Electrochemical Iodination through the In Situ Generation of Iodinating Agents: A Promising Green Approach.

The synthesis of iodinated compounds using cheap, simple, and green strategies is of fundamental importance. Iodination reactions are mainly used to synthesize useful intermediates, especially in the pharmaceutical field, where they are employed for the production of contrast media or of iodinated active pharmaceutical ingredients. Traditional synthetic methods suffer from the use of erosive, toxic, or hazardous reactants. Approaches which involve the use of molecular iodine as an iodinating agent require the addition of an oxidizing agent, which is often difficult to handle. Electrochemistry can offer a valid and green alternative by avoiding the addition of such oxidizing agents, transforming the iodine source in the active species through the use of electrons as the main reactants. Herein, we report the electrochemical iodination with the generation of iodinating species in situ in water by using iodides as the source of iodine atoms. First of all, the electrochemical behavior of iodide and iodine in water on carbonaceous anodes was studied and, after selecting the suitable potential, in situ electrochemical iodination was successfully applied to 5-hydroxyisophthalic acid and 5-sulfosalicylic acid, comparing the iodinating power of I2 and iodonium species.

Modelling the transport mechanism of organic molecules into cell membranes: The role of organic solvents.

Substrate availability is a key issue in the process design for chemicals production through enzymatic reactions. In whole cell bioconversions, the introduction of an organic solvent provides an efficient and flexible way to control substrate availability. Although successful, this route has often been based on trial and error experiments, and very little is known about the role of the solvent in the whole process. Modeling the transport mechanism of substrate molecules into cell membranes and the solvent-membrane interaction represents a first step toward a better understanding of the mechanisms underlying bioconversion efficiency. Hereafter, we report and discuss the results of such a modeling activity, which we carried out by means of molecular dynamics simulations. To better approach real-world experimental settings, we explicitly accounted for the possibility of solvent stirring. Our results are in agreement with the experimental data, paving the way to the application of molecular modeling in this recent and fast growing area of organic syntheses.

Identification of a Novel Biosurfactant with Antimicrobial Activity Produced by Rhodococcus opacus R7.

Rhodococcus members excrete secondary metabolites, especially compounds which act as biosurfactants. In this work, we demonstrated the ability of Rhodococcus opacus R7 to produce a novel bioactive compound belonging to the class of biosurfactants with antimicrobial properties during the growth on naphthalene. Chemical and biochemical analyses of the isolated compound demonstrated that the biosurfactant could be classified as a hydrophobic peptide. The ESI-full mass spectrometry revealed that the isolated biosurfactant showed a molecular weight of 1292 Da and NMR spectra evidenced the composition of the following amino acid residues: Ala, Thr, Asp, Gly, Ser. Surfactant activity of the R. opacus R7 compound was quantified by the critical micelle dilution (CMD) method and the critical micelle concentration (CMC) was estimated around 20 mg L-1 with a corresponding surface tension of 48 mN m-1. Moreover, biological assays demonstrated that R. opacus R7 biosurfactant peptide exhibited antimicrobial activity against Escherichia coli ATCC 29522 and Staphylococcus aureus ATCC 6538 with the minimum inhibition growth concentration (MIC) values of 2.6 mg mL-1 and 1.7 mg mL-1, respectively. In this study for the first time, a hydrophobic peptide with both biosurfactant and antimicrobial activity was isolated from a bacterium belonging to Rhodococcus genus.

Identification of viable TCDD access pathways to human AhR PAS-B ligand binding domain.

Unintentionally released in the environment as by-products of industrial activities, dioxins, exemplified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), represent a primary concern for human health. Exposure to these chemicals is known to produce a broad spectrum of adverse effects, including cancer. The main mechanism of action of TCDD in humans involves binding to the Aryl hydrocarbon Receptor (AhR). Although qualitatively established, TCDD capture by the AhR remains poorly characterized at the molecular level. Starting from a recently developed structural model of the human AhR PAS-B domain, in this work we attempt the identification of viable TCDD access pathways to the human AhR ligand binding domain by means of molecular dynamics. Based on the result of metadynamics simulations, we identify two main regions that may potentially serve as access paths for TCDD. For each path, we characterize the residues closely interacting with TCDD, thereby suggesting a possible mechanism for TCDD capture. Our results are reviewed and discussed in the light of the available information about Human AhR structure and functions.

Extraction and Characterization of Inulin-Type Fructans from Artichoke Wastes and Their Effect on the Growth of Intestinal Bacteria Associated with Health.

Globe artichoke is an intriguing source of indigestible sugar polymers such as inulin-type fructans. In this study, the effect of ultrasound in combination with ethanol precipitation to enhance the extraction of long chain fructans from artichoke wastes has been evaluated. The inulin-type fructans content both from bracts and stems was measured using an enzymatic fructanase-based assay, while its average degree of polymerization (DP) was determined by HPLC-RID analysis. Results show that this method provides artichoke extracts with an inulin-type fructans content of 70% with an average DP between 32 and 42 both in bracts and in stems. The prebiotic effect of long chain inulins from artichoke extract wastes was demonstrated by its ability to support the growth of five Lactobacillus and four Bifidobacterium species, previously characterized as probiotics. Besides, we considered the possibility to industrialize the process developing a simpler method for the production of inulin-type fructans from the artichoke wastes so that the artichoke inulin preparation could be suitable for its use in synbiotic formulations in combination with different probiotics for further studies including in vivo trials.

Identification of a bacteriocin-like compound from Lactobacillus plantarum with antimicrobial activity and effects on normal and cancerogenic human intestinal cells.

In this paper, we demonstrate that the antimicrobial activity of L. plantarum PBS067 strain against antagonist microorganisms was mediated by the production of a bacteriocin-like compound secreted at the stationary phase of the growth. The novel bacteriocin-like compound, designed plantaricin P1053, was identified by using sorption-desorption method, butanol extraction and SEC-HPLC. The molecular mass of plantaricin P1053 was shown to be 1053 Da by ESI-MS analysis. Plantaricin P1053 exhibited a broad-spectrum antimicrobial activity against Gram-positive bacteria as S. aureus and Gram-negative bacteria as E. coli. In addition to the antimicrobial activity, the isolated bacteriocin-like compound showed effects on normal and cancerogenic epithelial intestinal cell lines through an enhancing of viability of healthy cells and a proliferation reduction of cancer cells. Moreover, in this paper we demonstrate that the isolated bacteriocin-like compound acts on healthy cells through the epidermal growth factor receptor (EGFR) pathways. In conclusion, plantaricin P1053 isolated from L. plantarum PBS067 strain could represent one of the first multifunctional bacteriocin-like compound acting on human epithelial intestinal cells.

Synthesis and biological evaluation of new 3-amino-2-azetidinone derivatives as anti-colorectal cancer agents.

Several synthetic combretastatin A4 (CA-4) derivatives were recently prepared to increase the drug efficacy and stability of the natural product isolated from the South African tree Combretum caffrum. A group of ten 3-amino-2-azetidinone derivatives, as combretastatin A4 analogues, was selected through docking experiments, synthesized and tested for their anti-proliferative activity against the colon cancer SW48 cell line. These molecules, through the formation of amide bonds in position 3, allow the synthesis of various derivatives that can modulate the activity with great resistance to hydrolytic conditions. The cyclization to obtain the 3-aminoazetidinone ring is highly diastereoselective and provides a trans biologically active isomer under mild reaction conditions with better yields than the 3-hydroxy-2-azetidinone synthesis. All compounds showed IC50 values ranging between 14.0 and 564.2 nM, and the most active compound showed inhibitory activity against tubulin polymerization in vitro, being a potential therapeutic agent against colon cancer.

Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7.

BACKGROUND: Bacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o-xylene degradation in R. opacus strain R7 through a genome-based approach. RESULTS: Using genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb, phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o-xylene oxidation was evaluated. The involvement of the akb genes in o-xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o-xylene and by the selection of the R7-50 leaky mutant. Although the akb genes are specifically activated for o-xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o-xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o-xylene and R7 growth on dimethylphenols. CONCLUSIONS: These results suggest that R7 strain is able to degrade o-xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o-xylene degradation pathway in R7 strain.

Polyphenol Polymerization by an Alternative Oxidative Microbial Enzyme and Characterization of the Biological Activity of Oligomers.

The recombinant catalase-peroxidase HPI from E. coli was used as an alternative enzyme in polymerization reactions for the production of (-) epicatechin oligomers and their biological activity was characterized. The enzyme was prepared in two forms: a purified and an immobilized form. Both were tested for their activity in oxidative polymerization reactions, and their stability and reusability were assessed. The polymerization reactions were followed by SEC-HPLC analyses, and the substrate was completely converted into one or more polymerization products depending on the reactions conditions. Results showed that the utilized conditions allowed for the isolation of some oligomers of different molecular weight: the oligomers containing 6 and 7 units of epicatechin substrate are the heaviest ones. Epicatechin was also used in reactions catalyzed by HRP in the same reaction conditions for comparison. In addition, one selected oligomer obtained by HPI enzyme catalysis was shown to act as in vitro inhibitor of tumor cell growth, like one oligomer deriving from epicatechin by HRP catalysis. These data confirm that epicatechin oligomeric form is more effective than its monomer in biological activity and suggest the use of HPI as an alternative enzyme in reactions for the production of epicatechin oligomers.

Integrating computational methods to predict mutagenicity of aromatic azo compounds.

Azo dyes have several industrial uses. However, these azo dyes and their degradation products showed mutagenicity, inducing damage in environmental and human systems. Computational methods are proposed as cheap and rapid alternatives to predict the toxicity of azo dyes. A benchmark dataset of Ames data for 354 azo dyes was employed to develop three classification strategies using knowledge-based methods and docking simulations. Results were compared and integrated with three models from the literature, developing a series of consensus strategies. The good results confirm the usefulness of in silico methods as a support for experimental methods to predict the mutagenicity of azo compounds.

From dioxin to dioxin congeners: understanding the differences in hydrophobic aggregation in water and absorption into lipid membranes by means of atomistic simulations.

Translocation of small molecules through a cell membrane barrier is a fundamental step to explain the response of cells to foreign molecules. Investigating the mechanisms through which this complex process takes place is especially important in the study of the adverse effects of toxicants. In this work, we start from the results of a previous simulation study of the mechanism of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) absorption into a model membrane, and extend it to four structural congeners of dioxin. The new molecules have been chosen taking into consideration the structural features that characterize dioxin: aromaticity, planarity, the presence of chlorine and oxygen atoms, and hydrophobicity. Our results for the absorption mechanism confirm our expectations based on the chemical structures, but also reveal some interesting differences in single-molecules and especially in cooperative actions underlying cluster absorption. The analysis of key parameters, such as free energies of transfer and translocation times, supports the idea that dioxin, more than its congeners investigated here, likely accumulates in cell membranes.

Carcinogenicity prediction of noncongeneric chemicals by augmented top priority fragment classification.

Carcinogenicity prediction is an important process that can be performed to cut down experimental costs and save animal lives. The current reliability of the results is however disputed. Here, a blind exercise in carcinogenicity category assessment is performed using augmented top priority fragment classification. The procedure analyses the applicability domain of the dataset, allocates in clusters the compounds using a leading molecular fragment, and a similarity measure. The exercise is applied to three compound datasets derived from the Lois Gold Carcinogenic Database. The results, showing good agreement with experimental data, are compared with published ones. A final discussion on our viewpoint on the possibilities that the carcinogenicity modelling of chemical compounds offers is presented.

Purification of recombinant catalase-peroxidase HPI from E. coli and its application in enzymatic polymerization reactions.

In this paper, a recombinant catalase-peroxidase HPI from Escherichia coli was prepared, purified, and used in enzymatic polymerization reactions for the production of several oligomeric products. We tested the enzyme on four different substrates, chosen as representative of phenols and anilines: phenol, 3-methoxyphenol, catechol, and aniline. The polymerization reactions were followed by SEC-HPLC analysis, and except for aniline, all the other substrates were completely converted into one or more polymerization products. Results showed that reactions performed with phenol and 3-methoxyphenol allowed the isolation of some oligomers of different weight: a 27-monomeric unit oligomer and a 23-U oligomer are the heaviest ones. Experiments performed with catechol showed the formation of oligomers of 7 U in the reaction with HPI. HPI polymerization reactions performed with aniline allowed the identification of two different oligomers, one of 4 U and one of 10 U. All the substrates have been also used in reactions catalyzed by HRP in the same reaction conditions. Several products were common to the two enzymes. This work suggests the use of HPI as an alternative enzyme in peroxidatic reactions for the production of different oligomers from phenols and other compounds.

Aldol reactions of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) from Pseudomonas fluorescens N3.

In this paper, a recombinant trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) of Pseudomonas fluorescens N3 was used as a new catalyst for aldol condensation reactions. The reaction of some aldehydes with a different electronic activation catalyzed by tHBP-HA is presented and discussed together with some hints on the product structure. The enzyme is strictly pyruvate-dependent but uses different aldehydes as acceptors. The structure of the products is highly dependent on the electronic characteristics of the aldehyde. The results are interesting for both their synthetic importance and the mechanism of the formation of the products. Not only the products obtained and the recognition power are reported, but also some characteristics of its mechanism are analyzed. The results clearly show that the enzyme is efficiently prepared, purified, and stored, that it recognizes many different substrates, and that the products depend on the substrate electronic nature.

Determination of toxicant mode of action by augmented top priority fragment class.

Theoretical models can be an efficient tool to assess compound toxicity as an alternative to experimental determinations. Their application must follow some requirements that include the possibility of understanding the rationale that supports the prediction; here, the determination of the mode of action (MOA) is important. A combination of similarity and reactivity analysis has been applied to group chemical compounds with the aim at selecting groups that share structure and electronic state. The model is not based on experimental data but only on structural features. The result is a number of groups that contains similar compounds with similar reactivity and, possibly, similar MOA. The comparison of these groups to the experimentally determined MOAs available for the EPAFHAM database permits the discussion of the validity of both the model and the experimental data.

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner.

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.

Identification of toxifying and detoxifying moieties for mutagenicity prediction by priority assessment.

The search for structural subunits that affect compound toxicity cannot be manually performed on large databases. In addition, the a priori definition of important groups is impossible. Structural diversity requires the analysis of the complete data space and the selection of the details there present. A single substructure cannot be considered sufficient when assigning compound toxicity. In contrast, if we consider all the substructures in the database as the elements of a complete collection and if we can build a working hierarchy, the identification of the best feasible result using the available data is possible. If the database includes several significant examples, the results will be valuable. The use of a fragment-based description of a mutagenicity database together with the realization of a general hierarchy allows for the identification of the moieties that control the toxifying/detoxifying action of each compound.

Microbial enzymes for aromatic compound hydroxylation.

Redox enzymes are ubiquitous in all living organisms. In fact, oxidation and reduction reactions are fundamental for the transformation of cellular and external compounds both for cell reproduction and for energy production. Redox enzymes share a common characteristic that is the capacity of transferring electrons to and from molecules. In addition, microorganisms contain many oxidative enzymes, and because they are relatively easier to cultivate and study, they have been investigated in details, in particular for potential use in biotechnological field. One important reaction that oxidative enzymes perform is the introduction of one or two oxygen atoms on aromatic compounds. The most representative classes of enzymes that perform this reaction are oxygenases/hydroxylases, peroxidases, and laccases; they differ in many aspects: the metal present in the active site, the used reductive cofactor, the final oxidant, and the number of electrons transferred in each step. Their essential features and mechanisms of action have been the subject of several studies, together with some structural analyses. This review reports recent developments and summarizes some of the most interesting results concerning both structural requirements and mechanisms implicated in aromatic hydroxylation.

Characterization of the aldol condensation activity of the trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) cloned from Pseudomonas fluorescens N3.

The gene encoding trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA) was isolated from Pseudomonas fluorescens N3, an environmental strain able to degrade naphthalene. This enzyme is an aldolase of class I that reversibly catalyzes the transformation of the trans-o-hydroxybenzylidenepyruvate (t-HBP), releasing pyruvate and salicylaldehyde. The enzyme was expressed in Escherichia coli as a recombinant protein of 38kDa with a His6-Tag at its N-terminus. The recombinant protein His-tHBP-HA was purified by affinity chromatography and we present here the biochemical characterization of its activity in the aldol condensation reaction. The aldol condensation reaction parameters were determined using as acceptors both salicylaldehyde, which is the natural substrate taking part to the naphthalene degradative pathway, and benzaldehyde. In both cases, His-tHBP-HA shows similar apparent K(m) and apparent V(max) values. Further analyses showed that the optimal pH and temperature of His-tHBP-HA activity are 7.0 and 30°C, respectively. The tHBP-HA catalytic rates and the availability of an efficient system to produce large amounts of purified protein are relevant from a biotechnological point of view.

Definition and detection of outliers in chemical space.

Detection of outliers is a complex and challenging area of research in chemical theory. Among current notions, that of outliers in the chemical space--descriptors--is meaningful with multiple applications in the field of drug discovery and predictive modeling. Presented here is a new framework for outlier detection, relying on a discrete, fragment-based representation of the molecular structures. From this starting point, a recursive method is developed that quantifies the contribution of fragments to compound description and identifies outliers in chemical structure databases according to a novel definition. In contrast to existing detection routes, this approach avoids the use of thresholds usually required to quantify outlying behavior. Three chemical databases are investigated to demonstrate its generality and flexibility. The result reveals a new species of outliers, compounds with no specific structural features, rather than unique ones.