Enantiomer‐Specific In Vitro Biotransformation of Select Pharmaceuticals in Rainbow Trout (Oncorhynchus mykiss)

The occurrence of pharmaceuticals in the environment represents a challenge of emerging concern. Many pharmaceuticals are chiral compounds; however, few studies have examined the relative toxicity of pharmaceutical enantiomers to wildlife. Further, our understanding of stereospecific pharmacokinetics remains largely informed by research on humans and a few well‐studied laboratory test animals, and not by studies conducted with environmentally relevant species, including fish. The objective of this study was to investigate whether rainbow trout display stereospecific in vitro metabolism of three common chiral pharmaceuticals. Metabolism by trout liver S9 fractions was evaluated using a substrate depletion approach, which provides an estimate of intrinsic hepatic clearance (CL_{IN VITRO,INT}). No biotransformation was observed for rac‐, R‐, or S‐fluoxetine. Ibuprofen, including both enantiomers and the racemic mixture, appeared to undergo slow metabolism, but the resulting substrate depletion curves did not differ significantly from those of inactive controls. Contrary to relative clearance rates in humans, S(?)‐propranolol was more rapidly cleared than the R(+)‐ enantiomer. This work demonstrates that relative clearance rates and the effects of racemic mixtures in trout could not have been predicted based on human data. Additional research describing species differences and exploring tools for species extrapolation in biomedical and environmental studies is needed. Chirality 25:763–767, 2013, © 2013 Wiley Periodicals, Inc.     

Determination of absolute configuration in 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones by high performance liquid chromatography and CD spectroscopy

The absolute configuration of three 4‐aryl‐3,4‐dihydro‐2(1H)‐pyrimidones (Biginelli compounds, DHPMs) was established by comparison of the typical circular dichroism (CD) spectra of individual enantiomers with reference samples of known absolute configuration. The enantiomers were obtained by semipreparative separation of racemic mixtures on a Chiralcel OD‐H chiral stationary phase. The method was used to establish the enantiopreference of various lipases in biocatalytic kinetic resolution experiments employing activated DHPM esters. Chirality 11:659–662, 1999. © 1999 Wiley‐Liss, Inc.     

Lipase‐Catalyzed Kinetic Resolution of Novel Antifungal N‐Substituted Benzimidazole Derivatives

A series of new N‐substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase‐catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). Chirality 28:347–354, 2016. © 2016 Wiley Periodicals, Inc.     

Whole‐cell biocatalytic of Bacillus cereus WZZ006 strain to synthesis of indoxacarb intermediate: (S)‐5‐Chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester

In this study, a newly isolated strain screened from the indoxacarb‐rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester was obtained by bio‐enzymatic resolution. After the 36‐hour hydrolysis in 50‐mM racemic substrate under the optimized reaction conditions, the e.e._s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high‐level ability to produce (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.     

Disruption of trans‐pityol‐mediated attraction by racemic trans‐conophthorin in twig beetle Pityophthorus pubescens

Twig beetle Pityophthorus pubescens (Coleoptera: Curculionidae: Scolytinae) has been previously associated with the Fusarium circinatum (Hypocreales: Nectriaceae), the pathogen causing pitch canker disease, in P. radiata stands of the Basque Country (Northern Spain). Laboratory and field studies were conducted to evaluate the response of the insect to the racemic mixture of the spiroacetal trans‐7‐methyl‐1,6‐dioxaspiro[4.5]decane, also known as conophthorin. In walking bioassays, addition of 10 and 100 ng of racemic trans‐conophthorin to 1 ng of (±)‐trans‐pityol elicited a negative response in males, whereas females did not show any significant preference. Catches of males in attractant‐baited traps were strongly reduced by racemic trans‐conophthorin at all release rates tested (0.5, 1.0, 1.5 and 2.0 mg/day). In contrast, catches of females were not affected by any of the (±)‐trans‐conophthorin release rate.     

Precolumn o‐Phthalaldehyde‐N‐acetyl‐L‐cysteine Derivatization Followed by RP‐HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

An indirect reversed‐phase high‐performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP‐18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50–5000 ng/ mL for both enantiomers. The intra‐ and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments. Chirality 25:883–889, 2013. © 2013 Wiley Periodicals, Inc.     

Tetramethyl‐Bis(ethylenedithio)‐Tetrathiafulvalene (TM‐BEDT‐TTF) Revisited: Crystal Structures,Chiroptical Properties,Theoretical Calculations,and a Complete Series of Conducting Radical Cation Salts

The (S,S,S,S) and (R,R,R,R) enantiomers of tetramethyl‐bis(ethylenedithio)‐tetrathiafulvalene (TM‐BEDT‐TTF) show equatorial conformation for the four methyl groups in the solid state, according to the single‐crystal X‐ray analyses. Theoretical calculations at the Density Functional Theory (DFT) and time‐dependent (TD) DFT levels indicate higher gas phase stability for the axial conformer than the equatorial one by 1.25 kcal · mole^‐1 and allow the assignment of the UV–vis and circular dichroism transitions. A complete series of radical cation salts of 1:1 stoichiometry with the triiodide anion I₃^‐ was obtained by electrocrystallization of both enantiopure and racemic forms of the donor. In the packing the donors are organized in dimers that further interact through S · · · S intermolecular contacts and the triiodide anions lie parallel to pairs of oxidized donors. The conductivity of the racemate, which adopts the same, but disordered, structural type, is considerably lower, with much higher activation energy. Chirality 25:466–474, 2013.© 2013 Wiley Periodicals, Inc.     

New chiral stationary phases based on (R)‐1‐naphthylethylamine bound to 2,4,5,6‐tetrachloro‐1,3‐dicyanobenzene

Chiral functionalization of 2,4,5,6‐tetrachloro‐1,3‐dicyanobenzene (1) by regioselective nucleophilic substitution of one or two chlorine atoms by optically pure (R)‐(+)‐1‐naphthylethylamine (NEA), or by a glycine unit as a spacer to (R)‐NEA, enables the preparation of brush‐type chiral selectors (2, 3, 9, 13). By the introduction of the 3‐aminopropyltriethoxysilyl (APTES) group, reactive intermediates 4a/b, 5, 10a/b, and 14a/b are obtained ( a/b indicate a mixture of regioisomers with APTES in 6‐ and 2‐position). Binding of these to silica gel afforded four novel chiral stationary phases (CSPs) 6, 7, 15, and 16. HPLC columns containing CSPs with (R)‐NEA directly linked to polysubstituted aromatic ring (6, 7) are not very effective in resolution of most of the 23 racemic analytes, whereas the columns with distant π‐basic subunits (15, 16) exhibited higher resolving efficacy, in particular towards the isopropyl esters of racemic N‐3,5‐dinitrobenzoyl‐α‐amino acids. Effective resolution of test racemates reveals the importance of the presence of the hydrogen bond donor amido group and the distance between the persubstituted benzene ring in 1 and the π‐basic naphthalene ring of (R)‐NEA. Chirality 11:722–730, 1999. © 1999 Wiley‐Liss, Inc.     

The resolution of acyclic P‐stereogenic phosphine oxides via the formation of diastereomeric complexes: A case study on ethyl‐(2‐methylphenyl)‐phenylphosphine oxide

As an example of acyclic P‐chiral phosphine oxides, the resolution of ethyl‐(2‐methylphenyl)‐phenylphosphine oxide was elaborated with TADDOL derivatives, or with calcium salts of the tartaric acid derivatives. Besides the study on the resolving agents, several purification methods were developed in order to prepare enantiopure ethyl‐(2‐methylphenyl)‐phenylphosphine oxide. It was found that the title phosphine oxide is a racemic crystal‐forming compound, and the recrystallization of the enantiomeric mixtures could be used for the preparation of pure enantiomers. According to our best method, the (R)‐ethyl‐(2‐methylphenyl)‐phenylphosphine oxide could be obtained with an enantiomeric excess of 99% and in a yield of 47%. Complete racemization of the enantiomerically enriched phosphine oxide could be accomplished via the formation of a chlorophosphonium salt. Characterization of the crystal structures of the enantiopure phosphine oxide was complemented with that of the diastereomeric intermediate. X‐ray analysis revealed the main nonbonding interactions responsible for enantiomeric recognition.     

Stereoselective metabolism of propranolol glucuronidation by human UDP‐glucuronosyltransferases 2B7 and 1A9

Stereoselective metabolism of propranolol side‐chain glucuronidation was studied for two recombinant human uridine diphosphate glucuronosyltransferases (UGTs), UGT1A9 and UGT2B7. The S‐ and R‐propranolol side‐chain glucuronides produced in the incubation mixtures were assayed simultaneously by RP‐HPLC with fluorescent detector. The excitation and emission wavelengths were set at 310 nm and 339 nm, respectively. UGT1A9 prefers catalyzing S‐enantiomer to R‐enantiomer and the intrinsic clearance (CL_int) ratios of S‐enantiomer to R‐enantiomer are 3.8 times and 6.5times for racemic propranolol and individual enantiomers, respectively. UGT2B7, however, catalyzes slightly less S‐enantiomer than R‐enantiomer and the CL_int ratio of S‐enantiomer to R‐enantiomer is 0.8 times. The high concentration of racemic propranolol (>0.57 mmol/l) and individual enantiomers (>0.69 mmol/l) exhibited substrate inhibition of glucuronidation for UGT2B7, but only the S‐enantiomer (>0.44 mmol/l) in racemic propranolol exhibited substrate inhibition for UGT1A9. The substrate inhibition constants (K_si) were all similar (P > 0.05). Drug–drug interactions were also found between S‐ and R‐enantiomer glucuronidation metabolisms by UGT1A9 and UGT2B7. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Construction of Quaternary Stereocenters: Asymmetric α‐Amination of Branched Aldehydes Catalyzed by Monoimide Substituted Cyclohexane‐1,2‐Diamines

A highly efficient enantioselective α‐amination of branched aldehydes catalyzed by chiral imide monosubstituted 1,2‐diamine derivatives was reported to afford the quaternary stereogenic centers in excellent yields (up to 99%) and enantioselectivities (up to 97% ee). Chirality 25:668–672, 2013. © 2013 Wiley Periodicals, Inc     

Structural characterization of 2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase (PcpA) from Sphingobium chlorophenolicum,a new type of aromatic ring‐cleavage enzyme

PcpA (2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase) from Sphingobium chlorophenolicum, a non‐haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p‐hydroquinone and its substituted variants, is a member of the recently discovered p‐hydroquinone 1,2‐dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co‐ordination sphere of the Fe(II) centre forms an extensive hydrogen‐bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p‐hydroquinones access to the Fe(II) centre. The p‐hydroxyl group is essential for the substrate‐binding, thus phenols and catechols, lacking a p‐hydroxyl group, do not bind to PcpA. Site‐directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p‐hydroquinone 1,2‐dioxygenases.     

Synthesis of enantiopure planar chiral bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophanes

A new type of planar chiral (R_p)‐ and (S_p)‐4,7,12,15‐tetrasubstituted [2.2]paracyclophanes was prepared from racemic 4,7,12,15‐tetrabromo[2.2]paracyclophane as the starting substrate. Regioselective lithiation and transformations afforded racemic bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophane (4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophane). Its optical resolution was performed by the diastereomer method using a chiral camphanoyl group as the chiral auxiliary. The diastereoisomers were readily isolated by simple silica gel column chromatography, and the successive hydrolysis afforded (R_p)‐ and (S_p)‐bis‐(para)‐pseudo‐meta‐type [2.2]paracyclophanes ((R_p)‐ and (S_p)‐4,15‐dibromo‐7,12‐dihydroxy[2.2]paracyclophanes). They can be used as pseudo‐meta‐substituted chiral building blocks.     

Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

β‐Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β‐aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β³‐ and/or β²‐amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β‐peptide substrates, β‐aminopeptidases have potential to be used as biocatalysts for β‐peptide synthesis and for the resolution of enantiomerically pure β‐amino acids from racemic substrate mixtures. β‐Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N‐terminus of the newly formed β‐polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N‐terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β‐aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β‐aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β‐peptide conversion.     

Theoretical Study of N‐Heterocyclic Carbenes‐Catalyzed Cascade Annulation of Benzodienones and Enals

Growing attention in developing new N‐heterocyclic carbene (NHC)‐mediated reactions involving homoenolate intermediates has prompted our interest in exploring the mechanistic details of the related reactions. In this work, we carried out a detailed theoretical study for the NHC‐catalyzed annulation reaction of cinnamaldehyde ( A ) and benzodi(enone) ( B ) in the presence of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU). By performing density functional theory calculations, we show clearly the detailed reaction mechanism and rationalize the experimental observation. The reaction of A and B falls into two stages: the formation of homoenolate intermediate and the annulation of homoenolate with B . In the homoenolate formation stage, three possible paths are characterized. The pathway involving the DBU‐assisted 1,2‐proton transfer with a stepwise mechanism is kinetically more favorable, and the DBU‐assisted C1 proton departure is the rate‐determining step of the total reaction. The annulation of homoenolate with B involves four elementary steps. The conformational difference of homoenolate (cis and trans) leads to two slightly different reaction processes. In the total reaction, the process involving cis‐conformation of A is kinetically more feasible. This can be clearly understood through the frontier molecular orbital analysis and the electronic inductive effect. The calculated results are expected to offer valuable information for further design and development of NHC‐mediated reactions. Chirality 25:521‐528, 2013. © 2013 Wiley Periodicals, Inc.     

Nonaqueous Capillary Electrophoretic Enantioseparation of Water Insoluble Tröger's Base Derivatives Using β‐Cyclodextrin as Chiral Selector

Racemic mixtures of six Tröger's base derivatives were separated by chiral nonaqueous capillary electrophoresis. The separation protocol was optimized first for suitable solvents. Then the applicability of various salts dissolved in organic solvents and their mixtures was evaluated. As chiral selectors β‐cyclodextrin and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin at various concentrations were used. The best enantioselectivity for the studied analytes was obtained utilizing formamide as organic nonaqueous solvent containing a mixture of sodium citrate and tris(hydroxymethyl)aminomethane acetate as electrolytes, and β‐cyclodextrin as chiral additive. The experimental results demonstrated the feasibility of nonaqueous capillary electrophoresis for enantioseparation of Tröger's base derivatives. This technique represents a suitable alternative to more commonly used capillary electrophoresis in aqueous environment. Chirality 25:810–813, 2013. © 2013 Wiley Periodicals, Inc.     

Chiral Separation of Cathinone and Amphetamine Derivatives by HPLC/UV Using Sulfated ß‐Cyclodextrin as Chiral Mobile Phase Additive

In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP‐18e, 250 x 4 mm, 5 µm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated ß‐cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated ß‐cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP‐8e column. Chirality 26:411–418, 2014. © 2014 Wiley Periodicals, Inc.     

High‐throughput FTIR spectroscopy of intact HepG2 cells reveals additive and non‐additive effects of individual fatty acids when given as mixtures

In the present study we investigated the ability of high‐throughput FTIR spectroscopy in combination with multivariate data analysis to reveal if any combinatory effects of fatty acids in mixture are present in liver HepG2 cell cultures after three days of exposure. For this investigation we used an experimental mixture design containing three different octadecenoic acids (oleic acid: C18 : 1 cis‐ 9, elaidic acid: C18 : 1 trans‐ 9 and vaccenic acid: C18 : 1 trans‐ 11) of a total concentration of 100 μM. The results obtained revealed both additive and non‐additive effects of individual fatty acids when combined in mixtures. Furthermore, we demonstrate by use of scanning electron microscopy that cells are preserved as intact structures ensuring that FTIR measurements are obtained on whole cell keeping cell compounds in their natural surroundings during measurements. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)