Chiral ß-lactam-based integrin ligands through Lipase-catalysed kinetic resolution and their enantioselective receptor response.

Obtainment and testing of pure enantiomers are of great importance for bioactive compounds, because of the assessed implications of enantioselectivity in receptor-mediated responses. Herein we evaluated the use of biocatalysis to obtain enantiomerically pure ß-lactam intermediates further exploited in the synthesis of novel integrin ligands as single enantiomers. From a preliminary screening on a set of commercially available hydrolases, Burkholderia Cepacia Lipase (BCL) emerged as a suitable and highly performing enzyme for the kinetic resolution of a racemic azetidinone, key intermediate for the synthesis of novel agonists of integrins. Upon optimization of the biocatalytic protocol in terms of enzymes, acylating agents and procedures, the two ß-lactam enantiomers were obtained in excellent enantiomeric excesses (94% and 98% ee). Synthetic elaborations on the separated enantiomers allowed the synthesis of four chiral ß-lactams which were evaluated in cell adhesion assays on Jurkat cell line expressing α4ß1 integrin, and K562 cell line expressing α5ß1 integrin. Biological tests revealed that only (S)-enantiomers maintained the agonist activity of racemates with a nanomolar potency, and a specific enantio-recognition by integrin receptors was demonstrated.

Inter- and Intraspecific Variability of Nitrogenated Compounds in Gorgonian Corals via Application of a Fast One-Step Analytical Protocol.

Gorgonian corals play a structural role in temperate and tropical biogenic reefs, forming animal forests and creating biodiversity hot spots. In the Mediterranean Sea, slow-growing and long-lived gorgonian species are threatened by human disturbances and global environmental changes and concern about their conservation is rising. Alkaloid metabolites have proven to be essential in protecting these species from environmental stressors. Traditional profiling methodologies to detect these metabolites require a large quantity of living tissue. Here, the chemodiversity of gorgonian alkaloids was investigated by applying a fast and effective protocol combining extraction and derivatization using small-scale tissue samples and GC/MS analysis. The method was effective in identifying and quantifying alkaloids and guanine-based compounds. Eight N-heterocyclic compounds were found in six Mediterranean gorgonians differing for types and quantity. The metabolomic profile was conservative in species of the Eunicella genus, with three species sharing the same pattern. Conversely, Paramuricea clavata displayed a noticeable spatial pattern of variation among colonies collected in different locations. The analytical approach presented here proved to be effective, allowing rare, endangered, and small-sized species to be screened rapidly for detection of new compounds in order to explore their biological and ecological functions.

Ionic liquids effects on the permeability of photosynthetic membranes probed by the electrochromic shift of endogenous carotenoids.

Ionic liquids (ILs) are promising materials exploited as solvents and media in many innovative applications, some already used at the industrial scale. The chemical structure and physicochemical properties of ILs can differ significantly according to the specific applications for which they have been synthesized. As a consequence, their interaction with biological entities and toxicity can vary substantially. To select highly effective and minimally harmful ILs, these properties need to be investigated. Here we use the so called chromatophores--protein-phospholipid membrane vesicles obtained from the photosynthetic bacterium Rhodobacter sphaeroides--to assess the effects of imidazolinium and pyrrolidinium ILs, with chloride or dicyanamide as counter anions, on the ionic permeability of a native biological membrane. The extent and modalities by which these ILs affect the ionic conductivity can be studied in chromatophores by analyzing the electrochromic response of endogenous carotenoids, acting as an intramembrane voltmeter at the molecular level. We show that chromatophores represent an in vitro experimental model suitable to probe permeability changes induced in cell membranes by ILs differing in chemical nature, degree of oxygenation of the cationic moiety and counter anion.

Chemical and ecotoxicological properties of three bio-oils from pyrolysis of biomasses.

In view of the potential use of pyrolysis-based technologies, it is crucial to understand the environmental hazards of pyrolysis-derived products, in particular bio-oils. Here, three bio-oils were produced from fast pyrolysis of pine wood and intermediate pyrolysis of corn stalk and poultry litter. They were fully characterized by chemical analysis and tested for their biodegradability and their ecotoxicity on the crustacean Daphnia magna and the green alga Raphidocelis subcapitata. These tests were chosen as required by the European REACH regulation. These three bio-oils were biodegradable, with 40-60% of biodegradation after 28 days, and had EC50 values above 100mgL(-1) for the crustacean and above 10mgL(-1) for the alga, showing low toxicity to the aquatic life. The toxic unit approach was applied to verify whether the observed toxicity could be predicted from the data available for the substances detected in the bio-oils. The predicted values largely underestimated the experimental values.

Carbon dot/polylactic acid nanofibrous membranes for solar-mediated oil absorption/separation: Performance, environmental sustainability, ecotoxicity and reusability.

Carbon dots (CDs) are promising photothermal nanoparticles that can be utilized in environmental treatments. They exhibit favorable physicochemical properties, including low toxicity, physical and chemical stability, photo-dependant reversible behaviour, and environmentally friendly synthesis using benign building blocks. Here, we synthesized innovative CDs/polylactic acid (PLA) electrospun composite membranes for evaluating the removal of hydrophobic compounds like long-chain hydrocarbons or oils in biphasic mixtures with water. The ultimate goal was to develop innovative and sustainable solar-heated oil absorbents. Specifically, we fabricated PLA membranes with varying CD contents, characterized their morphology, thermal, and mechanical properties, and assessed the environmental impact of membrane production according to ISO 14040 and 14044 standards in a preliminary "cradle-to-gate" life cycle assessment study. Solar radiation experiments demonstrated that the CDs/PLA composites exhibited greater uptake of hydrophobic compounds compared to pure PLA membranes, ascribable to the CDs-induced photothermal effect. The adsorption and regeneration capacity of the new CDs/PLA membrane was demonstrated through multiple uptake/release cycles. Ecotoxicity analyses confirmed the safety profile of the new adsorbent system towards freshwater microalgae, further emphasizing its potential as an environmentally friendly solution for the removal of hydrophobic compounds in water treatment processes.

Membrane interactions of ionic liquids: possible determinants for biological activity and toxicity.

Ionic liquids (ILs) are a class of diverse organic salts with relatively low melting points (below 100°C) which have attracted considerable interest as a promising "green" substitute for organic solvents. The broad solvation properties of ILs and their high solubility in water, however, present health risks, in particular since it was shown that many ILs exhibit cytotoxic properties. In this context, interactions of ILs with the cellular membrane are believed to constitute a primary culprit for toxicity. We present a comprehensive biophysical and microscopy study of membrane interactions of a series of ILs having different side-chain compositions and lengths, and cationic head-group structures and orientations. The experimental data reveal that the ILs studied exhibit distinct mechanisms of membrane binding, insertion, and disruption which could be correlated with their biological activities. The results indicate, in particular, that both the side chain composition and particularly the head-groups of ILs constitute determinants for membrane activity and consequent cell toxicity. This work suggests that tuning membrane interactions of ILs should be an important factor for designing future compounds with benign environmental impact.

Shaping calcite crystals by means of comb polyelectrolytes having neutral hydrophilic teeth.

Comb polyelectrolytes (CPs) having neutral hydrophilic teeth, similar to double hydrophilic block copolymers, are a powerful tool to modify the chemical-physical properties of inorganic crystalline materials. One of their main applications is in concrete technology, where they work as superplasticizers, particle-dispersing agents. Here, CPs, having the same poly(acrylic acid) (PAA) backbone chain and differing in the grafting with methoxy poly(ethylene glycol) chains (MPEG) of two molecular weights, were used to investigate the influence of tooth chains in polymer aggregation and in control on morphology and aggregation of calcite particles. These polymers aggregate, forming interpolymer hydrogen bonds between carboxylic groups and ether oxygen functionalities. The presence of calcium ions in solution further enhances aggregation. Crystallization experiments of calcite in the presence of CPs show that the specificity of interactions between polymers and crystal planes and control on aggregation and size of particles is a function of the content and chain length of the MPEG in the PAA backbone. These parameters limit and can make specific the electrostatic interactions with ionic crystalline planes. Moreover, the mechanism of crystallization, classical or nonclassical, is addressed by the CP structure and concentration. These findings have implications in the understanding of the complex chemical processes associated to concrete superplasticizers action and in the study of the biomineralization processes, where biological comb polyelectrolytes, the acidic glycoproteins, govern formation of calcitic structures.

Extraction of astaxanthin from Haematococcus pluvialis with hydrophobic deep eutectic solvents based on oleic acid.

Three novel hydrophobic deep eutectic solvents (DESs) based on oleic acid and terpenes (thymol, dl-menthol, and geraniol) were prepared, characterized, and used to extract astaxanthin from the microalga Haematococcus pluvialis without any pre-treatment of the cells. The three DES were composed of Generally Recognized As Safe (GRAS) and edible ingredients. All the tested DESs gave astaxanthin recovery values of about 60 and 30% in 6 h if applied on freeze-dried biomass or directly on algae culture, respectively. The carotenoid profile was qualitatively identical to what was obtained by using traditional organic solvents, regardless of the DES used; the monoesters of astaxanthin with C18-fatty acids were the main compounds found in all the carotenoid extracts. The thymol:oleic acid DES (TAO) could preserve astaxanthin content after prolonged oxidative stress (40% of the astaxanthin initially extracted was still present after 13.5 h of light exposure), thanks to the superior antioxidant properties of thymol. The capacity of improving astaxanthin stability combined with the intrinsic safety and edibility of the DES components makes the formulation astaxanthin-TAO appealing for the food ingredients/additives industry.

Effects of imidazolium ionic liquids on growth, photosynthetic efficiency, and cellular components of the diatoms Skeletonema marinoi and Phaeodactylum tricornutum.

This article describes the toxic effects of imidazolium ionic liquids bearing alkyl (BMIM), monoethoxy (MOEMIM), and diethoxy (M(OE)(2)MIM) side chains toward two marine diatoms, Skeletonema marinoi and Phaeodactylum tricornutum. MOEMIM and M(OE)(2)MIM cations showed a lower inhibition of growth and photosynthetic efficiency with respect to their alkyl counterpart, with both algal species. However, a large difference in sensitivity was found between S. marinoi and P. tricornutum, the first being much more sensitive to the action of ionic liquids than the second one. The effects of salinity on BMIM Cl toxicity toward S. marinoi revealed that a decrease from salinity 35 to salinity 15 does not influence the biological effects toward the alga. Finally, Fourier transform infrared (FT-IR) microscopy of algal cells after ionic liquids exposure allowed us to detect an alteration of the organic cellular components related to silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentatively ascribed to different silica uptake and organization in outer cell walls.

Recovery of Polyhydroxyalkanoates From Single and Mixed Microbial Cultures: A Review.

An overview of the main polyhydroxyalkanoates (PHA) recovery methods is here reported, by considering the kind of PHA-producing bacteria (single bacterial strains or mixed microbial cultures) and the chemico-physical characteristics of the extracted polymer (molecular weight and polydispersity index). Several recovery approaches are presented and categorized in two main strategies: PHA recovery with solvents (halogenated solvents, alkanes, alcohols, esters, carbonates and ketones) and PHA recovery by cellular lysis (with oxidants, acid and alkaline compounds, surfactants and enzymes). Comparative evaluations based on the recovery, purity and molecular weight of the recovered polymers as well as on the potential sustainability of the different approaches are here presented.

Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass-1 d-1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L-1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L-1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.

Grape Pomace for Topical Application: Green NaDES Sustainable Extraction, Skin Permeation Studies, Antioxidant and Anti-Inflammatory Activities Characterization in 3D Human Keratinocytes.

Food waste is a global problem due to its environmental and economic impact, so there is great demand for the exploitation of new functional applications. The winemaking process leads to an incomplete extraction of high-value compounds, leaving the pomace still rich in polyphenols. This study was aimed at optimising and validating sustainable routes toward the extraction and further valorisation of these polyphenols, particularly for cosmeceutical applications. New formulations based on red grape pomace polyphenols and natural deep eutectic solvents (NaDESs) were here investigated, namely betaine combined with citric acid (BET-CA), urea (BET-U) and ethylene glycol (BET-EG), in which DESs were used both as extracting and carrying agents for polyphenols. The flavonoid profile determined by HPLC-MS/MS analysis showed similar malvidin content (51-56 µg mL-1) in the DES combinations, while BET-CA gave the best permeation performance in Franz cells, so it was further investigated in 3D human keratinocytes (HaCat spheroids) injured with the pro-oxidant agent menadione. BET-CA treatment showed good intracellular antioxidant activity (IC50 0.15 ± 0.02 µg mL-1 in malvidin content) and significantly decreased (p < 0.001) the release of the pro-inflammatory cytokine IL-8, improving cell viability. Thus, BET-CA formulation is worthy of investigation for potential use as a cosmetic ingredient to reduce oxidative stress and inflammation, which are causes of skin aging.

Chemoenzymatic synthesis of (2S)-2-arylpropanols through a dynamic kinetic resolution of 2-arylpropanals with alcohol dehydrogenases.

We applied Horse Liver Alcohol Dehydrogenase (HLADH) to the enantioselective synthesis of six (2S)-2-arylpropanols, useful intermediates in the synthesis of Profens. The influence of substrate structure and reaction conditions on yields and enantioselectivity were investigated. The high yields and high enantioselectivity towards the (S)-enantiomer obtained in the bioreduction of 2-arylpropionic aldehydes, clearly indicate the achievement of a DKR process through a combination of an enzyme-catalyzed kinetic reduction with a chemical base-catalyzed racemization of the unreacted aldehydes. The racemization step is represented by the keto-enol equilibrium of the aldehyde and can be controlled by modulating pH and reaction conditions.

Introduction of oxygenated side chain into imidazolium ionic liquids: evaluation of the effects at different biological organization levels.

The biological effects of a class of oxygenated imidazolium ionic liquids were studied in comparison with alkyl imidazolium salts (BMIM BF4 and BMIM N(CN)2).The cellular and subcellular effects were evaluated on rat pheochromocytoma PC12 cell lines, through MTT test, lactate dehydrogenase release and acetylcholinesterase inhibition; the eco-toxicological responses were assessed through the acute toxicity tests towards Daphnia magna and Vibrio fischeri. The introduction of ethoxy moieties in the lateral chain of imidazolium cations reduced the biological effects in all the tests. The acute toxicity towards D. magna was not affected by the number of ethoxy units, but the crustacean seemed to be sensitive to the type of anion; on the contrary, a further addition of ethoxy moieties increased the toxicity towards V. fischeri, M(OE)4MIM N(CN)2 being the most toxic oxygenated ionic liquid. In the cytotoxicity assays the salts with oxygenated cations resulted ineffective compared to BMIMs, independently from the anion and the number of ethoxy units in the lateral chain. In order to estimate the influence on membrane fluidity, an analysis of fluorescence anisotropy was done and it indicated that BMIM BF4, the most toxic ionic liquid among the tested ones, led to a destabilization of the model membranes at any molarity.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

New polyphenolic beta-lactams with antioxidant activity.

Novel 4-alkylidene-beta-lactams with a polyphenolic side chain were synthesized and evaluated as radical scavengers. We have undertaken a detailed study of the antioxidant activity in vitro with chemical and biological testing of the new beta-lactams and of the corresponding methyl polyhydroxy benzoates. Antioxidant activity of beta-lactams and methyl benzoates was measured with the Briggs-Rauscher oscillating reaction, the TEAC (Trolox( Equivalent Antioxidant Capacity) assay, and as ability to inhibit ROS (=Reactive Oxygen Species) production on myoblast H9c2 cells. The results were discussed with regard to mechanism and correlated with structural parameters.

Highly efficient asymmetric reduction of arylpropionic aldehydes by horse liver alcohol dehydrogenase through dynamic kinetic resolution.

The enantioselective synthesis of (2S)-2-phenylpropanol and (2S)-2-(4-iso-butylphenyl)propanol ((S)-Ibuprofenol) has been achieved by means of Horse Liver Alcohol Dehydrogenase (HLADH) in buffered aqueous solution or buffered organic solvent mixtures; under the reaction conditions, a dynamic kinetic resolution (DKR) process was realized with good reaction yields and enantiomeric ratios.

Acute toxicity of oxygenated and nonoxygenated imidazolium-based ionic liquids to Daphnia magna and Vibrio fischeri.

Room-temperature ionic liquids (RTILs) recently have generated great interest as a result of their potential commercial applications. In particular, because of their negligible vapor pressure and low inflammability, they have been suggested as green alternatives to traditional organic solvents. The toxicity and potential environmental risk of this heterogeneous class of chemicals, however, are poorly understood. An alkyl-substituted RTIL, 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]), is one of the most widely used cations of RTILs, and information regarding its toxicity is relatively extensive. On the other hand, oxygenated chain-substituted ionic liquids, 1-methoxyethyl-3-methylimidazolium salts (moemims), are a new class of RTILs that have been poorly studied. Here, we compared the acute toxicity of [bmim][BF(4)] and moemims to the crustacean Daphnia magna (end point, 48-h immobilization) and the bacterium Vibrio fischeri (end point, 15-min inhibition of bioluminescence). The concentrations of [bmim][BF(4)] resulting in 50% of the maximum adverse effect (EC50s) for D. magna and V. fischeri were 5.18 and 300 mg/L, respectively, and were consistent with previously published values. The EC50s of the two moemims for D. magna are very similar, ranging from 209 to 222 mg/L in different experimental trials, and are higher by two orders of magnitude than the EC50 of [bmim][BF(4)]. The EC50s of 1-methoxyethyl-3-methylimidazolium tetrafluoroborate ([moemim][BF(4)]) and 1-methoxyethyl-3-methylimidazolium dicyanamide ([moemim][dca]) for V. fischeri are 3,196 and 2,406 mg/L, respectively. Results indicate that introduction of an oxygenated side chain in the imidazolium cation can greatly reduce the toxicity of RTILs and that these RTILs are less toxic than commonly used chlorinated solvents, such as tricloromethane, but are more toxic than nonchlorinated solvent, such as methanol and acetone.

Can Integrin Agonists Have Cards to Play against Cancer? A Literature Survey of Small Molecules Integrin Activators.

The ability of integrins to activate and integrate intracellular communication illustrates the potential of these receptors to serve as functional distribution hubs in a bi-directional signal transfer outside-in and inside-out of the cells. Tight regulation of the integrin signaling is paramount for normal physiological functions such as migration, proliferation, and differentiation, and misregulated integrin activity could be associated with several pathological conditions. Because of the important roles of integrins and their ligands in biological development, immune responses, leukocyte traffic, haemostasis, and cancer, their potential as therapeutic tools is now widely recognized. Nowadays extensive efforts have been made to discover and develop small molecule ligands as integrin antagonists, whereas less attention has been payed to agonists. In recent years, it has been recognized that integrin agonists could open up novel opportunities for therapeutics, which gain benefits to increase rather than decrease integrin-dependent adhesion and transductional events. For instance, a significant factor in chemo-resistance in melanoma is a loss of integrin-mediated adhesion; in this case, stimulation of integrin signaling by agonists significantly improved the response to chemotherapy. In this review, we overview results about small molecules which revealed an activating action on some integrins, especially those involved in cancer, and examine from a medicinal chemistry point of view, their structure and behavior.

4-Alkyliden-azetidinones modified with plant derived polyphenols: Antibacterial and antioxidant properties.

Antimicrobial resistance is one of the major and growing concerns in hospital- and community acquired infections, and new antimicrobial agents are therefore urgently required. It was reported that oxidative stress could contribute to the selection of resistant bacterial strains, since reactive oxygen species (ROS) revealed to be an essential driving force. In the present work 4-alkylidene-azetidinones, a new class of antibacterial agents, were functionalized with phytochemical polyphenolic acids such as protocatechuic, piperonyl, caffeic, ferulic, or sinapic acids and investigated as dual target antibacterial-antioxidant compounds. The best candidates showed good activities against multidrug resistant clinical isolates of MRSA (MICs 2-8 µg/mL). Among the new compounds, two revealed the best antioxidant capacity with TEAC-DPPH and TEAC-ABTS being significantly more active than Trolox®.