Performance evaluation of silica microspheres functionalized by different amine-ligands for hydrophilic interaction chromatography.

In this work, for the first time five amine-ligands including mono-amine, di-amine, tri-amine, secondary and tertiary amine, were functionalized on mesoporous micro-silicas and developed as stationary phases for hydrophilic interaction liquid chromatography (HILIC). The investigations about the retention mechanisms, effects of different chromatographic conditions and stability were systematically conducted. Three kinds of polar and hydrophilic compounds (saccharides, sulfonamides, nucleosides and nucleobases) were selected as probe molecules to evaluate their separation performances. Among the five stationary phases, only aminopropyl-bonded silica has already gained wide developments and applications. Whereas, there are no related researches about the other four to be utilized as separation media. By a series of chromatographic evaluations, the results revealed the other four mesoporous micro-silica materials functionalized with di-amine, tri-amine, secondary and tertiary amine, had great potential to be explored as novel stationary phases of HILIC. Particularly, the two stationary phases functionalized with di-amine and tri-amine exhibited outstanding separation and retention abilities. This work offered some insights on the understanding of retention in HILIC mode and provided us possibility to explore other amine-based HILIC stationary phases.

Cyclodextrin Porous Liquid Materials for Efficient Chiral Recognition and Separation of Nucleosides.

Porous liquids are porous materials that have exhibited unique properties in various fields. Herein, we developed a method to synthesize the type I porous liquids via liquefaction of cyclodextrins by chemical modification. The cyclodextrin porous liquids were characterized by Fourier-transform infrared (FTIR) spectroscopy, NMR, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and UV-vis spectroscopy. The measured ionic conductivity of the γ-cyclodextrin porous liquid was 500 times as great as that of its reactants, which was found to be the first instance with such great conductivity for a type I porous liquid. What is more, the γ-cyclodextrin porous liquid had been demonstrated experimentally to have outstanding chiral recognition ability toward pyrimidine nucleosides in water, which was further confirmed by computational simulations. Additionally, enantiomeric excess of the extracted nucleoside was achieved up to 84.81% by convenient extraction from the mixture of racemic nucleosides and γ-cyclodextrin porous liquid. The great features of the novel cyclodextrin porous liquids could bring opportunities in many fields, including the preparation of chiral separation materials, development of new drug screening mechanisms, and construction of chiral response materials.

Protective Effects of Nucleosides-Rich Extract from Cordyceps cicadae against Cisplatin Induced Testicular Damage.

Cisplatin (CISP) is an efficacious anticancer agent used in chemotherapy, however, the constraint to its clinical utility is the stray organ toxicity including testicular damage linked to oxidative and inflammatory cascades. This study aimed to explore the protective effect of nucleosides-rich extract from Cordyceps cicadae (NRCE) against CISP-induced testicular damage in rats. Rats were subjected to prophylactic oral administration of NRCE (50, 100 and 400 mg/kg body weight/day) for 7 days prior to testicular toxicity induced by CISP (10 mg/kg, ip) and were sacrificed after 72 h post-CISP injection. Cisplatin caused significant deficits in sperm count, viability and motility, testosterone and follicle stimulating hormone (FSH) compared to normal control. It depressed testicular activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), total antioxidant content (TAC), whereas malondialdehyde (MDA) increased remarkably. CISP considerably increased tumor necrosis factor-alpha (TNF-α) and interleukin-one beta (IL-1ß) with alterations in testis histology compared to normal control. Interestingly, NRCE pretreatment inhibited the CISP-induced alterations in reproductive indices, restored the antioxidant activities in testes as well as inflammatory mediators and histology comparable to control. Our findings demonstrate that NRCE could prevent CISP testicular damage via inhibition of oxidative stress and pro-inflammation in rats.

A facile process for the preparation of organic gel-assisted silica microsphere material for multi-mode liquid chromatography.

Organic gel (OG) has excellent characteristics, including a large surface area, adjustable pore/channel size, and good chemical stability, and has attracted great attention in the field of materials. However, the OG packed column is difficult to pack due to the weak mechanical strength and poor monodispersity. Herein, 1-allyl-3-methyl imidazolium hexafluorophosphate-co-1-dodecanethiol ([AMIm]PF6-co-TDDM) was prepared on the silica microsphere for chromatographic packing available in multimode liquid chromatography (LC) mode with the good mechanical properties of silica microspheres through a simple OG synthesis method. [AMIm]PF6-co-TDDM@SiO2 hybrid microspheres with uniform particles and narrow particle size distribution are used as stationary phases of LC. These microspheres are used in anion-exchange (IEC), reversed-phase (RP), and hydrophilic interaction (HILIC) mode for the separation of different analytes. Such microspheres can also be used for the preliminary qualitative analysis of active ingredients in actual samples in addition to organic acids, alkylbenzenes, and nucleoside bases. The [AMIm]PF6-co-TDDM@SiO2 chromatography packing also has good reproducibility and stability. The adhesive properties of organogels and the adsorption properties of silica gel simplify the synthesis of stationary phase materials. This simple and effective strategy for preparing [AMIm]PF6-co-TDDM@SiO2 composite microspheres by one-pot method can expand the application of OG as a functional additive on silica microspheres in LC.

A novel imidazolium bonding stationary phase derived from N-(3-aminopropyl)-imidazole for hydrophilic interaction liquid chromatography.

In this work, a novel imidazolium bonding method was proposed for the synthesis of hydrophilic interaction liquid chromatography (HILIC) stationary phases. One obtained stationary phase (SilprAprImCl) was derived from direct reaction between N-(3-aminopropyl)-imidazole and 3-chloropropylated silica gel. Other two materials (SilprAprImBF4 and SilprAprImTf2N) were obtained from SilprAprImCl by ion exchange reaction, respectively. Fourier-transform infrared spectroscopy and elemental analysis afforded the proofs of successful imidazolium immobilization and satisfied bonding efficiency. Various polar compounds such as saccharides, nucleosides, and nucleobases were utilized to evaluate the retention behaviours of these materials in HILIC mode. Different effects from mobile composition, column temperature, imidazolium unite and paired anions (Cl-, BF4-, and Tf2N-) in imidazolium were proved and discussed. Separation mechanism and the role of the imidazolium ions were also investigated in mobile phases with different pH. Moreover, chromatographic stability was evaluated by consecutive injections. Finally, the reliability of these stationary phases was demonstrated by the separation of oligosaccharides in real fructooligosaccharides samples.

Boronic acid-decorated metal-organic frameworks modified via a mixed-ligand strategy for the selective enrichment of cis-diol containing nucleosides.

Functional metal-organic frameworks (MOFs) constructed via a pre-installation strategy of introducing mixed organic ligands have attracted considerable interest in various fields. In the present study, boronic acid decorated magnetic Zr-MOFs were successfully synthesized by introducing 3-carboxyphenylboronic acid ligands as fragments. The prepared material was used as an adsorbent for the enrichment of cis-diol-containing nucleosides. The adsorbent has excellent performance with regard to the enrichment and separation of the nucleosides. This may be attributed to its abundant boronic acid functional groups, and the convenience of magnetic separation it provides. The obtained material was chemically stable over a large pH range. The degree of linearity of the nucleosides was excellent (0.02-10 µg mL-1), and the detection and quantification limits were low (0.006-0.016 µg mL-1 and 0.02-0.05 µg mL-1, respectively). Furthermore, it was possible to attain adsorption equilibrium within 10 min. The high efficiency of this method makes it suitable for the successful extraction of nucleosides from human urine samples, with satisfactory recoveries of 88-146%, and 1.7-9.4% precision. We believe that the fabricated functional magnetic MOFs have great potential for the analysis of other cis-diol-containing target, and the pre-installation strategy could be adapted for the wider application of MOFs.

A new nucleoside and two new pyrrole alkaloid derivatives from Cordyceps militaris.

A new nucleoside, a new natural product nucleoside, and two new pyrrole alkaloids derivatives with eight known compounds were isolated from the fruiting body of Cordyceps militaris. The structures of the new compounds were elucidated through extensive analysis of spectroscopic data including 1D and 2D NMR, HRESIMS, IR and UV. All the isolated compounds were detected for their bioactivities against LPS-induced NO production in RAW 264.7 cells. Unfortunately, all the isolates have shown no obvious activity.

Two copolymer-grafted silica stationary phases prepared by surface thiol-ene click reaction in deep eutectic solvents for hydrophilic interaction chromatography.

Two new copolymer-grafted silica stationary phases were prepared and employed in hydrophilic interaction chromatography (HILIC). 2-(Dimethylamino)ethyl methacrylate (DMAEMA) are copolymerized with itaconic acid (IA) and acrylic acid (AA) respectively, via thiol-ene click reaction on silica surface with deep eutectic solvents (DES) as new solvents. The obtained poly(DMAEMA-co-itaconic acid)-grafted silica (Sil-PDM-PIA) and poly(DMAEMA-co-acrylic acid)-grafted silica (Sil-PDM-PAA) were characterized by Fourier transform infrared spectroscopy, elemental analysis and solid-state 13C NMR spectra. Their hydrophilic interaction performances were evaluated by separating nucleosides, nucleobases, saccharides, and amino acids. Compared with previous reported poly(itaconic acid)-grafted silica (Sil-PIA) and poly(acrylic acid)-grafted silica (Sil-PAA) stationary phases, these two new copolymer-grafted silica performed higher selectivity and better separation for polar analytes in HILIC.

D-2-allylglycine embedded imidazolium-bridged polyhedral oligomeric silsesquioxane hybrid monolithic column for efficient separation of both small molecules and macromolecules.

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms.

Determination of 107 Pesticide Residues in Wolfberry with Acetate-buffered Salt Extraction and Sin-QuEChERS Nano Column Purification Coupled with Ultra Performance Liquid Chromatography Tandem Mass Spectrometry.

A multi-residue method for the determination of 107 pesticide residues in wolfberry has been developed and validated. Similar pretreatment approaches were compared, and the linearity, matrix effect, analysis limits, precision, stability and accuracy were validated, which verifies the satisfactory performance of this new method. The LODs and LOQs were in the range of 0.14-1.91 µg/kg and 0.46-6.37 µg/kg, respectively. The recovery of analytes at three fortification levels (10 µg/kg, 50 µg/kg, 100 µg/kg) ranged from 63.3-123.0%, 72.0-118.6% and 67.0-118.3%, respectively, with relative standard deviations (RSDs) below 15.0%. The proposed method was applied to the analysis of fifty wolfberry samples collected from supermarkets, pharmacies and farmers' markets in different cities of Shandong Province. One hundred percent of the samples analyzed included at least one pesticide, and a total of 26 pesticide residues was detected in fifty samples, which mainly were insecticides and bactericide. Several pesticides with higher detection rates were 96% for acetamiprid, 82% for imidacloprid, 54% for thiophanate-methyl, 50% for blasticidin-S, 42% for carbendazim, 42% for tebuconazole and 36% for difenoconazole in wolfberry samples. This study proved the adaptability of the developed method to the detection of multiple pesticide residues in wolfberry and provided basis for the research on the risks to wolfberry health.

Modulation of electroosmotic flow in capillary electrophoresis by plant polyphenol-inspired gallic acid/polyethyleneimine coatings: Analysis of small molecules.

Plant polyphenols can form functional coatings on various materials through self-polymerization. In this paper, a series of modified capillary columns, which possess diversity of charge characteristics for modulating electroosmotic flow (EOF), were prepared by one-step co-deposition of gallic acid (GA), a plant-derived polyphenol monomer, and branched polyethyleneimine (PEI). The physicochemical properties of the prepared columns were characterized by Fourier transform infrared spectroscopy (FT-IR), UV-Vis spectroscopy and scanning electron microscopy (SEM). The magnitude and direction of EOF of GA/PEI co-deposited columns were modulated by changing a series of coating parameters, such as post-incubation of FeCl3, co-deposition time, and deposited amounts of GA and PEI with different relative molecular mass (PEI-600, PEI-1800, PEI-10000, and PEI-70000). Furthermore, the separation efficiencies of the prepared GA/PEI co-deposited columns were evaluated by separations of small molecules, including organic acids, polar nucleotides, phenols, nucleic acid bases and nucleosides. Results indicated that modulating of EOF plays an important role in enhancing the separation performance and reversing the elution order of the analytes. Finally, the developed method was successfully applied to quantitative analysis of acidic compounds in four real samples. The recoveries were in the range of 73.5%-85.8% for citric acid, benzoic acid, sorbic acid, salicylic acid and ascorbic acid in beverage and fruit samples, 101.6%-104.9% for cinnamic acid, vanillic acid, and ferulic acid in Angelica sinensis sample, while 84.6%-97.8% for guanosine-5'-monophosphate, uridine-5'-monophosphate, cytosine-5'- monophosphate and adenosine-5'-monophosphate in Cordyceps samples. These results indicated that the co-deposition of plant polyphenol-inspired GA/PEI coatings can provide new opportunities for EOF modulation of capillary electrophoresis.

Characterization of C-nucleoside Antimicrobials from Streptomyces albus DSM 40763: Strepturidin is Pseudouridimycin.

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.

Development of a new in-line coupling of a miniaturized boronate affinity monolithic column with reversed-phase silica monolithic capillary column for analysis of cis-diol-containing nucleoside compounds.

In-line coupling of capillary columns is an effective means for achieving miniaturized and automated separation methods. The use of multimodal column designed to allow the direct integration of a sample preparation step to the separation column is one example. Herein we propose a novel in-line coupling at the capillary scale between a boronate affinity capillary column (µBAMC unit) and a reversed-phase separation column. This has been made possible due to the elaboration of a new and efficient µBAMC unit. A thiol-activated silica monolithic capillary column was functionalized through thiol-ene photoclick reaction. This simple and fast reaction allows to prepare stable µBAMC units having grafting densities of 1.93 ± 0.17 nmol cm-1. This grafting strategy increases the surface density by a factor 4 compared to our previous strategies and opens the frame to in-line coupling with reversed-phase capillary column. Proof of concept of the in-line coupling was done by coupling a 1-cm length µBAMC unit to a 7-cm length reversed phase capillary column. The conditions of loading, elution and separation were optimized for cis-diol nucleosides analysis (uridine, cytidine, adenosine, guanosine). A loading volume (at pH 8.5) of up to 21 hold volume (i.e 1 µl) of the µBAMC unit can be loaded without sample breakthrough. For the least retained nucleoside (uridine) a limit of detection of 50 ng mL-1 was estimated. Elution and full separation of the four nucleosides was triggered by flushing the multimodal column with an acetic acid (50 mM) / methanol (98/2, v/v) mobile phase.

Comparative Analysis of Carbohydrates, Nucleosides and Amino Acids in Different Parts of Trichosanthes kirilowii Maxim. by (Ultra) High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry and Evaporative Light Scattering Detector Methods.

Trichosanthes kirilowii Maxim. is one of the original plants for traditional Chinese medicines Trichosanthis Fructus, Trichosanthis Semen, Trichosanthis Pericarpium and Trichosanthis Radix. Amino acids, nucleosides and carbohydrates are usually considered to have nutritional value and health-care efficacy. In this study, methods involving high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD), UV-visible spectrophotometry and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) were established for quantifying carbohydrates (fructose, glucose, stachyose, raffinose and polysaccharide), fourteen nucleosides and twenty one amino acids. Moreover, sixty-three samples from nine different parts, including pericarp, seed, fruit pulp, stem, leaf, main root, main root bark, lateral root and lateral root bark of T. kirilowii from different cultivated varieties were examined. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. The results showed that the average content of total amino acids in roots (15.39 mg/g) and root barks (16.38 mg/g) were relatively higher than for others. Contents of nucleosides in all parts of T. kirilowii were below 1.5 mg/g. For carbohydrates, fruit pulp has a higher content than others for glucose (22.91%), fructose (20.63%) and polysaccharides (27.29%). By using partial least-squared discriminate analysis (PLS-DA), Variable importance in the projection (VIP) plots and analysis of variance (ANOVA) analysis, the characteristic components of the different organs (fruit, stems and leaves, roots) were found. This analysis suggested there were potential medicinal and nutritive health care values in various parts of the T. kirilowii, which provided valuable information for the development and utilization of T. kirilowii.

Polyethyleneimine-functionalized carbon dots and their precursor co-immobilized on silica for hydrophilic interaction chromatography.

A new strategy based on synergistic effect of the carbon dots and their precursor was proposed to enhance the selectivity of hydrophilic interaction liquid chromatography (HILIC). In this work, polyethyleneimine (PEI) and PEI-functionalized carbon dots (PEICDs) mix-grafted silica packing material was prepared to act as a novel HILIC stationary phase. Both inner and outer surface of the porous silica are decorated with the mixture of PEI and carbon dots. This stationary phase, namely Sil-PEI/CDs, demonstrate enhanced retention ability and selectivity toward polar analytes, with which 11 nucleosides and nucleobases and 9 ginsenosides can be nicely separated. The Sil-PEI/CDs (RSD 0.12% - 0.54%) exhibited even better stability than the traditional PEI modified stationary phase (RSD 0.39% - 0.87%) within 40 h of continuously elution. And excellent column efficiency was observed from Sil-PEI/CDs (∼65,000 plates/m for cytidine). The strategy of mixed stabilization revealed a new method to prepare good performanced carbon dots based chromatographic column.

Nucleoside and N-acetyldopamine derivatives from the insect Aspongopus chinensis.

Two new nucleoside derivatives, named asponguanosines A and B (1 and 2), three new N-acetyldopamine analogues, aspongamides C-E (3-5), one new sesquiterpene, aspongnoid D (6), and three known compounds were isolated from the medicinal insect Aspongopus chinensis. Their structures including absolute configurations were assigned by using spectroscopic methods and ECD and 13C NMR calculations. Biological activities of compounds 3-7 towards human cancer cells, COX-2, ROCK1, and JAK3 were evaluated.

Targeted discovery and identification of novel nucleoside biomarkers in Apostichopus japonicus viscera using metabonomics.

In this study, we investigated the metabonomic profiles of Apostichopus japonicus using an LC-MS-based method in conjunction with multivariate data analysis. Based on the PLS-DA model, 85 differential metabolites (VIP value >1.0) were obtained from viscera and body wall samples. The MS/MS and NMR experiments were used for the qualitative identification of the characteristic peaks. Sphingoid-based nucleoside analogues were the main components in Chinese A. japonicus viscera. Our findings demonstrate that A. japonicus viscera contain a large number of compounds that may have applications as nutraceuticals or pharmaceuticals.

Development and validation of an HPLC-DAD-MS method for determination of four nucleoside compounds in the New Zealand native mushroom Hericium sp.

To identify and quantify the content of nucleoside compounds in the New Zealand native edible mushroom Hericium sp., a high-performance liquid chromatography coupled with a triple quadrupole detector mass method was developed and validated. Four nucleoside substitutes, namely cytidine, uridine, adenosine, and guanosine, were identified. Optimization was conducted to study the effect of extraction method type, solvent pH, and extraction time. The optimal conditions were obtained using ultrasonic treatment in water at pH 3.8 for 30 min. For chromatographic separation, a C18 column was applied using 0.1% formic acid (pH 3.4) as the mobile phase with detection at 260 nm. The total concentration of the four nucleoside compounds was high, at 10.7 mg/g dry weight, indicating a potential benefit for human health. The excellent validation results based on selectivity, linearity, precision, accuracy and robustness revealed the reliability of the newly developed analytical method, which could be applied routinely in research laboratories.

Preparation and evaluation of a reversed-phase/hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase functionalized with dopamine-based dendrimers.

In this work, a novel dendritic stationary phase was synthesized by the repeated grafting of 1,4-butanediol diglycidyl ether (BDDE) and dopamine (DA) on the surface of silica for performing mixed-mode high-performance liquid chromatography (MHPLC). Elemental analysis (EA), thermogravimetric analysis (TGA) and Fourier transform infrared spectrometry (FT-IR) showed the successful preparation of the dendritic stationary phase. The prepared stationary phase showed the retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and ion-exchange chromatography (IEC) under different mobile phase conditions. In detail, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and hydrophobic positional isomers were separated successfully in the RPLC mode. The baseline separation of nucleobases, nucleosides and flavonoids was achieved under HILIC mode, respectively. Meanwhile, some acidic and basic analytes were used to evaluate the IEC mode. The effects of different chromatographic conditions, such as acetonitrile content, salt concentration and pH in the mobile phase, on the different chromatographic modes were also investigated. In addition, the application of the mixed-mode dendritic stationary phase was demonstrated by the analysis of traditional Chinese medicine (TCM), including Carthamus tinctorius L. and Abelmoschus manihot (Linn.) Medicus. Interestingly, the stationary phase also has the ability for the capture and separation of boric acids. These meaningful applications confirmed that the mixed-mode dendritic stationary phase can be potentially applied in the analysis of complex samples.

Nucleobases and corresponding nucleosides display potent antiviral activities against dengue virus possibly through viral lethal mutagenesis.

Dengue virus affects millions of people worldwide each year. To date, there is no drug for the treatment of dengue-associated disease. Nucleosides are effective antivirals and work by inhibiting the accurate replication of the viral genome. Nucleobases offer a cheaper alternative to nucleosides for broad antiviral applications. Metabolic activation of nucleobases involves condensation with 5-phosphoribosyl-1-pyrophosphate to give the corresponding nucleoside-5'-monophosphate. This could provide an alternative to phosphorylation of a nucleoside, a step that is often rate limiting and inefficient in activation of nucleosides. We evaluated more than 30 nucleobases and corresponding nucleosides for their antiviral activity against dengue virus. Five nucleobases and two nucleosides were found to induce potent antiviral effects not previously described. Our studies further revealed that nucleobases were usually more active with a better tissue culture therapeutic index than their corresponding nucleosides. The development of viral lethal mutagenesis, an antiviral approach that takes into account the quasispecies behavior of RNA viruses, represents an exciting prospect not yet studied in the context of dengue replication. Passage of the virus in the presence of the nucleobase 3a (T-1105) and corresponding nucleoside 3b (T-1106), favipiravir derivatives, induced an increase in apparent mutations, indicating lethal mutagenesis as a possible antiviral mechanism. A more concerted and widespread screening of nucleobase libraries is a very promising approach to identify dengue virus inhibitors including those that may act as viral mutagens.