Efficient synthesis of unnatural dipeptides based on cis-2,5-disubstituted pyrrolidine

The well-defined unnatural dipeptides based on cis-2,5-disubstituted pyrrolidine backbone were synthesized from commercially available starting materials meso-diethyl-2,5-dibromoadipate, (S)-(?)-1-phenylethylamine, and phenylalanine. The configurations of all the chiral centers in these unnatural dipeptides are confirmed by X-ray crystal diffraction analysis.     

Stereospecific synthesis of α-methylated amino acids

Summary. Both 2,5-trans and 2,5-cis disubstituted 2-tert-butyl-5-(indol-3-yl)methylimidazolidin-4-ones were synthesised and their enolates were prepared using LDA. While the enolate of the 2,5-trans disubstituted derivative could not be methylated, the enolate of the cis-2,5-disubstituted derivative was successfully methylated with methyl iodide to a product which on hydrolysis gave enantiomerically pure α-methyl-L-tryptophan. Received October 31, 1998, Accepted July 23, 1999     

Synthesis and evaluation of in vitro antioxidant properties of novel 2,5-disubstituted 1,3,4-oxadiazoles

2,5-Disubstituted 1,3,4-oxadiazole compounds are one of the most attractive heterocyclic compounds for researchers due to their biological activities. In the undertaken research, a number of potential 2,5-disubstituted 1,3,4-oxadiazole analogues were synthesized through multi step reaction and characterized by FT-IR, ¹H NMR, mass spectra, and also by elemental analysis. Further benzophenone tagged indole acetohydrazides and 2,5-disubstituted 1,3,4-oxadiazoles were evaluated for antioxidant potential, through different in vitro models such as DPPH, nitric oxide and hydrogen peroxide methods. In the series of compounds some of them had shown good to moderate in vitro antioxidant potential compare to the standard drug ascorbic acid in all the above three methods.     

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.     

Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745

Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi. The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity. We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes. versicolor. We identified 13 oligomers formed from 2,5-xylidine. (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25×10^–5 M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture. Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.     

Facile synthetic route to enantiopure unsymmetric cis-2,5-disubstituted pyrrolidines

The (+/-)-cis-5-arylcarbamoyl-2-ethoxycarbonylpyrrolidines 6a-g were firstly synthesized in 53-64% yields by using meso-diethyl-2,5-dibromoadipate 3 and (S)-(-)-1-phenylethylamine in three steps. The diastereomeric mixture (S;2S,5R)-(-)-7 and (S;2R,5S)-(+)-8 were prepared by the Grignard reaction and separated by a flash column chromatography in 29 and 52% yields. The absolute configurations of (+)-8 was confirmed by X-ray crystallographic analysis and the enantiopure pyrrolidines (2S,5R)-(-)-9/(2R,5S)-(+)-9 and (2S,5R)-(-)-10/(2R,5S)-(+)-10 were obtained in good yields.     

Derivatives of 2,5-anhydroallitol and 2,5-anhydroaltritol

Two routes to protected derivatives of 2,5-anhydroallitol were investigated. The first route, involving a two-step reduction of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allonitrile (4), gave a mixture of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-altritol (7) and a lesser amount of the desired 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allitol (6). Isomerization was shown to occur in the first reduction step—treatment of the nitrile 4 with Raney nickel, sodium hypophosphite, and acetic acid. The second route gave isomerically pure 2,5-anhydro-3,4,6-tri-O-benzyl-D-allitol (21) via reduction of the corresponding ethyl allonate (18).     

Substituted benzoic and picolinic acids as foliar sprays against potato common scab

Glasshouse tests on potato cv. Arran Banner measured the effects of single early foliar sprays of 18 substituted benzoic and two dichloropicolinic acids on the severity of common scab, caused by soil-borne Streptomyces scabies. The monosubstituted benzoic acids tested (25 mM) did not affect scab. However, anti-scab action was shown by some 2,5-disubstituted acids. Decreases in scab severity were about 35% from 1·6 mM 5-bromo-2-chloro- and 2,5-dimethylbenzoic acids, and over 50% from 1·6 mM 2,5-dichloro-, 2,5-dibromo- and 5-chloro-2-nitrobenzoic acids and from 0·05 mM 3,6-dichloropicolinic acid (clopyralid), which is structurally similar to 2,5-dichlorobenzoic acid. None of the spray treatments affected yield or shape of tubers. No visible effects on foliage were caused by two of the benzoic acids active against scab (5-chloro-2-nitro- and 2,5-dimethyl-), but the other active acids caused some distortion. In tests of the six dichlorobenzoic acids against S. scabies in culture, the 2,5-isomer (which was the most effective against the disease in plants) was one of the least toxic. With other 2,5-disubstituted benzoic acids, it probably decreased scab indirectly by altering the response of the host to infection.     

Metabolism of 2,5-dichlorobenzoic acid inPseudomonas stutzeri

4-Chroropyrocatechol is formed as a results of the oxidation of 2,5-dichlorobenzoate byPseudomonas stutzeri. 3-Chloro-cis,cis-muconic acid is the product of the oxidation of 4-chloropyrocatechol. Pyrocatechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, but not pyrocatechol 2,3-dioxygenase or protocatechuate 3,4-dioxygenase activities were found in cell-free extracts. Theortho cleavage activity for catechols appeared to involve induction of isoenzymes with different stereospecificity towards chlorocatechols. A catablic pathway for the degradation of 2,5-dichlorobenzoate by a newly isolated strain ofP. stutzeri was proposed.     

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied.

Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days.

Results: Administration of 0.5% 2,5-HD significantly (P?<?0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats.

Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.     

Crystal and molecular structure of L-cis-3,6-dimethyl-2,5-piperazinedione (L-alanyl-L-alanyl-2,5-diketopiperazine)

The crystal structure of L -cis-3, 6-dimethyl-2,5-piperazinedione (L -alanyl-L -alanyl-2,5-diketopinerazine) has been determined. The unit cell is triclinic with a = 8.05, b = 6.08, c = 5.15 Å, α = 131.7°, β = 82.4°, γ = 106.6°. The space group is P1 with one molecule per unit cell. The six-membered ring is found to be nonplanar with an angle between the two amide group planes of 26°. One amide group deviates slightly from planarity. The experimental molecular model is discussed in terms of its flexibility.     

Kinetic resolution and isomerization of 2,5-disubstituted pyrrolines

The efficient kinetic resolution of 2,5-disubstituted pyrrolines was accomplished by employing hydrosilylation with a chiral catalyst, (EBTHI)TiF2 (EBTHI = ethylenebis(tetrahydroindenyl)). An interesting isomerization reaction of the cyclic imines catalyzed by the same chiral catalyst was also discovered.     

Degradation of 2,5-dichlorobenzoic acid byPseudomonas aeruginosa JB2 at low oxygen tensions

From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h^–1 and a dissolved oxygen concentration of 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K _m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (< 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.Abbreviations BA benzoic acid - 2,5-DBA 2,5-dichlorobenzoic acid - QO ₂ ^max maximum specific respiration rate     

2,5-Diketogluconate formation byChromobacterium

Chromobacterium lividum oxidizes gluconate to 2,5-diketogluconate via 2-ketogluconate. The conversion by either growing or resting cells proceeds best in an aerated medium at pH 5.9. The yield is nearly 50%. Diketogluconate is not further decomposed. The preparation of chromatographically pure diketogluconate using eitherChromobacterium orGluconobacter is described. Several properties of the diketo compound are described.     

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

Synthesis of a 2-Amino-2,5-dideoxy-D-ribofuranoside Analogue

Benzyl 2-benzamido-2,5-dideoxy-α-D-ribofuranoside was synthesized via benzyl 2-amino-3-C-(branched-chain)-2,3,5-trideoxy-α-d-furanoside from an optically active 3-oxa-6-azabicyclo[3.2.0]-heptan-7-one derivative.     

Synthesis of a 2-acetamido-5-amino-2,5-dideoxy- -xylopyranosyl derivative

2-Acetamido-5-amino-2,5-dideoxy- -xylopyranosyl hydrogensulfite (11) has been synthesized from benzyl 2-(benzyloxycarbonylamino)-2-deoxy-5,6-O-isopro-pylidene-β- -glucofuranoside (1). O-Deisopropylidenation of 1 gave the triol 2, which was converted, via oxidative cleavage at C-5-C-6 and subsequent reduction, into the related benzyl β- -xylofuranoside derivative (3). Catalytic reduction of benzyl 2-(benzyloxycarbonylamino)-2-deoxy-5-O-tosyl-β- -xylofuranoside, derived from 3 by selective tosylation, and subsequent N-acetylation, afforded benzyl 2-acetamido-2-deoxy-5-O-tosyl-β- -xylofuranoside, which was treated with sodium azide to give the corresponding 5-azido derivative (6). (Tetrahydropyran-2-yl)ation of the product formed by hydrolysis of 6 gave 2-acetamido-5-azido-2,5-dideoxy-1,3- di-O-(tetrahydropyran-2-yl)- -xylofuranose (9). Treatment of 2-acetamido-5-amino-2,5-dideoxy-1,3-di-O-(tetrahydropyran-2-yl)- -xylofuranose, derived from 9 by reduction, with sulfur dioxide in water gave 11. Hydrogenation of 6 and subsequent acetylation yielded 3-acetamido-4,5-diacetoxy-1-acetyl-xylo-piperidine. Evidence in support of the structures assigned to the new derivatives is presented.     

Synthesis and anticonvulsant activity of new N-mannich bases derived from benzhydryl- and isopropyl-pyrrolidine-2,5-dione

Synthesis and anticonvulsant properties of 26 new N-Mannich bases of 3-benzhydryl-(5–17) and 3-isopropyl-pyrrolidine-2,5-diones (18–30) have been described. Initial anticonvulsant screening for these compounds was evaluated in mice after intraperitoneal administration in the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizures tests. The acute neurological toxicity was determined by applying the rotorod test. The in vivo results in mice showed that the majority of 3-benzhydryl-pyrrolidine-2,5-dione derivatives revealed effectiveness, while 3-isopropyl-pyrrolidine-2,5-dione derivatives were practically devoid of activity. The quantitative evaluation in both tests revealed that the most active were N-[{4-(3-chlorophenyl)-piperazin-1-yl}-methyl]-3-benzhydryl-pyrrolidine-2,5-dione (9) with ED_{5 0} value?=42.71?mg/kg (MES), ED_{5 0} value?>150?mg/kg (scPTZ), and N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-benzhydryl-pyrrolidine-2,5-dione (13) with ED_{5 0} value?=101.46?mg/kg (MES) and ED_{5 0} value?=72.59?mg/kg (scPTZ). These molecules showed higher potency and lower neurotoxicity than the reference antiepileptic drugs (ethosuximide and valproic acid). To explain the probable mechanism of action of selected active derivatives (9 and 13), their influence on Na_v1.2 and l-type calcium channel was evaluated in vitro.     

2,5-Diketo- D-gluconate production by a mixed culture of two newly-isolated strains: Flavimonas oryzihabitans and Pseudomonas cepacia

A mixed culture of two Gram-negative bacteria isolated from soil converted 50 g d-glucose l^–1 to 2,5-diketo-d-gluconate (2,5 DKG) in 92% yield within 150 h. The first strain, producing 2-keto-d-gluconate (2 KDG) from d-glucose via d-gluconate (DG), was classified as Flavimonas oryzihabitans. The second strain, that converts 2 KDG to 2,5 DKG, was identified as Pseudomonas cepacia. This approach presents a new possibility to produce ascorbic acid by microbial transformation, including the use of other, more convenient substrates.     

Acetate as a source of reducing equivalents in the reductive dechlorination of 2,5-dichlorobenzoate

Desulfomonile tiedjei is the key dechlorinating organism in a three-tiered bacterial consortium that grows on the methanogenic degradation of 3-chlorobenzoate. 2,5-Dichlorobenzoate, however, is only converted to 2-chlorobenzoate and is not a methanogenic substrate for the consortium. The dechlorinator uses hydrogen produced from benzoate by the benzoate degrading member of consortium as its source of reducing equivalents for the dechlorination reaction. Incubation of 3-chlorobenzoate grown consortium cells with 2,5-dichlorobenzoate resulted in the consumption of acetate concurrent with the formation of 2-chlorobenzoate indicating that acetate can serve as an alternative source of reducing equivalents for reductive dechlorination. This interpretation was confirmed by the finding that the formation of ¹⁴CO₂ from 2-¹⁴C-labeled acetate was stoichiometric. The addition of hydrogen to 2,5-dichlorobenzoate metabolizing cells resulted in (i) an 2.7-fold increase in the rate of dechlorination, and (ii) a drop in the amount of label recovered as CO₂+CH₄ from methyl ¹⁴C-labeled acetate, indicating that hydrogen was the preferred source of reducing equivalents for reductive dechlorination. Benzoate, an indirect source of H₂ in the consortium, also inhibited the oxidation of acetate, while glucose, methanol, and butyrate did not affect labeled gas production and therefore were not suitable electron donors. Concomittant to dechlorination of 2,5-dichlorobenzoate 3- and 4-methoxybenzoate were converted to 3- and 4-hydroxybenzoate respectively. These conversions stimulated the rate of dechlorination 2-fold. Demethylation of 4-methoxybenzoate stimulated, but demethylation of 3-methoxybenzoate inhibited the oxidation of benzoate during the dechlorination of 2,5-dichlorobenzoate, suggesting that these isomers are metabolized through different pathways. Experiments with benzoate, 3-chlorobenzoate and 2,5-dichlorobenzoate metabolizing cells amended with ¹⁴CO₂ showed that actively dechlorinating cells catalyzed an exchange reaction between CO₂ and acetate.