Studies of Protein Binding to Biomimetic Membranes Using a Group of Uniform Materials Based on Organic Salts Derived From 8-Anilino-1-naphthalenesulfonic Acid.

Tuning the 8-anilino-1-naphthalenesulfonic acid (ANS) structure usually requires harsh conditions and long reaction times, which can result in low yields. Herein, ANS was modified to form an ANS group of uniform materials based on organic salts (GUMBOS), prepared with simple metathesis reactions and distinct cations, namely tetrabutylammonium (N4444), tetrahexylammonium (N6666), and tetrabutylphosphonium (P4444). These ANS-based GUMBOS were investigated as fluorescent probes for membrane binding studies with four proteins having distinct physicochemical properties. Liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine were employed as membrane models as a result of their ability to mimic the structure and chemical composition of cell membranes. Changes in fluorescence intensity were used to monitor protein binding to liposomes, and adsorption data were fitted to a Freundlich-like isotherm. It was determined that [N4444][ANS] and [P4444][ANS] GUMBOS have enhanced optical properties and lipophilicity as compared to parent ANS. As a result, these two GUMBOS were selected for subsequent protein-membrane binding studies. Both [N4444][ANS] and [P4444][ANS] GUMBOS and parent ANS independently reached membrane saturation within the same concentration range. Furthermore, distinct fluorescence responses were observed upon the addition of proteins to each probe, which demonstrates the impact of properties such as lipophilicity on the binding process. The relative maintenance of binding cooperativity and maximum fluorescence intensity suggests that proteins compete with ANS-based probes for the same membrane binding sites. Finally, this GUMBOS-based approach is simple, rapid, and involves relatively small amounts of reagents, making it attractive for high-throughput purposes. These results presented herein can also provide relevant information for designing GUMBOS with ameliorated properties.

Protein discrimination using erythrosin B-based GUMBOS in combination with UV-Vis spectroscopy and chemometrics.

GUMBOS (Group of Uniform Materials Based on Organic Salts) have recently emerged as interesting materials for protein analysis due to their unique features and high tunability. In this regard, four novel erythrosin B (EB)-based GUMBOS were synthesized and their potential to discriminate among proteins with distinct properties (e.g., size, charge, and hydrophobicity) was assessed. These solid-phase materials were prepared using a single-step metathesis reaction between EB and various phosphonium and ammonium cations, namely tetrabutylphosphonium (P4444+), tributylhexadecylphosphonium (P44416+), tetrabutylammonium (N4444+), and benzyldimethylhexadecylammonium (BDHA+). Subsequently, the effect of pH (3.0, 4.5, and 6.0) and reaction time (5, 10, and 15 min) on the discriminatory power of synthesized GUMBOS was evaluated. Absorption spectra resulting from the interaction between EB-based GUMBOS and proteins were analyzed using partial least squares discriminant analysis (PLSDA). Unlike time, the pH value was determined to have influence over GUMBOS discrimination potential. Correct protein assignments varied from 86.5% to 100.0%, and the best discriminatory results were observed for [P4444]2[EB] and [N4444]2[EB] at pH 6.0. Additionally, these two GUMBOS allowed discrimination of protein mixtures containing different ratios of albumin and myoglobin, which appeared as individualized clusters in the PLSDA scores plots. Overall, this study showcases EB-based GUMBOS as simple synthetic targets to provide a label-free, cost-effective, rapid, and successful approach for discrimination of single proteins and their mixtures.

Development of an automated yeast-based spectrophotometric method for toxicity screening: Application to ionic liquids, GUMBOS, and deep eutectic solvents.

Saccharomyces cerevisiae has been used as a eukaryotic model organism for studying the toxic effects of various compounds. In this context, an automated spectrophotometric method based on the enzymatic reduction of methylene blue dye to a colorless product by living yeast cells was implemented in a sequential injection analysis system. Loss of yeast viability/impaired metabolic activity was monitored by an increase in optical density at 664 nm. To prove the usefulness of this approach, the toxicity of ILs (ionic liquids), GUMBOS (group of uniform materials based on organic salts), and DESs (deep eutectic solvents) was examined. Differences obtained between IC50 values confirmed the impact of structural elements on each compounds' toxicity. While DESs appeared to be less toxic than ILs, GUMBOS were found to be among the most toxic compounds to yeast cells and thus can be viewed as promising antimicrobial candidates. The automated methodology showed satisfactory repeatability and reproducibility (RSD < 9%), which is in good agreement with Green Chemistry principles. In fact, the method required consumption of only 40 µL of reagents and produced less than 2 mL of effluents per cycle. Thus, the developed assay can be used as an alternative tool for toxicity screening.

GUMBOS and nanoGUMBOS in chemical and biological analysis: A review.

GUMBOS (group of uniform materials based on organic salts) is a novel class of materials that exhibits similar features to those of ionic liquids, but have melting points between 25 and 250 °C. GUMBOS can be easily converted into nanomaterials (nanoGUMBOS), with advantages of working at nanoscale. Due to the huge number of possible cation-anion combinations, these materials can be multifunctional and designed for a specific task. This review highlights the possibility of fine-tuning GUMBOS physical and chemical properties in view of changing their ionic counterparts. Their outstanding potential for analytical applications is shown through recent developments in areas such as sensing, and solid-phase extraction. Available methods for synthesis of nanoGUMBOS, and their different outcomes in shapes and optical properties are described, with pros and cons being outlined. Finally, an analysis is made of opportunities and challenges faced by this class of organic ionic materials.

Environmental Impact of Ionic Liquids: Recent Advances in (Eco)toxicology and (Bio)degradability.

This Review aims to integrate the most recent and pertinent data available on the (bio)degradability and toxicity of ionic liquids for global and critical analysis and on the conscious use of these compounds on a large scale thereafter. The integrated data will enable focus on the recognition of toxicophores and on the way the community has been dealing with them, with the aim to obtain greener and safer ionic liquids. Also, an update of the most recent biotic and abiotic methods developed to overcome some of these challenging issues will be presented. The review structure aims to present a potential sequence of events that can occur upon discharging ionic liquids into the environment and the potential long-term consequences.

Assessment of ionic liquids' toxicity through the inhibition of acylase I activity on a microflow system.

Acylase I (ACY I) plays a role in the detoxication and bioactivation of xenobiotics as well in other physiological functions. In this context, an automated ACY I assay for the evaluation of ionic liquids' (ILs) toxicity was developed. The assay was implemented in a sequential injection analysis (SIA) system and was applied to eight commercially available ILs. The SIA methodology was based on the deacetylation of N-acetyl-l-methionine with production of l-methionine, which was determined using fluorescamine. ACY I inhibition in the presence of ILs was monitored by the decrease of fluorescence intensity. The obtained results confirmed the influence of ILs' structural elements on its toxicity and revealed that pyridinium and phosphonium cations, longer alkyl side chains and tetrafluoroborate anion displayed higher toxic effect on enzyme activity. The developed methodology proved to be robust and exhibited good repeatability (RSD < 1.3%, n = 10), leading also to a reduction of reagents consumption and effluents production. Thus, it is expected that the proposed assay can be used as a novel tool for ILs' toxicity screening.

Automated evaluation of protein binding affinity of anti-inflammatory choline based ionic liquids.

In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API). In each cycle, 100 µL of HSA and 100 µL of IL-API (variable concentration) were aspirated at a flow rate of 1 mL min(-1) and then sent through the reaction coil to the detector where the fluorescence intensity was measured. In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants (Kd) were calculated by means of models of drug-protein binding in the equilibrium. The calculated Kd showed that all the compounds bind strongly to HSA (Kd<100 µmol L(-1)) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding. The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%).

Imidazolium ionic liquids as solvents of pharmaceuticals: influence on HSA binding and partition coefficient of nimesulide.

In this work, the influence of imidazolium ionic liquids (ILs) on bio-chemical parameters that influence the in vivo behavior of nimesulide was evaluated. In this context, the binding of nimesulide to human serum albumin (HSA), in IL media, was studied. In parallel, the evaluation of the interaction of drug-IL systems, with micelles of hexadecylphosphocholine (HDPC), enabled the calculation of partition coefficients (K(p)). Both assays were performed in buffered media in the absence and in the presence of emim [BF(4)], emim [Ms] and emim [TfMs] 1%. Even though there was an increase of the dissociation constant (K(d)) in IL media, nimesulide still binds to HSA by means of strong interactions. The thermodynamic analysis indicates that the interaction is spontaneous for all the tested systems. Moreover, the studied systems exhibited properties that are favorable to the interaction of the drug with biological membranes, with K(p) values 2.5-3.5 higher than in aqueous environment. The studied nimesulide-IL systems presented promising characteristics regarding the absorption and distribution of the drug in vivo, so that the studied solvents seem to be good options for drug delivery.