Multi-Omic Profiling of a Newly Isolated Oxy-PAH Degrading Specialist from PAH-Contaminated Soil Reveals Bacterial Mechanisms to Mitigate the Risk Posed by Polar Transformation Products.

Polar biotransformation products have been identified as causative agents for the eventual increase in genotoxicity observed after the bioremediation of PAH-contaminated soils. Their further biodegradation has been described under certain biostimulation conditions; however, the underlying microorganisms and mechanisms remain to be elucidated. 9,10-Anthraquinone (ANTQ), a transformation product from anthracene (ANT), is the most commonly detected oxygenated PAH (oxy-PAH) in contaminated soils. Sand-in-liquid microcosms inoculated with creosote-contaminated soil revealed the existence of a specialized ANTQ degrading community, and Sphingobium sp. AntQ-1 was isolated for its ability to grow on this oxy-PAH. Combining the metabolomic, genomic, and transcriptomic analyses of strain AntQ-1, we comprehensively reconstructed the ANTQ biodegradation pathway. Novel mechanisms for polyaromatic compound degradation were revealed, involving the cleavage of the central ring catalyzed by Baeyer-Villiger monooxygenases (BVMO). Abundance of strain AntQ-1 16S rRNA and its BVMO genes in the sand-in-liquid microcosms correlated with maximum ANTQ biodegradation rates, supporting the environmental relevance of this mechanism. Our results demonstrate the existence of highly specialized microbial communities in contaminated soils responsible for processing oxy-PAHs accumulated by primary degraders. Also, they underscore the key role that BVMO may play as a detoxification mechanism to mitigate the risk posed by oxy-PAH formation during bioremediation of PAH-contaminated soils.

Metabolites in Milk after Enrofloxacin Treatment and Their Persistence to Temperature.

In this work, metabolomic profile changes in milk from cows affected by mastitis and treated with enrofloxacin (ENR) have been studied using LC-HRMS techniques. Principal component analysis was applied to the obtained results, and the interest was focused on changes affecting compounds without a structural relationship to ENR. Most of the compounds, whose concentrations were modified as a result of the pharmacological treatment and/or the pathological status, were related to amino acids and peptides. Compounds that may become possible biomarkers for either disease or treatment have been detected. Additionally, the alterations caused by thermal processes, such as those applied to milk before consumption, on the identified metabolites have also been considered.

Effects of supercritical fluid chromatography conditions on enantioselectivity and performance of polyproline-derived chiral stationary phases.

The chromatographic behaviour and performance of four polyproline-derived chiral stationary phases (CSPs) were tested using supercritical fluid chromatography (SFC). A series of structurally related racemic compounds, whose enantioseparation was proved to be sensitive to the type of mobile phase used in NP-HPLC, were chosen to be tested in the SFC conditions. Good enantioselection ability was shown by the CSPs for the analytes tested in the new conditions. Resolution, efficiency and analysis time, were considerably improved with respect to NP-HPLC when CO2/alcohol mobile phases were used. Monolithic columns clearly show enhanced chromatographic parameters and improved performance respect to their bead-based counterparts.

Monolithic silica columns functionalized with substituted polyproline-derived chiral selectors as chiral stationary phases for high-performance liquid chromatography.

In this study, two polyproline-derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead-based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.

Octaproline, a conformationally flexible chiral selector in liquid chromatographic enantioseparation.

A proline octapeptide-derived chiral selector (CS) end-capped using a pivaloyl group was covalently linked to a silica gel chromatographic matrix by the C-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) using different conditions was compared to those containing 3,5-dimethylphenylcarbamate residues on the proline units. An enantioseparation ability highly dependent on the mobile phase used is observed for these CSPs. When mixtures of alkane/alcohol or alkane/ether are used as mobile phase a similar enantioselectivity is obtained. Nevertheless, in the presence of chlorinated solvents, and without a hydrogen bonding donor in the mobile phase, enantioselectivity is extremely reduced. The reversibility of this phenomenon, attributed to a conformational change in the CS, is examined.

Huprine-tacrine heterodimers as anti-amyloidogenic compounds of potential interest against Alzheimer's and prion diseases.

A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the ß-amyloid peptide (Aß) and a prion peptide with a key role in the aggregation of the prion protein. Huprine-tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Aß aggregation, and ß-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimer's and prion diseases.

Retention of fluorinated chiral selectors in biphasic fluorinated solvent systems and its application to the separation of enantiomers by countercurrent chromatography.

Ethoxynonafluorobutane (ENFB) has been used as a component of new biphasic solvent mixtures. The suitability of several mixtures as solvent systems in countercurrent chromatography was tested. The applicability of the ENFB/2-PrOH/H(2)O mixture to the separation of enantiomers, in combination with a fluorinated chiral selector (CS), was evaluated. N-Perfluoroundecanoyl-l-proline-3,5-dimethylanilide (2), analogous to the previously used N-dodecyl-l-proline-3,5-dimethylanilide (1), was synthesized for this purpose. The capacity of the new solvent system to retain the fluorinated CS in the fluorinated phase used as stationary was examined. Chiral selector 1 was applied in analogous conditions for comparative purposes. Additionally, MTBE/phosphate buffer solvent system was also used with the two CSs. The ENFB/2-PrOH/H(2)O (25:35:40) mixture was found to be adequate in the enantioseparation of DNB-Leu and DNB-Leu-tBu. Enantioselectivity was maintained in the fluorinated solvent system without compromising eluting time. The complete separation of DNB-Leu-tBu was achieved and no leaks of CS to the mobile phase were detected.

Preparative enantioseparation of (+/-)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide by centrifugal partition chromatography.

The racemic compound (+/-)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide ((+/-)-1), an analogue of increased lipophilicity of the chiral selector (CS) contained in the Whelk-O HPLC chiral stationary phase (CSP) has been resolved into its enantiomers by applying centrifugal partition chromatography (CPC). Considering the known enantioselectivity of the Whelk-O CS for naproxen, and the reciprocity concept in enantioseparation, (S)-naproxen related compounds were tested as CSs. In the search for an adequate solvent system, the partition behaviour of the two solutes, CS and racemate, has been studied using several biphasic solvent mixtures. The optimal CS concentration and sample loading capacity were determined in the chosen solvent system. The search for an appropriate CS and solvent system, the scale-up and optimization of the enantiomer separation by CPC, as well as the rationale for the unexpected elution order of enantiomers, are here described. The comparison of the preparative CPC separation achieved with that in HPLC, using a CSP containing an analogous CS, resulted favourable to the former in terms of loading capacity, solvent consumption and throughput.

Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)-naproxen derivative as chiral selector.

Countercurrent chromatography (CCC) is a liquid-liquid chromatographic technique without a solid support. Several alternative elution modes can be applied to take advantage of the special nature of the liquid stationary phase. Among these dual-mode (DM) and multiple dual-mode (MDM) consist of switching alternatively between Reversed and Normal Phase operation during the experiment (once for DM and several times for MDM). In this paper, MDM has been applied to the chiral CCC separations of two racemic mixtures, (+/-)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(+/-)-leucine, using (S)-naproxen N,N-diethylamide as chiral selector (CS). Although the behaviour of the two analytes differed, improved resolution factors were successfully obtained. Results are rationalized on the basis of the distinct partition behaviour of the CS/enantiomer complexes in the biphasic system.

The chromatographic separation of enantiomers through nanoscale design.

The pharmacological implications of chirality in drugs and the strict legislation in this regard have led to the development of efficient enantioselective technologies to obtain enantiomerically pure compounds and to analyze their quality. However, although the most popular technique for enantioselective analysis is HPLC, the advent of nanotechnologies opens up great possibilities regarding the development of high throughput methods and processes. This tutorial review describes the state of the art in the application of nanoparticles or nanostructured materials to enantioseparation. The possibilities for this kind of materials, previously modified to include conventional chiral selectors, or the use as chiral selectors of those intrinsically chiral are considered.

Selective monosulfoxidation of tetrathiafulvalenes into chiral TTF-sulfoxides.

Four inner tetrathiafulvalene-sulfoxides have been synthesized upon reaction of tetrathiafulvalene (TTF), tetramethyl-tetrathiafulvalene (TMTTF), tetrakis (thiomethyl)-tetrathiafulvalene (TMT-TTF), and bis(ethylenedithio)-tetrathiafulvalene (BEDT-TTF) with enantiopure (+) or (-)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine as oxidizing agent. Chiral HPLC studies indicate very weak enantioselectivities for TTF-SO 3 and TMTTF-SO 4, formation of racemic mixture in the case of TMT-TTF-SO 5, and a rather good selectivity, up to 44% ee, in the case of BEDT-TTF-SO 1. The solid state structures of TMTTF-SO 4 and TMT-TTF-SO 5 have been determined by single crystal X-ray diffraction. Both compounds crystallize as racemates in the centrosymmetric triclinic space group P-1. Theoretical calculations at DFT/B3LYP/6-31+G* level afford optimized geometries in good accordance with the experimental structures and emphasize the participation of the chiral sulfoxide group in HOMO and LUMO. Time-dependent DFT calculations corroborated with electronic circular dichroism spectra allow the assignment of the absolute configuration (R) for the major enantiomer of 1 when the (+)-sulfonyl-oxaziridine is used as oxygen transfer reagent. Preliminary semipreparative HPLC separation provided enantioenriched fractions up to 63% ee.

Polyproline derivatives as chiral selectors in high performance liquid chromatography: chromatographic and conformational studies.

A proline oligopeptide-derived chiral selector (CS), containing 3,5-dimethylphenylcarbamate residues on the 4 position of the pyrrolidine rings, was bonded to a silica gel chromatographic matrix by the N-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) was compared with that of a CSP containing the analogous monomeric CS and that resulting from bonding of the polyproline-derived CS by the carboxy-terminal group using several solvents as mobile phase. The CSs were also studied from the conformational point of view in solution using circular dichroism and 13C NMR. A relationship was found between the presence of an ordered conformation in the particular conditions used and increased enantioselectivity.

Optimisation of the derivatization in cellulose-type chiral selectors for enantioseparation by centrifugal partition chromatography.

Cellulose was chemically modified with hydrophobic dodecanoyl groups followed by 3,5-dimethylphenylcarbamoyl substituents forming mixed ester/carbamate derivatives in order to improve the solubility in lipophilic solvents compared to the corresponding homosubstituted cellulose tris(3,5-dimethylphenylcarbamate). Two mixed derivatives of different degree of substitution were prepared, tested as chiral selectors (CSs) in counter-current chromatography (CCC) and compared to the homo-substituted derivative. Alternatively, cellulose tris(3,5-dichlorophenylcarbamate), was synthesised and tested with the same purpose. The racemic drugs pindolol and warfarin were used as test compounds. Biphasic organic/aqueous solvent systems, composed of methyl isobutyl ketone, t-butyl methyl ether or ethyl acetate and aqueous ammonium acetate or sodium phosphate buffer, were used. Centrifugal partition chromatography, a variety of CCC, in the classical elution mode and the pH-zone-refining displacement mode was applied. The enantioseparation of pindolol and warfarin was achieved in the latter conditions. The presence of dodecanoyl chains on the CS increased solubility in the organic solvents used. The selectivity of the mixed dodecanoyl/3,5-dimethylphenylcarbamoyl cellulose derivative with low degree of dodecanoyl groups (degree of substitution 0.3/degree of substitution 2.6, respectively) was similar to that of the homo-substituted derivative. Furthermore, the loading capacity for pindolol was increased by a factor of three compared to cellulose tris(3,5-dimethylphenylcarbamate). Nevertheless, the increasing degree of substitution with dodecanoyl groups on the CS, although improved solubility in the stationary phase, contributed negatively to the enantioselectivity, where warfarin was more affected than pindolol.

A (4R)-hydroxy-L-proline-derived chiral scaffold and its oligomers as chiral selectors in liquid chromatography chiral stationary phases for enantioseparation.

The chromatographic behaviour of a poly-L-proline-derived chiral stationary phase (CSP) is compared to the corresponding single proline-derived CSP. Structurally diverse racemic test compounds and mobile phases, including normal- and RP conditions, were used. Although the application domain of the poly-L-proline-derived CSP (CSP-3) was considerably restricted, this CSP showed a higher retention and a slightly broader application domain than the monomeric analogue (CSP-1) when heptane/2-PrOH was used as mobile phase. The presence of an alcohol in the mobile phase was essential for enantioseparation in the poly-L-proline-derived CSP when normal-phase conditions were applied.

Application of cellulose and amylose arylcarbamates as chiral selectors in counter-current chromatography.

Here we studied the applicability of cellulose and amylose tris(3,5-dimethylphenylcarbamate) as chiral selectors for the separation of enantiomers by counter-current chromatography (CCC). A variety of organic/aqueous biphasic solvent systems and eight racemic analytes of acidic, neutral, and basic nature were tested for this purpose. Classical elution mode and pH-zone-refining displacement conditions were used. Partial enantioseparation of pindolol and warfarin was achieved in methyl isobutyl ketone (MIBK)/aqueous solution and methyl tert-butyl ether (MTBE)/aqueous solution, respectively. For these two racemates enantiomeric excess values from 84% to 97% were achieved under the best conditions tested. However, resolution by CCC was not achieved for propranolol, naproxen and N-(3,5-dinitrobenzoyl)-(+/-)-leucine, easily separated in HPLC by the same selectors.

Metabolism of fluoranthene by Mycobacterium sp. strain AP1.

The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.

Enantiomer separation by counter-current chromatography. Optimisation and drawbacks in the use of L-proline derivatives as chiral selectors.

Several L-proline and (4R)-hydroxy-L-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(+/-)-leucine and (+/-)-ketoprofen). Successful separations of DNB-(+/-)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.

Preparative enantiomer separation of dichlorprop with a cinchona-derived chiral selector employing centrifugal partition chromatography and high-performance liquid chromatography: a comparative study.

A countercurrent chromatography protocol for support-free preparative enantiomer separation of the herbicidal agent 2-(2,4-dichlorphenoxy)propionic acid (dichlorprop) was developed utilizing a purposefully designed, highly enantioselective chiral stationary-phase additive (CSPA) derived from bis-1,4-(dihydroquinidinyl)phthalazine. Guided by liquid-liquid extraction experiments, a solvent system consisting of 10 mM CSPA in methyl tert-butyl ether and 100 mM sodium phosphate buffer (pH 8.0) was identified as a suitable stationary/mobile-phase combination. This solvent system provided an ideal compromise among stationary-phase retention, enantioselectivity, and well-balanced analyte distribution behavior. Using a commercial centrifugal partition chromatography instrument, complete enantiomer separations of up to 366 mg of racemic dichlorprop could be achieved, corresponding to a sample load being equivalent to the molar amount of CSPA employed. Comparison of the preparative performance characteristics of the CPC protocol with that of a HPLC separation using a silica-supported bis-1,4-(dihydroquinidinyl)phthalazine chiral stationary phase CSP revealed comparable loading capacities for both techniques but a significantly lower solvent consumption for CPC. With respect to productivity, HPLC was found to be superior, mainly due to inherent flow rate restrictions of the CPC instrument. Given that further progress in instrumental design and engineering of dedicated, highly enantioselective CSPAs can be achieved, CPC may offer a viable alternative to CSP-based HPLC for preparative-scale enantiomer separation.

Enantiomer separation by countercurrent chromatography using cinchona alkaloid derivatives as chiral selectors.

Cinchona-derived anion-exchange-type chiral selectors have been adapted and employed in countercurrent chromatography (CCC) for the separation of enantiomers of N-derivatized amino acids and 2-aryloxypropionic acids. The accurate optimization of the enantioseparation in terms of solvent system composition, pH values, ionic strength, and CCC operating conditions was performed. A wide range of solvent mixtures was evaluated. Successful resolutions were achieved in systems such as ammonium acetate buffer/tert-amyl alcohol/methanol/heptane and especially ammonium acetate buffer/methyl isobutyl ketone or diisopropyl ether. Up to 300 mg (0.92 mmol) of N-(3,5-dinitrobenzoyl)-(+/-)-leucine was totally resolved in a single run using a 10 mM concentration of chiral selector in 122 mL of stationary phase. This amount could be increased up to 900 mg (2.77 mmol) when pH-zone-refining mode was applied. The results here presented account for the high potential of CCC as a preparative enantiomer separation technique.

Enantiomeric separation of drugs and herbicides on a beta-cyclodextrin-bonded stationary phase.

A chemically bonded beta-cyclodextrin chiral stationary phase for HPLC was prepared in a "one pot" process by the reaction of a phenylated beta-cyclodextrin with silica gel. Various racemic analytes such as drugs (aminoalcohol adrenergic beta-blockers, benzodiazepine anxiolytics, arylpropionic acid antiinflammatory agents) and herbicides (aryloxypropionic acids and esters) were separated on the prepared material. The column showed good chiral recognition ability for most of the solutes tested when using heptane and either 2-propanol or chloroform as organic mobile phase modifiers.