Chemo-enzymatic synthesis of (R)-(+)-aminoglutethimide by kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol

Chemo-enzymatic approaches for the synthesis of the family of aromatase inhibitory drug via lipase-catalyzed kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol (2) as appropriate precursors were described. Enzymatic transesterification of primary alcohol (±)-2 using Pseudomonas cepacia (Amano PS, PCL) provided the enantiopure alcohol (R)-(−)-2 with 99% ee at conversion of 86%, while that of (±)-2 using Pseudomonas fluorescens (Amano AK, LAK) provided the (S)-(+)-2 with 96% ee at conversion of 86%. Chemical transformation of substrate (R)-(−)-2 gave (R)-(+)-aminoglutethimide (1) in enantioselectively high yield.     

Transition metal complexes IX. Nickel complexes with a chiral azaphosphole ligand

The reaction of (−)-(R)-myrtenal and (+)-(R)-phenylethylamine gave a Schiff base 1 which was reacted with MePBr₂ in the presence of a base to give under dehydrohalogenation of an intermediate McCormack product a salt 2. Treatment of 2 with sodium led to the formation of the azaphosphole 4. η³-C₃H₅NiCl and 4 gave a 1:1 adduct 5 and nickel(0) gave a 1:4 complex 6. Compounds 4–6 were characterized by NMR spectroscopy as well as by single crystal X-ray structure determination.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Biotransformation of monoterpenoids, (−)- and menthols, terpinolene and carvotanacetone by Aspergillus species

Four monoterpenoids, (−)- and (+)-menthols, terpinolene and carvotanacetone were biotransformed by Aspergillus niger and several related species. Aspergillus niger converted (−)-menthol to 1-, 2-, 6-, 7-, and 9-hydroxymenthols and the mosquito repellent-active 8-hydroxymenthol. On the other hand, (+)-menthol was smoothly biotransformed by A. niger to give 7-hydroxymenthol. Aspergillus cellulosae biotransformed (−)-menthol specifically to 4-hydroxymenthol. Terpinolene and (−)-carvotanacetone were converted by A. niger to two , β-unsaturated ketones, a fenchane-type compound and diastereoisomeric p-menthane-2,9-diols and 8-hydroxycarvomenthol, respectively.     

Physical, chemical and microscopic characterization of a new starch from chayote (Sechium edule) tuber and its comparison with potato and maize starches

In this work, the chayote tuber starch (CHS) was isolated and its chemical composition and its physical and microscopic characteristics were determined, and compared with potato (PS) and maize (MS) starches. The starch content in chayote tubers (728 g kg⁻¹ dry weight) was similar to potato tubers (700 g kg⁻¹ dry weight), with a high level of purity (>98%), while its phosphorous content was higher (0.15%) than PS (0.08%) and MS (0.01%). Starch granules were oval, irregular, truncated and rounded with sizes between 7 and 50 μm with smooth surfaces. CHS dispersions (1% and 4%, w/w) showed higher viscosity (75 and 1715 mPa s), than PS (350% and 50% lower) and MS (715% and 600% lower). The gelatinization temperature (65 to 74 °C) was similar in CHS and PS. The pasting properties (RVA) of the starches suggest that CHS showed better characteristics than the commercial potato and maize starches. Therefore, CHS could be used as a thickening agent and a substitute to PS in food dispersions where a high viscosity is needed.     

The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p-hydroxyphenylpyruvate dioxygenase

The lichen secondary metabolite usnic acid exists as a (−) and a (+) enantiomer, indicating a or β projection of the methyl group at position 9b, respectively. (−)-Usnic caused a dose-dependent bleaching of the cotyledonary tissues associated with a decrease of both chlorophylls and carotenoids in treated plants whereas no bleaching was observed with the (+) enantiomer. (−)-Usnic acid inhibited protophorphyrinogen oxidase activity (I₅₀=3 μM), but did not lead to protoporphyrin IX accumulation. Bleaching appears to be caused by irreversible inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase by (−)-usnic acid (apparent IC₅₀=50 nM).     

Validated chiral high-performance liquid chromatographic method for the determination of trans-(−)-paroxetine and its enantiomer in bulk and pharmaceutical formulations

A stereospecific high-performance liquid chromatography method for the determination of trans-(−)-paroxetine and its enantiomer in bulk raw material and pharmaceutical formulations was developed and validated. The enantiomeric separation was achieved, without any derivatization, on a carbamate derivative-based column (Chiralpak AD). The effect of the organic modifiers, 2-propanol and ethanol, in the mobile phases was optimised to obtain enantiomeric separation. Limits of detection and quantitation of 2 and 6 ng, respectively, were obtained for both of the enantiomers. The linearity was established in the range of 5–41 μg for trans-(−)-paroxetine and in the range of 10–160 ng for trans-(+)-paroxetine. The accuracy of the method was 102.3% (mean value) for trans-(−)-paroxetine and 99.9% (mean value) for trans-(+)-paroxetine. For the precision (repeatability), a relative standard deviation value of 1.5% (mean value) for trans-(−)-paroxetine and of 2.1% (mean value) for trans-(+)-paroxetine was found. The method is capable of determining a minimum limit of 0.2% of trans-(+)-isomer in commercial samples.     

Correlations between prestalk-prespore tendencies and cAMP-related activities in Dictyostelium discoideum

Amoebae of the cellular slime mould Dictyostelium discoideum (strain Ax2) grown in axenic medium containing 86 mM glucose [G(+) cells] or no glucose [G(−) cells] were examined for the characteristics of aggregation, cAMP secretion rate, cAMP phosphodiesterase activity and cell surface cAMP-binding activity. (When G(+) and G(−) cells are mixed, G(+) cells preferentially differentiate into prespore cells and sort out to the posterior region of a slug.)Under the same conditions, G(−) cells aggregate later than G(+) cells and the formation of stable streams by G(−) cells was particularly delayed. The movements of G(−) cells during aggregation were less organized compared with the ordered behaviour of G(+) cells, yet G(−) cells seemed to be more sensitive to chemotactic signals. Furthermore, the cAMP-binding activity of G(−) cells was considerably higher than that of G(+) cells, a difference that is probably due to the difference in the number of the cAMP receptor sites. Also G(−) cells, compared to G(+) cells, secreted slightly more cAMP and showed higher activity of cell-bound phosphodiesterase activity at the aggregation stage, whereas the extracellular phosphodiesterase activity was lower, although these differences were minor.     

Lactones 7: 1. Enantioselective lactonization of racemic ethyl (5,5-dimethyl-2,3-epoxycyclohex-1-yl)acetate

Seven fungi strains were checked as biocatalysts of lactonization of ethyl (5,5-dimethyl-2,3-epoxycyclohex-1-yl)acetate. Two of them transformed the racemic substrate with high efficiency and enantioselectivity. Rhodotorula rubra transformed preferentially the (−) enantiomer of substrate, whereas Fusarium semitectum, the (+) one.     

Development of a reaction system for the selective conversion of (−)-trans-carveol to (−)-carvone with whole cells of Rhodococcus erythropolis DCL14

The present article addresses the development of a microbial reaction system for the transformation of carveol to carvone, using whole cells of Rhodococcus erythropolis DCL14. This strain contains a NAD-dependent carveol dehydrogenase (CDH) when grown on limonene or on cyclohexanol. When a mixture of (−)-cis and (−)-trans-carveol is supplied, only (−)-trans-carveol is converted. Thus, besides (−)-carvone, pure (−)-cis-carveol can be obtained as product.

Initial experiments were performed batchwise using an aqueous system. (−)-Trans-carveol conversion rate gradually decreased during successive reutilisation batches. After the third reutilisation, activity was completely lost. Cells grown on cyclohexanol showed a slightly higher activity as compared to cells grown on (+)-limonene. A production of 4.3 μmol (−)-carvone formed per mg protein was achieved. A significant improvement with respect to initial reaction rate and productivity was obtained with aqueous–organic two-phase systems. Using a 5 to 1 buffer/iso-octane system, a 40% increase in the initial rate and a 16-fold increase of the production was observed. A further improvement resulted from increasing the volume of solvent (1 to 1 buffer/dodecane ratio). An initial reaction rate of 26 nmol/(min*mg protein) was observed, while production increased to 208 μmol (−)-carvone formed per mg protein. As in the single-phase system, reaction rate gradually decreased along the successive cell reutilisation batches. Addition of co-substrates for the regeneration of NAD did not prevent this decay. A simple downstream process was developed for the recovery of carvone and cis-carveol.     

Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

A pterocarpan and two isoflavans from alfalfa

(−)6aR,11aR-Dihydro-3-hydroxy-9,10-dimethoxy-6H-benzofuro[3,2c] [1]-benzopyran (10-methoxymedicarpin), (+)-(2,3,4,-trimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7-hydroxy-2′,3′,4′-trimethoxyisoflavan) and (+)-(2,3,4-trimethoxy-5-hydroxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7,5′-dihydroxy-2′,3′,4′-trimethoxyisoflavan) were isolated for the first time from dried Medicago sativa hay. Structural assignments were based on ¹H NMR and mass spectra, X-ray crystallography, and optical rotations.     

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

Biotransformations of ortho-, meta- and para-aromatic nitrocompounds by strains of Aspergillus terreus: Reduction of ketones and deracemization of alcohols

Seven strains of the fungus Aspergillus terreus isolated from several provenances in Brazil, catalyzed biotransformations of ortho-, meta- and para-nitrophenyl compounds at different pH values. ortho-Nitroacetophenone and meta-nitroacetophenone were transformed into (S)-(+)-1-(ortho-nitrophenyl)ethanol and (S)-(−)-1-(meta-nitrophenyl)ethanol with high enantiomeric excess (e.e. ≥98%) and conversion (≥98%) by all the strains used. Deracemization of (RS)-1-(meta-nitrophenyl)ethanol was obtained with high selectivity (e.e. up to ≥98%) and good conversion (c 98%). The biotransformations in acidic medium using these fungus strains were more efficient than under basic or neutral conditions.     

A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 Aromatase)

In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the “I-helix model” by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (−)-R-vorozole, (−)-S-fadrozole, R-liarozole and (−)-R-aminoglutethimide. It is found that (+)-S-vorozole, (−)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu³⁰² and Asp³⁰⁹) and C-terminus (Ser⁴⁷⁸ and His⁴⁸⁰). The weak activity of (−)-R-aminoglutethimide might be attributed to a lack of interaction wit the C-terminus.     

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Temperature dependence study of five-coordinate complex formation of zinc(II) octaethyl and tetraphenylporphyrin

Five coordinate complex formation of zinc(II)–octaethylporphyrin (Zn(OEP)) and zinc(II)–tetraphenylporphyrin (Zn(TPP)) have been studied in the presence of seven systematically selected electron donor molecules. Stability constants in toluene were determined at various temperature ranged between 20 and 60°C by absorption and steady-state fluorescent measurements from which thermodynamic parameters were determined. Depending on the porphyrin and the axial ligand the entropy is changing between −127 and −61 J mol⁻¹ K⁻¹ while the enthalpy is ranging from −49 to −24 kJ mol⁻¹.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).