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ADP-ribosylation refers to the post-translational modification of protein substrates with monomers or polymers of the small molecule ADP-ribose. ADP-ribosylation is enzymatically regulated and plays roles in cellular processes including DNA repair, nucleic acid metabolism, cell death, cellular stress responses, and antiviral immunity. Recent advances in the field of ADP-ribosylation have led to the development of proteomics approaches to enrich and identify endogenous ADP-ribosylated peptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). A number of these methods rely on reverse-phase solid-phase extraction as a critical step in preparing cellular peptides for further enrichment steps in proteomics workflows. The anionic ion-pairing reagent trifluoroacetic acid (TFA) is typically used during reverse-phase solid-phase extraction to promote retention of tryptic peptides. Here we report that TFA and other carboxylate ion-pairing reagents are inefficient for reverse-phase solid-phase extraction of ADP-ribosylated peptides. Substitution of TFA with cationic ion-pairing reagents, such as triethylammonium acetate (TEAA), improves recovery of ADP-ribosylated peptides. We further demonstrate that substitution of TFA with TEAA in a proteomics workflow specific for identifying ADP-ribosylated peptides increases identification rates of ADP-ribosylated peptides by LC-MS/MS.

Enantiomers of cationic compounds of pharmaceutical relevance, namely tetrahydro-s-carboline and 1,2,3,4-tetrahydroisoquinoline analogs, were separated by high-performance liquid chromatography. Separations were performed on Cinchona-alkaloid-based zwitterionic ion exchanger type chiral stationary phases applied as cation exchangers using mixtures of methanol and acetonitrile or tetrahydrofuran as bulk solvent components containing triethylammonium acetate or ammonium acetate as organic salt additives. On the zwitterionic ZWIX(+) and ZWIX(-) columns investigated, retention and enantioseparation of the studied basic analytes were influenced by the nature and concentration of the organic components of the mobile phase. The effect of organic salt additives on the retention behavior of the studied analytes can be described by the stoichiometric displacement model related to the counterion concentration. Investigations on the structure-retention relationships were performed applying different mobile phase systems for the two types of cationic analytes. For the thermodynamic characterization, parameters such as changes in standard enthalpy (Δ(ΔH°)), entropy (Δ(ΔS°)), and free energy (Δ(ΔG°)) were calculated on the basis of van't Hoff plots derived from the ln α versus 1/T curves. In most cases, enthalpy-driven enantioseparations were observed, with a consistent dependence of the calculated thermodynamic parameters on the mobile phase composition. Elution sequences of the studied compounds were determined in all cases.

I have explored the structural features and the dynamics of triethylammonium acetate by means of semi-empirical (density functional tight binding, DFTB) molecular dynamics. I find that the results from the present simulations agree with recent experimental determinations with only few minor differences in the structural interpretation. A mixture of triethylamine and acetic acid does not form an ionic liquid, but gives rise to a very complex system where ionization is only a partial process affecting only few molecules (1 over 4 experimentally). I have also found that the few ionic couples are stable and remain mainly embedded inside the AcOH neutral moiety.

The speciation of protic ionic liquids (ILs) has attracted high attention in the recent years, due to its effect in the properties of the resulting liquids. In this work we analyze the behavior of mixtures between triethylamine (N222) and acetic acid (HOAc), a system that generates controversy about its speciation. Using different synthetic strategies, liquids were obtained with molar compositions that were always between 1:2 and 1:4 mol/mol N222:HOAc. However, the experimentally observed normal boiling points indicate that the actual IL has a molar composition of 1:4 N222:[HOAc]/[OAc]−. Furthermore, when the synthesized liquid is mixed with the aprotic IL 1‑ethyl‑3‑methylimidazolium acetate, more triethylamine is expelled, indicating that the excess N222 forms an [HN222]+‑N222 complex rather than becoming ionized by the remaining HOAc present in the system. Our experimental data emphasizes the consideration of these systems not as mere “mixtures of ions and non-ionized molecules,” but as complex systems of oligomeric ions, with distinctive and characteristic properties.

Ionic Liquid (ILs) in oil microemulsions (IL/O MEs) have emerged as a potential carrier for transdermal drug delivery, particularly for sparingly soluble drug molecules, such as acyclovir and methotrexate. However, less attention was paid in the selection of non-toxic and biodegradable ILs. In this paper, we report new IL/O MEs with ammonium acetate ILs, which can dissolve acyclovir and methotrexate significantly. Microemulsions were composed of a mixture of nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80), and sorbitan laurate (Span-20)/ILs/oil isopropyl myristate (IPM). Three ILs called 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), triethylammonium acetate ([TEA][OAc]) and diethylammonium acetate ([DEA][OAc]) were selected due to their non-toxic property. Based on the pseudo-ternary diagrams constructed, it was evident that Span-20 played the key role as a surfactant while Tween-80 acted as the co-surfactant, showing optimal dissolution of the ILs into the system prepared with a ratio of 1:3 (Tween 80:Span20). This binary mixture also depicted the lowest viscosity, which is favorable as a carrier agent in drug delivery. The incorporation of various ILs into microemulsions resulted in a particle size ranging from 11 to 39.7 nm at diverse drug loading capacity. The formulation with IL [TEA][OAc] and IL [DEA][OAc] displayed substantial drug carrying capacity and stability compared to the commercially available IL [EMIM][OAc] loaded system.

A nonaqueous reversed phase liquid chromatography-mass spectrometry (LC-MS) method has been developed for extremely hydrophobic MPCs (monolayer-protected clusters), and has been applied to the efficient separation of gold–dodecanethiolate (ddt) assemblies, leading to the identification of three dodecanethiolate-protected gold clusters, Au130(ddt)50, Au137(ddt)56, and Au144(ddt)60, as prominent components of a commercial product of nominally 2 nm (core-diameter) protected gold nanoparticles obtained from nanoComposix, Inc. Various components were separated, according to hydrophobic character, using a linear gradient of methanol–dichloromethane mobile phases, on a C18 HPLC column. Varying concentrations of mobile-phase modifier (triethylammonium acetate) were compared for effect on chromatographic peak shape and cluster retention. Positive electrospray ionization (ESI) was used to ionize all components in the sample. LC separation prior to inline + ESI-MS detection facilitated sample analysis via production...

The aim of this work is to introduce, model, and optimize a new non-acid-catalyzed system for a direct N $$=$$ N–C bond formation. By reacting naphthols or phenol with anilines in the presence of the sodium nitrite as nitrosonium ( $$\hbox {NO}^{+})$$ source and triethylammonium acetate (TEAA), a N $$=$$ N–C group can be formed in non-acid media. Modeling and optimization of the reaction conditions were investigated by response surface method. Sodium nitrite, TEAA, and water were chosen as variables, and reaction yield was also monitored. Analysis of variance indicates that a second-order polynomial model with F value of 35.7, a P value of 0.0001, and regression coefficient of 0.93 is able to predict the response. Based on the model, the optimum process conditions were introduced as 2.2 mmol sodium nitrite, 2.2 mL of TEAA, and 0.5 mL $$\hbox {H}_{2}\hbox {O}$$ at room temperature. A quadratic (second-order) polynomial model, by analysis of variance, was able to predict the response for a direct N=N–C group formation. Predicted response values were in good agreement with the experimental values. Electrochemistry studies were done to introduce new Michael acceptor moieties. Broad scope, high yields, short reaction time, and mild conditions are some advantages of the presented method.

Enantioseparation of nineteen s2-amino acids has been performed by liquid chromatography on chiral stationary phases based on native teicoplanin and teicoplanin aglycone covalently bonded to 2.7 µm superficially porous silica particles. Separations were carried out in unbuffered (water/methanol), buffered [aqueous triethylammonium acetate (TEAA)/methanol] reversed-phase (RP) mode, and in polar-ionic (TEAA containing acetonitrile/methanol) mobile phases. Effects of pH in the RP mode, acid and salt additives, as well as counter-ion concentrations on chromatographic parameters have been studied. The structure of selectands (s2-amino acids possessing aliphatic or aromatic side chains) and selectors (native teicoplanin or teicoplanin aglycone) was found to have a considerable influence on separation performance. Analysis of van Deemter plots and determination of thermodynamic parameters were performed to further explore details of the separation performance.

The pharmaceutical industry faces a challenge to find potential solvents for drug molecules that are sparingly soluble in water and conventional organic solvents. Recently, ionic liquids (ILs) have attracted great attention as pharmaceutical solvents owing to their unique physicochemical and biological properties. In this study, the solubility of the sparingly soluble drug molecule acyclovir (ACV) in a wide variety of ILs was investigated by conductor-like screening model for real solvents (COSMO-RS) calculations. The predicted solubilities were validated by experimental measurements, and good agreement was found between the predicted and experimental results. The solubility of ACV was greatly affected by the structure of ILs, particularly the anionic moiety. Among the various ILs tested, ACV showed excellent solubility in ammonium-based ILs with an acetate anion. In vitro cytotoxicity of ILs to the MCF-10 normal breast epithelial cell line and cancer cell lines (MDA MB 231 and MCF 7) was investigated. The ammonium-based ILs showed higher IC 50 values than the imidazolium-based ILs with the acetate anion. Biodegradability results showed that diethylammonium acetate, triethylammonium acetate, and choline acetate ILs have high levels of biodegradation under aerobic conditions and can be classified as readily biodegradable. These findings will be useful for the design of IL-based drug delivery carriers that can act as versatile and efficient drug delivery systems for sparingly soluble drug molecules.

A one-pot method has been described to synthesize benzopyran-annulated pyrano[2,3-c]pyrazoles, effectively by combining O-alkenyloxy/alkynyloxy-acetophenones with various pyrazolones in triethylammonium acetate (TEAA) under microwave irradiation. While combination of O-allyloxy- or O-prenyloxy-acetophenones with pyrazolones occurred efficiently, that of O-propargyloxy-acetophenones was found effective in the presence of ZnO catalyst, via a domino Knoevenagel-hetero-Diels-Alder (DKHDA) reaction. Aminobenzopyran frameworks were also synthesized, after nitro-containing products were reduced in tandem with iron(II) in an acidic medium. The in vitro antiproliferative activity of these compounds was measured and discussed against gram-positive, gram-negative and M. tuberculosis bacteria, fungi, and various representative human solid tumor cell lines, in addition to their ferric reducing antioxidant capability.

A mild and efficient catalytic method has been developed to synthesize 3,4-dihydropyrimidinones in high yield by one-pot three component Biginelli condensation in the presence of triethylammonium acetate (TEAA) which acts as catalyst/reaction medium. Further, we have studied the antioxidant and antibacterial activities of these synthesized 3,4-dihydropyrimidinones. All the synthesized compounds reveal the significant antioxidant properties, these properties have been studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and cupric reducing antioxidant capacity (CUPRAC) assays. In addition, to this, these compounds also show the good antibacterial activity against four human pathogenic bacteria. (C) 2014 King Saud University. Production and hosting by Elsevier B.V.

Room temperature ionic liquids (IL) and deep eutectic solvents (DES) are known to aid the conformational stability and activity of proteins and enzymes in aqueous solutions. They are popular alternatives to harsh organic protecting osmolytes offsetting the thermal/chemical denaturation of proteins due to their greener attributes and low costs. In this paper, using extensive molecular dynamics (MD) simulations we show that a small helix–rich protein, chicken villin headpiece subdomain (HP-36), is rendered stable by an IL, triethylammonium acetate (TEAA) in aqueous solutions containing very high concentrations of urea (8 M). For comparison, the protein is also simulated in a deep eutectic mixture composed of 4 M choline chloride (ChCl) and 8 M urea under identical simulation conditions. Our simulations clearly show that a sharp increase in the relative proportion of TEAA (from 1 : 5 to 1 : 2 with respect to the denaturant urea) does not have a pronounced structure-enhancing effect on the protein. In contrast, for ChCl to function as an efficient protein stabilizer against the harsh action of urea, deep eutectic conditions are mandatory. To the best of our knowledge, this is the first study investigating the comparative aspects of the microscopic properties of a protein in an IL and a DES having similar ammonium-based structural frameworks.

Rationale A method was needed to accomplish solid-phase extraction of a large urine volume in a convenient way where resources are limited, towards a goal of metabolome and xenobiotic exposome analysis at another, distant location. Methods A porous extraction paddle (PEP) was set up, comprising a porous nylon bag containing extraction particles that is flattened and immobilized between two stainless steel meshes. Stirring the PEP after attachment to a shaft of a motor mounted on the lid of the jar containing the urine accomplishes extraction. The bag contained a mixture of nonpolar and partly nonpolar particles to extract a diversity of corresponding compounds. Results Elution of a urine-exposed, water-washed PEP with aqueous methanol containing triethylammonium acetate (conditions intended to give a complete elution), followed by MALDI-TOF/TOF-MS, demonstrated that a diversity of compounds had been extracted ranging from uric acid to peptides. Conclusions The PEP allows the user to extract a large liquid sample in a jar simply by turning on a motor. The technique will be helpful in conducting metabolomics and xenobiotic exposome studies of urine, encouraging the extraction of large volumes to set up a convenient repository sample (e.g. 2 g of exposed adsorbent in a cryovial) for shipment and re-analysis in various ways in the future, including scaled-up isolation of unknown chemicals for identification. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis of pyrazole-linked triazolo-pyrimidine hybrids was achieved by employing Biginelli-type reaction methodology in an ionic liquid (triethylammonium acetate) under microwave irradiation. This method proved to be highly efficient and the ionic liquid employed was found recyclable for up to five consecutive cycles. The synthesized molecules were further screened for their antimalarial efficacy screening out the active scaffolds J15, J18, J21, J24, J27, and J30. The active molecules were evaluated in an enzyme inhibition study against the active Plasmodium falciparum dihydrofolate reductase (Pf-DHFR), computationally as well as in vitro, demonstrating their potency as DHFR inhibitors. The active entities were also investigated for their oral bioavailability by predicting ADME properties in silico, indicating good bioavailability.

Graphical abstract, Highlights • The use of nESI-MS to examine intact Antibody Drug Conjugates (ADC). • Use of TEAA as charge reducing agent improves cysteine-linked ADC characterization. • TEAA preserves the intact mAb and facilitate easy drug load determination by native MS. • This method is particularly beneficial for users of low resolution mass spectrometers., Antibody-drug-conjugates (ADC) are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC–MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA) preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS.

Over the past few years, a continuous alteration of the recreational drug market took place. Among other novel psychoactive drugs, new synthetic tryptamine derivatives appeared on the market. These compounds are mainly traded via the Internet, which has become an important marketplace for the sale of recreational drugs. The goal of our research was to check, if 13 new synthetic tryptamines obtained by test purchase via different online vendors meet the promised identity. Analysis was performed by GC-MS, using a common 30 m HP-5MS capillary column as stationary phase. Subsequently, a simple HPLC method for the separation of these tryptamines was developed. Therefore, the aim was to establish a method to separate a broad spectrum of trypamines simultaneously within short time. Measurements were performed by a LiChrospher® RP-18e column and a mobile phase consisting of 0.1% triethylammonium acetate buffer, methanol and acetonitrile. Both presented methods were found to be suitable for the identification as well as separation of tryptamines as the analysis times were short and the selectivity sufficient. Moreover, enantioseparation of 3 chiral tryptamine derivatives purchased via the Internet by HPLC-UV and an Astec® Cyclobond I™ 2000 as CSP was performed. All of them were sold as racemic mixtures.

The densities of glycine based peptides namely glycyl-glycine (digly), glycine-ւ-valine (gly-val), glycyl-ւ-leucine (gly-leu) and glycyl-glycyl-glycine (triglycine/trigly) in aqueous solutions containing ~0.20 mol·kg−1 of triethylammonium acetate [TEAA], triethylammonium glycolate [TEAG], triethylammonium pyruvate [TEAPy] and triethylammonium propionate [TEAP] protic ionic liquids (PILs) are reported at 298.15 K and at atmospheric pressure. Experimental density data obtained for these systems were used to estimate apparent molar volume (ϕV), the limiting apparent molar volume (ϕV0), partial molar volume of solute ( V ¯ 2 ) and partial molar volume of mixed solvent (PILs + water) ( V ¯ 1 ). The change in limiting apparent molar volume due to transfer (ΔtrϕV0) of peptides from water to aqueous ionic liquid solutions were also estimated. The outcomes obtained from all these thermodynamic parameters were discussed in terms of ion–peptide interactions, ion–ion interactions, hydrophobic solvation, peptide group contributions to limiting volumes, etc.

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA) – hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100 mM, pH 7.0), the combination of HFIP (100 mM) and TEA (8.6 mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5 nM for CTP, UTP, dATP, dCTP, and dTTP, at 1 nM for ATP, and at 5 nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (< 15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

Rationale Ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-LC/ESI-MS) has been widely used for the quality control of oligonucleotides. However, researchers are still looking to improve mobile phase systems for IP-RP-LC/ESI-MS analysis of oligonucleotides. This study compared the performance of six ion-pairing reagents with three different counter anions for IP-RP-LC/ESI-MS analysis of oligonucleotides. Methods The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT Premier XE mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing acetate, bicarbonate, and hexafluoroisopropanolate salts of six ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine), respectively, were optimized for IP-RP-LC/ESI-MS analysis of oligonucleotides, and then the optimized conditions were applied for the separation of oligonucleotides. Results Counter anions definitely play a role in IP-RP-LC/ESI-MS analysis of oligonucleotides. Buffer containing 30 mM diisopropylethylamine and 200 mM hexafluoroisopropanol provided the highest separation of unmodified heterogeneous oligonucleotides, but tripropylammonium hexafluoroisopropanolate achieved the most enhanced separation of sequence isomers. However, triethylammonium acetate and bicarbonate had equally the highest separation for positional isomers. Conclusions IP-RP-LC/ESI-MS separation of oligonucleotides is mainly sequence dependent, but it is also dependent on both the type of ion-pairing reagent and counter anion present in the mobile phase.

An organic-inorganic hybrid monolithic column was prepared within the confines of a capillary via a single-step in situ sol-gel approach using zirconium tetrabutoxide as a precursor to compose the inorganic backbone and 3-triethoxysilylpropyl carbamoylated derivative of erythromycin (TEOSPC-ERY) as a co-precursor to introduce the organic chiral selector moiety in the zirconia backbone. The resulting carbamoylated ERY-zirconia hybrid monolith (ERY-ZHM) showed homogeneous morphology with well-defined through pores and was tightly connected with the inner wall of the capillary. The column was employed for capillary electrochromatographic enantioseparation of six basic chiral drugs in mobile phases (MPs) consisting of acetonitrile (ACN) and triethylammonium acetate (TEAA) buffer. The effects of composition of MP and applied voltage on chiral separation were investigated by using propranolol as a representative analyte. The highest resolution (Rs=3.33) was obtained with a MP consisting of 10/90 (v/v) ACN/TEAA buffer (10mM, pH 7), 10 kV applied voltage and 25°C capillary temperature. The relative standard deviations for resolution values regarding run to run, day to day, column to column and batch to batch repeatability were 0.41%, 0.89%, 1.80% and 2.26% (for n=3), respectively, indicating satisfactory stability of columns and reproducibility of column preparation process.

The interactions between ionic liquids (ILs) and amino acids (AAs) can easily be quantified using the apparent transfer free energy (∆G′tr) values that can be used to interpret the stability of the proteins in the same solvent medium. In this study, ∆G′tr values of AAs such as phenylalanine (Phe), histidine (His), threonine (Thr), glutamine (Gln) and serine (Ser) from the water to ILs have been obtained from the solubility measurements, as a function of IL concentration at 298.15 K under atmospheric pressure. The investigated ILs contain diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). The results reveal that the IL effect was specific and independent for each AA in the aqueous medium. Apparently, we observed a positive as well as negative ∆G′tr values for Phe, His, Glu, Thr, and Ser AAs in the ammonium-based ILs.

Syntheses of some new pyrrolo-fused pyrrolo[1,2-a] indole derivatives have been achieved by combining N-allyl-indole-2-carbaldehyde with a variety of N-alkyl-glycine esters as well as tetrahydroisoquinolines in an ionic liquid, triethylammonium acetate (TEAA), a recyclable reaction medium, via intramolecular [3+2] cycloaddition reaction. This new method is highly efficient, and the ionic liquid employed is recyclable. The stereochemistry of all the compounds was confirmed by 2D NMR NOESY and in some cases single crystal X-ray diffraction data. The in vitro screening of all new candidates against various bacterial strains and representative human solid tumor cell lines, A549 (lung), HeLa (cervix), SW1573 (lung), T-47D (breast) and WiDr (colon), revealed that many of them have good antibacterial, antifungal and antitubercular and antiproliferative activities.

Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer’s disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities.

There are two challenges in oligonucleotide detection by liquid chromatography coupled with mass spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber, and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as an ion-pair reagent. The MS signal intensity was improved 600-fold (for standard oligonucleotide dT15) by the isopropanol vapor assisted electrospray, and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS with few adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced more than 10-fold on average compared with adding acids into the electrospray solution. Finally, oligonucleotides and peptides or histones were simultaneously detected in MS with little interference with each other. Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.

A new reconfigurable contained-electrospray (ES) ion source is described that can be operated in three unique modes (Types I, II and III) and was applied to control the charge state of proteins for subsequent online characterization by mass spectrometry. Using this device, proteins prepared in 100% water were highly charged after exposure to hydrochloric acid vapor. For myoglobin, the shift to a higher charge state occurred faster than the heme cofactor could escape the unfolding protein. This effect reflected in the detection of highly charged holo-myoglobin intermediates (+26), which suggests the modification process occurred on a millisecond time scale (Type I mode). By introducing a cavity in the contained-ES emitter (Type II mode), increased protein denaturation was observed where only apo-myoglobin ions were detected. A similar increase in the charge effect was observed for less pH sensitive proteins such as ubiquitin and cytochrome c. In a third type of experiment, acidified aqueous-based droplets containing unfolded proteins were briefly exposed to aqueous ammonium acetate/triethylammonium acetate solution mixtures to increase the droplet pH and to induce protein folding during electrospray ionization. Charge-state distributions showed protein folding occurred, including the appearance of charged reduced species that were not observed when sprayed in pure water. The three operational modes of contained-electrospray allow online modification of an analyte during charge droplet formation using both vapor and non-volatile liquid reagents, without a direct addition of the modifying reagent to the analyte solution.

The folding/unfolding equilibrium of proteins in aqueous medium can be altered by adding small organic molecules generally termed as co-solvents. Denaturants such as urea are instrumental in the unfolding of proteins while protecting osmolytes favour the folded ensemble. Recently, room temperature ionic liquids (ILs) have been shown to counteract the deleterious effect of urea on proteins. In this paper, using atomistic molecular dynamics we show that a ternary mixture containing a particular ammonium-based IL, triethylammonium acetate (TEAA), and urea (in 1 : 5 molar ratio) helps a small 15-residue S-peptide analogue regain most of its native structure, whereas a binary aqueous mixture containing a large amount of urea alone completely distorts it. Our simulations show that the denaturant urea directly interacts with the peptide backbone in the binary mixture while for the ternary mixture both urea as well as the IL are preferentially excluded from the peptide surface.

In this study, perphenylcarbamated cyclodextrin clicked chiral stationary phase (CSP) was developed with high column efficiency. The characteristics of the column were evaluated in terms of linearity, limit of detection and limit of quantification. The enantioselectivity of the as-prepared clicked CSP was explored with 26 recemates including aryl alcohols, flavanoids and adrenergic drugs in reversed phase high-performance liquid chromatography. The effect of separation parameters including flow rate, column temperature, organic modifier and buffer on the enantioselectivity of the clicked CSP was investigated in detail. The correlation study of the analytes structure and their chiral resolution revealed the great influence of analytes' structure on the enantioseparations with cyclodextrin CSP. Methanol with 1% of triethylammonium acetate buffer (pH 4) was proved to be the best choice for the chiral separation of basic enantiomers.

There is a growing demand for the separation and identification of short nucleic acid fragments, such as oligonucleotides. There were two main goals of the present investigation, namely, evaluation of the impact of stationary phase type and the influence of various ion-pair reagents on the retention behavior of oligonucleotides in ion-pair liquid chromatography. Three types of ion-pair reagents were studied: triethylammonium acetate, dimethylbuthylammonium acetate and mixtures of 1,1,1,3,3,3-hexafluoro-2-propanol and triethylamine. Two novel types of packing materials, namely, cholesterol and alkylamide were used for this purpose for the first time. The results indicate that the mechanism of oligonucleotides retention is determined by the hydrophobicity of ion-pair reagents and polar ligands localized on the surface of stationary phases. Oligonucleotides were most effectively separated with the use of alkylamide and cholesterol packings. These two stationary phases reduce the time of analysis in comparison with the octadecyl packing material. Moreover, separation was achieved under non-denaturating conditions.

In the present work, we describe a novel application of functionalized single-walled carbon nanotubes (SWCNTs) as a stationary phase for the liquid chromatographic separation of polycyclic aromatic hydrocarbons and structurally similar analogues. The SWCNTs were first oxidized to give carboxylic derivatives (SWCNT-COOH), afterwards these were covalently derivatized with hydroxypropyl-β-cyclodextrin (HP-β-CD). Then, the HP-β-CD-SWCNTs were bonded to silica gel with 3-(triethoxysilyl) propyl isocyanate, which were used as a stationary phase to separate the investigated solutes by HPLC. On this stationary phase, eight polycyclic aromatic hydrocarbons were separated using water/methanol (5:5, v/v) as the mobile phase and six structurally similar dipine drugs were also separated using (3:7, v/v) methanol/triethylammonium acetate buffer (0.1%, v/v, pH 4.1) as the mobile phase. The results showed that the HP-β-CD-SWCNTs stationary phase had stronger separation ability for the aromatic hydrocarbons and analogues compared with the HP-β-CD stationary phase. This method can be used to improve the separation efficiency of the β-CD stationary phases, and the HP-β-CD-SWCNTs column can be used for the determination of the aromatic hydrocarbons in wastewater.

Chiral separation of benzoxazine enantiomers was studied on click chemistry based beta-cyclodextrin stationary phases under reversed-phase high performance liquid chromatographic mode. The effects of the type and percentage of the organic modifier, the concentration of triethylammonium acetate buffer (TEAA), and pH on enantioselective separation were examined and studied. It was demonstrated that acetonitrile was better for the chiral separation of benzoxazine enantiomers than methanol. It was observed that the retention time and the resolution of benzoxazine enantiomers decreased with the increase of the volume ratio of TEAA from 0.1% to 1.0%. The separation of benzoxazine enantiomers was of the maximum resolution at pH 4.1. With the optimized mobile phase of the mixture of acetonitrile and 0. 1% TEAA (pH 4.1), all the enantiomers were separated at the baseline. The chiral recognition mechanism is also discussed. The separation was probably based on the inclusion complex interaction and the hydrogen bonding between enantiomers and chiral stationary phases. This work provided the experience for the intensive study of click beta-cyclodextrin bonded stationary phases, and it also illustrated the potential of click chemistry in the preparation of beta-cyclodextrin based chiral stationary phase.

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9 nM and 4.2±0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

Tandem mass spectrometry is a tool to dissect noncovalent protein complexes into smaller substructures for quaternary structure analysis. The commonly used activation method, collision induced dissociation (CID), often provides limited structural information from the typical dissociation pattern where unfolded monomers are ejected from the protein complex. In contrast, surface-induced dissociation (SID) has been shown to be very effective at dissociating protein complexes with less unfolding than CID. We present here SID of a large noncovalent tetradecamer protein, GroEL (801 kDa). A wide variety of products, including heptamers representative of the native topology, are released from the precursor upon SID, significantly different from the ubiquitous monomer ejection in CID. Enhanced dissociation into heptamers is observed when the charge states of the GroEL precursor are reduced by adding triethylammonium acetate into the spraying buffer. Ion mobility is utilized after SID to separate products overlapping in m/z to simplify the SID spectra. Compact heptamers from the charge-reduced tetradecamer are clearly distinguished from other overlapping species. SID can be very useful for quaternary structure studies of large noncovalent protein complexes, as manifested by the GroEL data where the tetradecamer dissociates into heptamers, reflecting the native topology of the complex.

An improved domino/Knoevenagel-hetero-Diels-Alder reaction of two new aldehyde substrates; 7-olefinoxy-coumarin-8-carbaldehyde and 2-alkensulfanyl-quinoline-3-carbaldehyde, with pyrazolones was studied in ionic liquid triethylammonium acetate (TEAA), affording a series of pyrazolopyran annulated-pyrano-fused coumarins, and thiopyrano-fused quinolones. Besides acting as catalyst, since no additional catalyst used, the ionic liquid TEAA also promised its easy recovery. In all new polyheterocycles, the cis-fusion of two pyranyl rings had been inferred from 2D NMR COSY and NOESY experiments. All are good antitubercular agents, as they are found active against Mycobacterium tuberculosis H37Rv, and antibacterial agents, as they are found active against three Gram-positive (Streptococcus pneumoniae, Clostridium tetani, Bacillus subtilis) and three Gram-negative (Salmonella typhi, Vibrio cholerae, Escherichia coli) bacteria.

In this work, we explore the anion effect on the thermophysical properties of protic ionic liquids (PILs) such as triethylammonium acetate [Et3NH][CH3COO] (TEAA), triethylammonium dihydrogen phosphate [Et3NH][H2PO4] (TEAP) and triethylammonium hydrogen sulfate [Et3NH][HSO4] (TEAS) with polar solvent such as dimethylsulfoxide (DMSO). To understand the molecular interactions between DMSO with ILs, densities (ρ), ultrasonic sound velocities (u) and viscosities (η) have been measured over whole range of compositions and at three temperatures, 298.15, 303.15, and 308.15 K under atmospheric pressure. A second order empirical polynomial and Arrhenius equations were used to correlate the ρ and η as function of temperature, respectively. Sound velocity mixing rules were used on experimental values. Further, the excess molar volume (VE), the deviation in isentropic compressibility (Δκs) and deviation in viscosity (Δη) were calculated using these properties. The obtained VE, Δκs and Δη values were correlated by using the Redlich–Kister polynomial equation.

Ammonium based ionic liquids (ILs) are biocompatible co-solvents that stabilize the native state of proteins. Experimentally, we have explored the stability of α-chymotrypsin (CT) in the presence of nine ILs, i.e., diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), diethylammonium dihydrogen phosphate (DEAP), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), trimethylammonium acetate (TMAA), trimethylammonium hydrogen sulfate (TMAS), trimethylammonium dihydrogen phosphate (TMAP). Thermodynamic folding properties such as transition temperature (Tm), Gibbs free energy change of unfolding (ΔGU), enthalpy change (ΔH) and heat capacity change (ΔCp) of CT in ILs are obtained by fluorescence spectra analysis. Fluorescence and circular dichroism (CD) spectroscopy experiments were performed to probe CT stabilization and structural changes in the presence of ILs. Our experimental results suggest that the ILs act as stabilizers for the CT structure and the stability of CT depends on the structural arrangement of the ions of ILs. Our experimental results reveal that ILs (DEAA, DEAS and DEAP) having more hydrophobic ammonium cations [DEA+] are weak stabilizers for CT, while trimethyl ammonium cations [TMA+] ILs having small alkyl chain length such as TMAA, TMAS and TMAP are strong stabilizers and therefore more biocompatible for the native structure of CT.

A solvent-cum catalyst, ionic liquid triethyl- ammonium acetate-mediated one-pot procedure for the synthesis of some new angular benzopyrano(3,4-c)pyrano- fused pyrazoles, all of which incorporate a tertiary ring junction carbon, has been developed. The stereochemistry of the products has been confirmed by single-crystal X-ray diffraction data.

A simple and efficient one-pot synthesis of heteroaryl-substituted dihydropyrano(c)chromenes and pyrano[2,3-d]pyrimidines has been developed. Reaction proceeds via initial Knoevenagel, subsequent Michael and final heterocyclization reactions of heteroaryl aldehyde, malononitrile, and barbituric acid/dimedone. Triethylammonium acetate acts as a green catalyst as well as reusable solvents for this reaction. Short reaction time, environment friendly procedure, reusability, and excellent yields are the main advantages of this procedure. All synthesized compounds have shown good antibacterial activity against different microbial stains but not active against cancer cell lines.