place from the methylene position to the anionic aryne ring. Third, the resultant ylide reacted with a second equivalent of the same Schiff base in situ or an electron-deficient alkene through a (3+2) cycloaddition. These sequential tandem 1,2-addition/(3+2) cycloaddition reactions led to the desired heterocycles in 63-88% yields.

Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.

We described a rapid site-selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2-cyanobenzothiazole (CBT)-cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT-solid support to immobilize the protein. According to microarray analysis, we found that glutathione S-transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N- and C-terminally immobilized maltose-binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT-Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site-selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.

Relationship between structure of conjugated oxime esters and their ability to cleave DNA.

The size and geometry of polycycles are critical to intercalation into DNA. This work involves the establishment of a new compound library that includes 35 O-benzoyl oxime esters with intercalators of five types. These conjugated compounds were synthesized by the condensation of substituted benzoyl chlorides (XC6H4COCl; X = H, Me, CN, F, and NO2) or naphthoyl chlorides with oximes of fluoren-9-one, 9,10-anthraquinone, xanthen-9-one, thioxanthen-9-one, and 9H-thioxanthen-9-one 10,10-dioxide to give the corresponding esters in 80-99% yields. All of these compounds could cleave DNA when photolyzed by UV light. Of these conjugates, 9,10-anthraquinone-O-9-(4-fluorobenzoyl)oxime with a binding constant of 4.49 × 10(4) M(-1) cleaved DNA most efficiently. Examination of the structure-activity relationship supports a conclusion that two factors affect DNA-cleaving potency. These are (1) the planarity of the intercalating moiety, and (2) the size and substituents of the benzoyl ring. The DNA-cleaving ability followed the order 9,10-anthraquinone > fluoren-9-one ≥ xanthen-9-one ∼ thioxanthen-9-one > 9H-thioxanthen-9-one 10,10-dioxide. The benzoyl-containing oxime ester conjugates were more active than the corresponding naphthoyl-containing conjugates. The potency that was associated with the different substituents on the benzoyl ring followed the order F > CN ≥ NO2 > Me ∼ H.

Silicon-induced phenanthrene formation from benzynes and allenylsilanes.

Now directly feasible: The silicon atom in allenylsilanes enabled their reaction with two equivalents of benzynes to generate (α-phenanthrenyl)vinylsilanes in good to excellent yields through an unprecedented [2+2+2] pathway (see scheme).

Deoxygenative olefination reaction as the key step in the syntheses of deoxy and iminosugars.

Just a spoonful of sugar! A new synthetic strategy involving the use of a deoxygenative olefination reaction as the key step was developed for the preparation of deoxy and iminosugars in their optically active form (see scheme). This strategy has been proven successful by the use of a pentose, hexose, heptose, and disaccharide as the starting materials. Furthermore, it was applied in a formal total synthesis of iminosugar (-)-1-deoxy-L-fuconojirimycin, which can inhibit α-L-fucosidase.

Synthesis of anti-HIV lithospermic acid by two diverse strategies.

An efficient and convergent route for the synthesis of the natural product (+)-lithospermic acid, which possesses anti-HIV activity, was accomplished. The (±)-trans-dihydrobenzo[b]furan core therein was prepared by two different strategies. The first strategy involved the use of a palladium-catalyzed annulation to generate an appropriately substituted benzo[b]furan ester followed by a stereoselective reduction of a carbon-carbon double bond with Mg-HgCl(2)-MeOH. The second strategy relied on an aldol condensation between a suitably substituted methyl arylacetate and 3,4-dimethoxybenzaldehyde, followed by cyclization. Finally, a total synthesis of (+)-lithospermic acid was completed via coupling of a trans-dihydrobenzo[b]furan cinnamic acid with an enantiomerically pure methyl lactate.

Oxime esters of 2,6-diazaanthracene-9,10-dione and 4,5-diazafluoren-9-one as photo-induced DNA-cleaving agents.

Two series of oxime esters containing the 2,6-diazaanthracene-9,10-dione bis-(O-benzoyloxime) and 4,5-diazafluoren-9-one O-9-benzoyloxime moieties have been synthesized and tested as photo-induced DNA cleaving agents. All these compounds were found to cleave DNA upon irradiation with 312 nm UV light. The structure-activity relationship of these molecules for DNA cleavage was established. A plausible reaction mechanism is also proposed.