Amphiphilic Sialic Acid Derivatives as Potential Dual-Specific Inhibitors of Influenza Hemagglutinin and Neuraminidase.

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

Molecular data storage using direct analysis in real time (DART) ionization mass spectrometry for decoding.

Molecular data storage is becoming a viable alternative to traditional information storage systems. Here, we propose a method where the presence or absence of a given molecule in a mixture of compounds represents a bit of information. As a novel approach, direct analysis in real time (DART) ionization mass spectrometry is used to recover and decode the information stored at the molecular level. Nicotinic acid derivatives were synthesized and used as the 'bit compounds'. Their volatility and ease of ionization make these molecules especially suitable for DART-MS detection. The application of DART-MS as a method with an ambient ionization technique, enables the re-reading of digital chemical codes embedded in the material of ordinary objects. Our method is designed to store and read back short pieces of digital information, up to several hundred bits. These codes can have the function of barcodes or QR codes, as shown in our proof-of-principle applications. First, modelling a QR code as a link to our university's website, three solutions were prepared, each representing 22 bits. Proceeding further, the bit compounds were incorporated into a polymer matrix that is suitable for 3D printing, and a toy ship was created with a hidden barcode. In addition, decoding software was developed to process the DART-MS spectra. The nicotinic acid components representing the bits dominated the DART-MS spectra and error-free decoding was achieved.

Enhanced Copolymer Characterization for Polyethers Using Gel Permeation Chromatography Combined with Artificial Neural Networks.

Gel permeation chromatography (GPC) is a generally applied method for the mass analysis of various polymers and copolymers, but it inherently fails to provide additional important information such as the composition of copolymers. However, we will show that GPC measurements using different solvents can yield not just the correct molecular weight but the composition of the copolymer. Accordingly, artificial neural networks (ANNs) have been developed to process the data of GPC measurements and determine the molecular weight and the chemical composition of the copolymers. The target values of the ANNs were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy. Our GPC-ANN method is demonstrated by the analysis of various poloxamers, i.e., poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO) block copolymers. Two ANNs were constructed. The first one (ANN_1) works in a wider mass range (from 900 to 12,500 dalton), while the second one (ANN_2) produces more output values. ANN_2 can thus predict seven characteristic copolymer parameters, namely, two average molecular weights, the average weight fraction of the EO unit, and four average numbers of the repeat units. The correlation between the experimentally obtained outputs and the predicted ones is high (r > 0.98). The accuracy of the ANNs is very convincing, and both ANNs predict the number-average molecular weight (Mn) with an accuracy below 5%. Furthermore, this work is the first step for creating an open database and applications extending the use of the GPC-ANN method for the analysis of copolymers.

Multiple mechanisms contribute to fluorometry signals from the voltage-gated proton channel.

Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements.

Isocyanonaphthol Derivatives: Excited-State Proton Transfer and Solvatochromic Properties.

Fluorescent probes that exhibit solvatochromic or excited-state proton-transfer (ESPT) properties are essential tools for the study of complex biological or chemical systems. Herein, the synthesis and characterization of a novel fluorophore that reveals both features, 5-isocyanonaphthalene-1-ol (ICOL), are reported. Various solvatochromic methods, such as Lippert−Mataga and Bilot−Kawski, together with time-dependent density functional theory (TD-DFT) and time-resolved emission spectroscopy (TRES), were applied to gain insights into its excited-state behavior. To make comparisons, the octyloxy derivative of ICOL, 5-isocyano-1-(octyloxy)naphthalene (ICON), was also prepared. We found that internal charge transfer (ICT) takes place between the isocyano and −OH groups of ICOL, and we determined the values of the dipole moments for the ground and excited states of both ICOL and ICON. Furthermore, in the emission spectra of ICOL, a second band at higher wavelengths (green emission) in solvents of higher polarities (dual emission), in addition to the band present at lower wavelengths (blue emission), were observed. The extent of this dual emission increases in the order of 2-propanol < methanol < N,N-dimethylformamide (DMF) < dimethyl sulfoxide (DMSO). The presence of the dual fluorescence of ICOL in these solvents can be ascribed to ESPT. For ICOL, we also determined ground- and excited-state pKa values of 8.4 ± 0.3 and 0.9 ± 0.7, respectively, which indicates a considerable increase in acidity upon excitation. The TRES experiments showed that the excited-state lifetimes of the ICOL and ICON spanned from 10.1 ns to 5.0 ns and from 5.7 ns to 3.8 ns, respectively. In addition, we demonstrated that ICOL can be used as an effective indicator of not only the critical micelle concentration (cmc) of ionic (sodium lauryl sulfate (SLS)) and nonionic surfactants (Tween 80), but also other micellar parameters, such as partition coefficients, as well as to map the microenvironments in the cavities of biomacromolecules (e.g., BSA). It is also pointed out that fluorescence quenching by pyridine can effectively be utilized for the determination of the fractions of ICOL molecules that reside at the water−micelle interface and in the interior spaces of micelles.

Quantification of Polyethylene Glycol 400 Excreted in the Urine by MALDI-TOF Mass Spectrometry.

Polyethylene glycol 400 (PEG 400) was used as a permeability probe to examine the gastrointestinal tract which can be involved in the pathogenesis of some inflammatory and autoimmune diseases. A novel methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The excretion ratios were determined for the most intense ions corresponding to nine PEG 400 oligomers. The relative error of accuracy was between -6.0% and 8.5%, and the relative standard deviation (RSD) of the precision was below 15%. Our method was successfully applied in a large-scale experimental study involving nearly two hundred volunteers. Due to the large number of measurements, detailed and reliable statistical analysis was performed. No significant difference was found between the male and female group of volunteers at 0.05 significance level, except the two largest PEG oligomers. However, the average excretion ratios of the male volunteers are greater than that of the women for all the nine PEG oligomers, suggesting a difference in the intestinal permeability between men and women.

Mass Spectral Filtering by Mass-Remainder Analysis (MARA) at High Resolution and Its Application to Metabolite Profiling of Flavonoids.

Flavonoids represent an important class of secondary metabolites because of their potential health benefits and functions in plants. We propose a novel method for the comprehensive flavonoid filtering and screening based on direct infusion mass spectrometry (DIMS) analysis. The recently invented data mining procedure, the multi-step mass-remainder analysis (M-MARA) technique is applied for the effective mass spectral filtering of the peak rich spectra of natural herb extracts. In addition, our flavonoid-filtering algorithm facilitates the determination of the elemental composition. M-MARA flavonoid-filtering uses simple mathematical and logical operations and thus, it can easily be implemented in a regular spreadsheet software. A huge benefit of our method is the high speed and the low demand for computing power and memory that enables the real time application even for tandem mass spectrometric analysis. Our novel method was applied for the electrospray ionization (ESI) DIMS spectra of various herb extract, and the filtered mass spectral data were subjected to chemometrics analysis using principal component analysis (PCA).

Tandem Mass-Remainder Analysis of Industrially Important Polyether Polyols.

The characteristics of the polyalkylene oxide polyether polyols highly influence the properties of final polyurethane products. As a novel approach, in order to gain structural information, the recently invented data mining procedures, namely the Mass-remainder analysis (MARA) and the Multistep Mass-remainder analysis (M-MARA) are successfully applied for the processing of tandem mass spectrometry (MS/MS) data of various industrially important polyether polyols. M-MARA yields an ultra-simplified graphical representation of the MS/MS spectra and sorts the product ions based on their double bond equivalent (DBE) values. The maximum DBE values unambiguously differentiate among the various polyether polyols. Accordingly, the characteristic DBE values were 0, 1 for the linear diol polyethers, 0, 1, 2 for the three-arm, and 0, 1 2, 3, 4 for the six-arm polyether polyols. In addition, it was also found that the characteristic collision energy necessary for the optimum fragmentation yield depended linearly on the molecular weight of the polyols. This relationship offers an easy way for instrument tuning to gain structural information.

Encoding Information into Polyethylene Glycol Using an Alcohol-Isocyanate "Click" Reaction.

In this article, the capability of encoding information using a homologous series of monodisperse monomethoxypolyethylene glycols (mPEG), with a number of ethylene oxide units ranging from nEO = 5 to 8, and monodisperse linear aliphatic isocyanates containing a number of CH2 units from 3 to 7, is demonstrated. The "click" reaction of the two corresponding homologous series yielded 20 different isocyanate end-capped polyethylene glycol derivatives (mPEG-OCONHR) whose sodiated adduct ion's nominal m/z values spanned from 360 to 548, providing an average ca. 8 m/z unit for the storage of one-bit information. These mPEG-OCONHR oligomers were then used to encode information in binary sequences using a 384-well MALDI sample plate and employing the common dried-droplet sample preparation method capable of encoding 20 bit, i.e., 2.5 byte information in one spot, was employed. The information stored in the spots was read by MALDI-TOF MS using the m/z value of the corresponding mPEG-OCONHR oligomers. The capability of the method to store data was demonstrated by writing and reading a text file, visualizing a small picture and capturing a short audio file written in Musical Instrument Digital Interface (MIDI) sequence. Due to the very large similarities in the chemical structures of the encoding oligomers and their "easy to be ionized" property, as well as their very similar ionization efficiencies, the MALDI-TOF MS signal intensities from each compound was so strong and unambiguous that complete decoding could be performed in each case. In addition, the set of the proposed encoding oligomers can be further extended to attain higher bit "densities".

Mass Spectrometric Characterization of Epoxidized Vegetable Oils.

Matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry (MALDI-MS and ESI-MS) were used for the characterization of epoxidized soybean and linseed oils, which are important raw materials in the biopolymer production. The recently invented data mining approach, mass-remainder analysis (MARA), was implemented for the analysis of these types of complex natural systems. Different epoxidized triglyceride mass spectral peak series were identified, and the number of carbon atoms and epoxide groups was determined. The fragmentation mechanisms of the epoxidized triglyceride (ETG) adducts formed with different cations (such as H⁺, Na⁺, Li⁺, and NH4⁺) were explored. As a novel approach, the evaluation of the clear fragmentation pathways of the sodiated ETG adducts enabled the estimation of the epoxidized fatty acid compositions of these types of oils by MS/MS.

Mass-Remainder Analysis (MARA): An Improved Method for Elemental Composition Assignment in Petroleomics.

Data processing and visualization methods have an important role in the mass spectrometric study of crude oils and other natural samples. The recently invented data mining procedure, Mass-Remainder Analysis (MARA), was further developed for use in petroleomics. MARA is based on the calculation of the remainder after dividing by the exact mass of a base unit, in petroleomics by the mass of the CH2 group. The two key steps in the MARA algorithm are the separation of the monoisotopic peaks from the other isotopic peaks and the subsequent intensity correction. The effectiveness of our MARA method was demonstrated on the analysis of lubricating mineral oil and crude oil samples by ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry experiments. MARA is able to handle a huge portion of the overlapped peaks even in a moderate resolution mass spectrum. With use of MARA, effective chemical composition assignment and visual representation were achieved for complex mass spectra recorded by a time-of-flight analyzer with a limited resolution of 40 000 at m/ z 400. In the absence of an ultra-high-resolution mass analyzer, MARA can provide a closer look on the mass spectral peaks, like a digital zoom in a simple camera.

Rapid mapping of various chemicals in personal care and healthcare products by direct analysis in real time mass spectrometry.

Residual chemicals on personal care and healthcare products, such as sanitary articles, sterile gauze bandages, nappies, plasters, were studied by direct analysis in real time mass spectrometry (DART-MS). We have identified around 40 compounds in seventeen different commercially available items. The tentative identification was further supported for about half of the chemicals by tandem mass spectrometric experiments (DART MS/MS). The most notable hazardous substances were tributyl phosphate, tris(2,4-di-tert-butylphenyl) phosphite, phthalic acid esters, erucamide, and nonylphenol ethoxylates (NPEs). In addition, we developed an efficient DART-MS analysis to determine the concentration of NPE in a swab sample. The quantitative result obtained by DART-MS was confirmed by liquid chromatography with mass spectrometric detection (LC-MS).

Mass-Remainder Analysis (MARA): a New Data Mining Tool for Copolymer Characterization.

A new data mining method is proposed for the determination of the copolymer composition from moderate/low resolution complex mass spectra. The Mass-remainder analysis (MARA) does not require a "Kendrick-like" transformation to a new mass scale, it is simply based on the calculation of the remainder after dividing by the exact mass of one of the repeat units of the copolymer (e.g., B of an A/B copolymer). Plotting the remainder of this division (MR) versus m/ z the homologous series differing only by a number of base units (e.g., B unit) can be visualized. The number of A units ( nA) and subsequently nB is assigned to the m/ z peaks using the bijective nA, MR mapping. Simultaneously, our algorithm removes the isotopes from the peak list. However, the intensities of the monoisotopes are increased to the value corresponding, approximately, to the total intensity of their isotope peaks. The correction of the mass spectral peak intensities enables the accurate calculation of the usual polymer and copolymer quantities: the molecular weight-average, the number-averaged molecular weight of A and B units, the composition drift, or the bivariate distribution, among others. Our Mass-remainder analysis method was demonstrated by the analysis of various ethylene oxide/propylene oxide copolymers.

Rapid qualitative analysis of 2 flavonoids, rutin and silybin, in medical pills by direct analysis in real-time mass spectrometry (DART-MS) combined with in situ derivatization.

Direct analysis in real-time mass spectrometry (DART-MS) with in situ silylation was used for the rapid analysis of the flavonoids silybin ((2R,3R)-3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenyl)-2-hydroxymethyl-2,3-dihydrobenzo[1,4]dioxin-6-yl]chroman-4-one) and rutin (quercetin-3-O-rutinoside). Three different derivatization reagents, hexamethyldisilazane/trimethylchlorosilane/pyridine (HMDS/TMCS/pyridine), N,O-bis(trimethylsilyl)acetamide/trimethylchlorosilane/N-trimethylsilyimidazole (BSA/TMCS/TMSI), and N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS), were applied. Silybin and rutin were detected with various degrees of silylation, and the formation of dimers with pyridine and imidazole was also observed. HMDS/TMCS/pyridine was the best choice for the DART-MS analysis of silybin, and BSA/TMCS/TMSI was the most effective for the detection of rutin. The effects of the DART source temperature on desorption, ionization, in-source fragmentation, dimer formation, and hydrolysis of the trimethylsilyl groups were also studied. In addition, the collision-induced dissociation properties of the derivatized silybin and rutin were explored. With our in situ silylation method, the derivatized bioactive compounds in intact medical pills could also be detected by DART-MS.

Self-Nanoemulsifying Drug Delivery Systems Containing Plantago lanceolata-An Assessment of Their Antioxidant and Antiinflammatory Effects.

The most important components of Plantago lanceolata L. leaves are catalpol, aucubin, and acteoside (=verbascoside). These bioactive compounds possess different pharmacological effects: anti-inflammatory, antioxidant, antineoplastic, and hepatoprotective. The aim of this study was to protect Plantago lanceolata extract from hydrolysis and to improve its antioxidant effect using self-nano-emulsifying drug delivery systems (SNEDDS). Eight SNEDDS compositions were prepared, and their physical properties, in vitro cytotoxicity, and in vivo AST/ALT values were investigated. MTT cell viability assay was performed on Caco-2 cells. The well-diluted samples (200 to 1000-fold dilutions) proved to be non-cytotoxic. The acute administration of PL-SNEDDS compositions resulted in minor changes in hepatic markers (AST, ALT), except for compositions 4 and 8 due to their high Transcutol contents (80%). The non-toxic compositions showed a significant increase in free radical scavenger activity measured by the DPPH test compared to the blank SNEDDS. An indirect dissolution test was performed, based on the result of the DPPH antioxidant assay; the dissolution profiles of Plantago lancolata extract were statistically different from each SNEDDS. The anti-inflammatory effect of PL-SNEDDS compositions was confirmed by the ear inflammation test. For the complete examination period, all compositions decreased ear edema as compared to the positive (untreated) control. It can be concluded that PL-SNEDDS compositions could be used to deliver active natural compounds in a stable, efficient, and safe manner.

Screening of additives and other chemicals in polyurethanes by direct analysis in real time mass spectrometry (DART-MS).

Direct analysis in real time mass spectrometry (DART-MS) was used to characterize commercial polyurethane (PUR) samples without sample pretreatment. More than 50 substances, such as catalysts, stabilizers, antioxidants, flame retardants, plasticizers, chain extenders, chain terminators, polyols, solvents, degradation products and contaminants, a few of them presumably toxic, were detected and identified in 18 PUR items. The identification of 16 compounds was further confirmed by DART MS/MS experiments. Catalysts were the largest class of compounds detected in the PURs by DART-MS. In each of the 18 PUR samples, at least one catalyst residue was identified. In addition, DART-MS was able to detect the migration of hazardous chemicals from the PURs to other objects. The collision-induced dissociation (CID) properties of two PUR catalysts, such as the protonated bis[2-(dimethylamino)ethyl] ether (DMAEE) and the protonated 2,2-dimorpholinodiethylether (DMDEE), as well as those of two PUR antioxidants (Antioxidant 1135 and Antioxidant 1076), were explored.

An approach to estimate the activation energies of fragmentation occurring in quadrupole collision cell of the mass spectrometer.

The classical semi-quantitative Rice-Ramsperger-Kassel (RRK) theory was used for the calculation of the internal energy dependent reaction rate coefficient of the collision-induced dissociation (CID) reaction in tandem mass spectrometry (MS/MS). The survival yield (SY) was determined by the reaction rate equation for the unimolecular dissociation of the precursor ion. The parameters of the rate equation and the RRK model were approximated based on the instrumental conditions. We used the RRK equation for the description of the basic behavior of the fragmentation reactions and for the estimation of the internal energy of the precursor ion. The critical energies for fragmentation (Eo ) of various molecules were estimated and compared with those reported in the literature. The model was extended by taking into account the initial internal energy distribution of the ions created in the ion source. It must be emphasized that our approach provides only a crude estimate for Eo .

Rapid Discrimination of Closely Related Seed Herbs (Cumin, Caraway, and Fennel) by Direct Analysis in Real Time Mass Spectrometry (DART-MS).

Direct analysis in real time mass spectrometry (DART-MS) was applied as a rapid method for the discrimination of the spices and traditional medicines cumin (Cuminum cyminum L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.). The seeds of these plants were analyzed without sample preparation by DART ion source coupled with quadrupole time-of-flight (QTOF) tandem mass spectrometry. The relatively clean DART spectra showed characteristic patterns, fingerprints, for each herb. It was found that a marker compound can be assigned to each species that can identify unambiguously these plants. Principal component analysis has also been used to analyze the DART-MS data of these seed herbs. Crispanone, carvone, and fenchone are the dominant compounds in the positive DART spectra of cumin, caraway, and fennel, respectively. Crispanone was first time identified as a constituent of cumin. Furthermore, the collision-induced dissociation (CID) behavior of the [M+NH4]+ ion of crispanone was also described.

Gas-Phase Interaction of Anions with Polyisobutylenes: Collision-Induced Dissociation Study and Quantum Chemical Modeling.

The gas-phase interaction of anions including fluoride, chloride, bromide, iodide, ethyl sulfate, chlorate, and nitrate with polyisobutylene (PIB) derivatives was studied using collision-induced dissociation (CID). The gas-phase adducts of anions with PIBs ([PIB + anion](-)) were generated from the electrosprayed solution of PIBs in the presence of the corresponding anions. The so-formed adducts subjected to CID showed a loss of anion at different characteristic collision energies, thus allowing the study of the strength of interaction between the anions and nonpolar PIBs having different end-groups. The values of characteristic collision energies (the energy needed to obtain 50% fragmentation) obtained by CID experiments correlated linearly with the binding enthalpies between the anion and PIB, as determined by density functional theory calculations. In the case of halide ions, the critical energies for dissociation, that is, the binding enthalpies for [PIB + anion](-) adducts, increased in the order of I(-) < Br(-) < Cl(-) < F(-). Furthermore, it was found that the binding enthalpies for the adducts formed with halide ions decreased approximately with the square radius of the halide ion, suggesting that the strength of interaction is mainly determined by the "surface" charge density of the halide ion. In addition, the characteristic collision energy versus the number of isobutylene units revealed a linear dependence.