Chiral metal complexes. Part 22. Stereoisomers of the dichloro [3,6-diaza-2S,7S-di(2-pyridyl)octane] - cobalt(III) ion and related species

The reaction of 3,6-diaza-2S,7S-di(2-pyridyl)- octane, S,S-peaen, with Co(II) and O₂ in aqueous solution yields a mixture, from which may be isolated three chelate diastereomers after the addition of HCl and HClO₄. These are δ-α-[Co(S,S-peaen)Cl₂]- ClO₄ and its Λ-α and Δ-β analogues. Previous workers had reported that a second β-diastereoisomer could be obtained but it has been shown that this is in fact an isomeric mixture of both Δ-α- and Λ-α- [Co(S,S-peaen)Cl₂]ClO₄. All three isomers react with oxalate anions to form an apparently unique product Λ-α-[Co(S,S-peaen)ox]⁺ in aqueous solution and which can be crystallized as its perchlorate salt. Two of the reactions reported represent unusual examples of octahedral inversions.     

R-(−)-mandelic acid production from racemic mandelic acids by Pseudomonas polycolor with asymmetric degrading activity

The microbial asymmetric degradation of S-(+)-mandelic acid was investigated in order to develop a practical process for R-(−)-mandelic acid production from racemic mandelic acids. Among the 790 culture strains tested, microorganisms belonging to the Brevibacterium, Pseudomonas, Rhodococcus, Rhodotorula, Rhodosporidium, Sporobolomyces and Gibberella genera exhibited high S-(+)-mandelic acid degrading activity. Pseudomonas polycolor IFO 3918 was determined to be the best strain and used as a biocatalyst for eliminating the S-(+)-isomer. The maximum rate of S-(+)-isomer degradation was obtained at 30°C and pH 7.0. Under these optimal conditions, the S-(+)-isomer in a racemic mandelic acid 45 g/l mixture was completely degraded within 24 h, with 20 g of R-(−)-mandelic acid per liter remaining in the reaction mixture. Crystalline R-(−)-mandelic acid with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess was obtained at a yield of 35% by acidification of the reaction mixture, extraction with ethyl acetate and subsequent concentration.     

High yield production of S-adenosyl-l-homocysteine with microbial cells as the catalyst

A simple and efficient enzymatic process for the production of S-adenosyl-l-homocysteine through catalysis by bacterial S-adenosylhomocysteine hydrolase (EC 3.3.1.1) is described. Washed cells of several actinomycetes, and Alcaligenes and Pseudomonas strains were found to be favorable sources of the enzyme. The reaction is carried out in potassium phosphate, pH 8.0, containing adenosine and l-homocysteine as the substrates, and washed cells of Alcaligenes faecalis AKU 101, which contain a high level of the enzyme, as the catalyst. With concentrations of adenosine and l-homocysteine of up to 200 mM, the molar conversion to S-adenosyl-l-homocysteine after 4–21 h at 37°C was nearly 100%. The maximum yield of 199 mM (76.5 mg ml⁻¹) was attained with a reaction mixture containing 300 mM substrates. S-Adenosyl-l-homocysteine of a high purity was easily isolated from the reaction mixture by simple gel-filtration on Sephadex G-10 with good recovery.     

Synthesis of four stereoisomers of 4-amino-2-(hydroxymethyl)tetrahydrofuran-4-carboxylic acid

The 5-benzyl ether, 15, of a 1,2,4,5-pentanetetrol of known 2S configuration was made by a multistep synthesis from d-ribose. Ring-closure of the 1-O-tosyl derivative, 17, with retention of configuration, followed by oxidation, gave the 2S enantiomer, 22, of 2-benzyloxymethyl-4-oxotetrahydrofuran. The latter was converted by a hydantion synthesis into the 4-amino-4-carboxylic acid (mixture of 2S,4R and 2S,4S isomers, 28 and 29). Spontaneous lactonization of the 2S,4R diastereomer proved it to have the “cis” configuration. The remaining, 2S,4S diastereomer then must be “trans” it is identical with a natural compound recently isolated from an acid hydrolyzate of diabetic urine. In a parallel synthesis, the 4-O-mesyl derivative (de-O-isopropylidenated 19) was cyclized, with inversion at ring-position 2, leading after oxidation to the 2R enantiomer, 25, of the 4-oxotetrahydrofuran. The hydantoin synthesis this time yielded a mixture of the 2R,4R and 2R,4S amino-acids. Spontaneous lactonization of the latter showed it to have the “cis” configuration. Absolute configurations were assigned to the four optically active products, based on the known absolute configuration of d-ribose and the known mechanisms of the synthetic reactions.     

Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio (α) and enantiomeric excess of (S)-ibuprofen (ee_S).     

Enantioselective Synthesis of S-Equol from Dihydrodaidzein by a Newly Isolated Anaerobic Human Intestinal Bacterium

A newly isolated rod-shaped, gram-negative anaerobic bacterium from human feces, named Julong 732, was found to be capable of metabolizing the isoflavone dihydrodaidzein to S-equol under anaerobic conditions. The metabolite, equol, was identified by using electron impact ionization mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectroscopy, and UV spectral analyses. However, strain Julong 732 was not able to produce equol from daidzein, and tetrahydrodaidzein and dehydroequol, which are most likely intermediates in the anaerobic metabolism of dihydrodaidzein, were not detected in bacterial culture medium containing dihydrodaidzein. Chiral stationary-phase high-performance liquid chromatography eluted only one metabolite, S-equol, which was produced from a bacterial culture containing a racemic mixture of dihydrodaidzein. Strain Julong 732 did not show racemase activity to transform R-equol to S-equol and vice versa. Its full 16S rRNA gene sequence (1,429 bp) had 92.8% similarity to that of Eggerthella hongkongenis HKU10. This is the first report of a single bacterium capable of converting a racemic mixture of dihydrodaidzein to enantiomeric pure S-equol.     

Enantiomeric Composition of the trans-Dihydrodiols Produced from Phenanthrene by Fungi

The trans-dihydrodiols produced during the metabolism of phenanthrene by Cunninghamella elegans, Syncephalastrum racemosum, and Phanerochaete chrysosporium were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the trans-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the trans-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene trans-1,2-dihydrodiol produced by C. elegans was a mixture of the 1R,2R and 1S,2S enantiomers in variable proportions. The phenanthrene trans-3,4-dihydrodiol produced by P. chrysosporium was the optically pure 3R,4R enantiomer, but that produced by S. racemosum was a 68:32 mixture of the 3R,4R and 3S,4S enantiomers. The phenanthrene trans-9,10-dihydrodiol produced by P. chrysosporium was predominantly the 9S,10S enantiomer, but those produced by C. elegans and S. racemosum were predominantly the 9R,10R enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.     

Two novel phytotoxic substances from Leucas aspera

Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 μM, respectively. The concentration required for 50% growth inhibition (I₅₀) of the test species ranges from 31 to 80 μM, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract.     

Induction of enantio-selective apoptosis in human leukemia HL-60 cells by (S)-erypoegin K,an isoflavone isolated from Erythrina poeppigiana

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a chiral carbon at the C-2′′ position of its furan ring and naturally occurs as a racemic mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-erypoegin K from genistein and separated the optical isomers by HPLC using a chiral column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and apoptosis-inducing activity, with an IC₅₀ value of 90 nM, about 50% lower than that of its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity on HL-60 cells.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Thiiranium ion intermediates in the formation and reactions of S-(2-haloethyl)-l-cysteines

Thiiranium ions formed from the cysteine or glutathione conjugates of 1,2-dihaloethanes are believed to be responsible for the genotoxicity of the parent alkyl halides. The conversions of specifically deuterated β-hydroxyethyl sulfides to the corresponding β-haloethyl sulfides are studied to provide direct evidence for the involvement of thiiranium ions in the reactions of the cysteine conjugates of 1,2-dihaloethanes. S-(2-Hydroxyethyl-1, 1-d₂)-l-cysteine is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of the corresponding S-(2-haloethyl)-l-cysteines in concentrated hydrochloric, hydrobromic, or hydroiodic acids without detectable formation of the 2,2-d₂ isomer of the parent hydroxyethyl derivative. Dissolution of S-(2-hydroxyethyl)-l-cysteine in trifluoromethanesulfonic acid yields a compound with the NMR spectral properties of S-(l-cysteinyl)ethyl thiiranium trifluoromethanesulfonate. The organosoluble S-(2-hydroxyethyl-1,1-d₂) benzyl sulfide is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of S-(2-chloroethyl) benzyl sulfide by thionyl chloride or triphenylphosphine: carbon tetrachloride. These results demonstrate the involvement of thiiranium ion intermediates in the conversion of 2-hydroxyethyl sulfides to 2-haloethyl sulfides in halogen acids and a similar symmetrical intermediate in the chlorination reactions effected by thionyl chloride or triphenylphosphine: carbon tetrachloride.     

Three dihydroisocoumarin glucosides from Hydrangea macrophylla subsp. serrata

Three new dihydroisocoumarin glucosides, macrophyllosides A, B and C were obtained from dry leaves of Hydrangea macrophylla subsp. serrata, together with the previously known hydrangenol 8-β-glucoside. Using NMR and CD techniques and some chemical transformation, the structures were elucidated as (3S)-3′,4′,5′-trimethoxyphenyl-8-β-D-glucopyranosyl dihydroisocoumarin, (3R and (3S)-3′,5′-dimethoxy-4′-hydroxyphenyl-8-β-D-glucopyranosyl dihydroisocoumarins. CD experiments indicated that hydrangenol 8-β-glucoside was the mixture of 3S- and 3R- stereoisomers.     

Enantiomerism: A characteristic of the proanthocyanidin chemistry of the monocotyledonae

Proanthocyanidin polymers containing 2,3-cis-procyanidin units with a partly racemic mixture of 2R (the normal configuration) and 2S units are widespread in the Monocotyledonae, being present in several families in the Arecidae, Commelinidae and Lilliidae.     

Synthesis of 1,6-thioanhydro-D-glucitol

Treatment of 2,4-O-benzylidene-1,6-di-O-tosyl-D-glucitol (1) with potassium thiolbenzoate afforded the 6-S-benzoyl compound 2 and its 5-benzoate 4, the structure of which was proved chemically. When 1 was acetylated and then treated with the thiolate, the acetylated 6-S-benzoyl compound 19 was obtained in good yield in addition to some 1,6-di-S-benzoyl derivative 21. Treatment of 19 with acetic anhydride-acetic acid-sulfuric acid afforded 2,3,4,5-tetra-O-acetyl-6-S-acetyl-1-O-tosyl-D-glucitol (26), which was converted by sodium methoxide into a mixture of 1,5-anhydro-6-thio-D-glucitol (28) and 1,6-thioanhydro-D-glucitol (29). These two compounds were isolated as their acetates (30 and 31) by column chromatography, or by converting 28 into its S-trityl derivative (32).     

Reactions of ethyl 3,5,6-tri-O-acetyl-2-S-ethyl-1,2-dithio-α-D-mannofuranoside with halogens

Ethyl 3,5,6-tri-O-acetyl-2-S-ethyl-1,2-dithio-α-D-mannofuranoside (5) reacted with bromine to give the very unstable glycosyl bromide 4, which with water gave a mixture of the 1-hydroxyl analogue (8) and the nonreducing α-D-(1→1)-linked disaccharide derivative 9. When the bromide 4 was treated with mercuric acetate or potassium acetate, 1,3,5,6-tetra-O-acetyl-2-S-ethyl-2-thio-α-D-mannofuranose (7) was obtained, but silver acetate in carbon tetrachloride gave 7 in admixture with its β-anomer (10). Methanol reacted with 4 to give an anomeric mixture of the glycofuranosides (11 and 12). An excess of chlorine converted the dithio derivative 5 into a 3,5,6-tri-O-acetyl-2-chloro-2-S-ethyl-2-thio-D-manno(or gluco)furanosyl chloride (13), whereas a lower proportion of chlorine appeared to give the 1-chloro analogue of 4. Treatment of the dichloro derivative 13 with methanol led to a mixture of three methyl glycosides, one (14) retaining the chlorine atom at C-2, and the other two (15 and 16) resulting from exchange of both chlorine atoms by methoxyl groups.     

Regioselective glutathione conjugation of the carcinogen, 7,12-dihydroxymethylbenz[α]anthracene,via reactive 7-hydroxymethyl sulfate ester in rat liver cytosol

Potent mutagenicity of 7,12-dihydroxymethylbenz[α]anthracene (DHBA) toward Salmonella typhimurium TA 98 in the presence of rat liver cytosol fortified with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) was completely retarded by the addition of glutathione (GSH). The reactive and intrinsically mutagenic metabolite, DHBA 7-sulfate, formed by hepatic cytosolic sulfotransferase disappeared from the incubation mixture by the addition of GSH. Non-mutagenic S-(12-hydroxymethylbenz[α]anthracen-7-yl)methylglutathione was isolated from the incubation mixture consisting of the hepatic cytosol, DHBA, PAPS, and GSH and proved to be formed by GSH S-transferase directly from DHBA 7-sulfate as an obligatory intermediate.     

Stereochemical aspects of the conversion of cyclopeptine into dehydrocyclopeptine by cyclopeptine dehydrogenase from Penicillium cyclopium

Cyclopeptine dehydrogenase, an enzyme from Penicillium cyclopium, catalyses the reversible transformation of the benzodiazepine alkaloids cyclopeptine and dehydrocyclopeptine. By the dehydrogenation of cyclopeptine two hydrogen atoms are removed from the positions 3 and 10. It was demonstrated that, from the two optical isomers of cyclopeptine, only the naturally occurring 3S-compound was used as substrate by cyclopeptine dehydrogenase. To test the stereospecificity of the enzyme with respect to the second hydrogen which is eliminated from C-10 a mixture of cyclopeptine-3S-[10R-³H₁] and cyclopeptine-3R-10S-³H₁] was prepared. The 3S-isomer was transformed by the enzyme into radioactively labelled dehydrocyclopeptine. This demonstrated that cyclopeptine dehydrogenase removes the 10-proS hydrogen atom from the cyclopeptine molecule. Because the formed dehydrocyclopeptine has the trans-configuration it is probable that a synperiplanar elimination takes place. The hydride ion removed from cyclopeptine is transferred to the 4-proR-position of NAD⁺. Cyclopeptine dehydrogenase thus belongs to the A-specific dehydrogenases.     

Production of S-adenosyl-l-homocysteine by bacterial cells with a high content of S-adenosylhomocysteine hydrolase: Utilization of a racemic mixture of homocysteine as the substrate

Alcaligenes faecalis cells contain a high level of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) and are the useful catalyst for the preparative production of S-adenosyl-l-homocysteine (Shimizu et al., 1984, Eur. J. Biochem. 141, 385–392; 1986, J. Biotechnol. 4, 81–90). A problem with this production was that the d-isomer of homocysteine was not utilized. To solve this problem, we screened for microbial strains capable of synthesizing S-adenosyl-l-homocysteine from d-homocysteine and adenosine, and found that Pseudomonas putida cells catalyze this conversion. Although P. putida S-adenosylhomocysteine hydrolase catalyzed only the condensation of l-homocysteine with adenosine, the bacterium also produced a racemase which interconverts the d- and l-homocysteine isomers. For practical purposes. A. faecalis was still superior in showing high S-adenosylhomocysteine hydrolase activity and low adenosine deaminase activity. Therefore, P. putida was used just as a source of the racemase. With 200 mM adenosine and 200 mM dl-homocysteine, the molar conversion to S-adenosyl-l-homocysteine was 86%, when a mixture of A. faecalis cells (36 mg ml⁻¹) and P. putida cells (4 mg ml⁻¹) was used as the catalyst.     

Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each), and boosted twice (25 μg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10⁵ CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10⁵ CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.     

Formation of 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene and inhibition of aminoazo dye-nucleic acid binding in vitro by reaction of glutathione with metabolically-generated N-methyl-4-aminoazobenzene-N-sulfate

The reaction of glutathione (GSH) with metabolically-formed N-methyl-4-aminoazobenzene-N-sulfate (MAB-N-sulfate), a presumed ultimate carcinogenic metabolite of N,N-dimethyl-4-aminoazobenzene (DAB), was investigated using a hepatic sulfotransferase incubation mixture containing GSH and the proximate carcinogen, N-hydroxy-N-methyl-4-aminoazobenzene (N-HO-MAB). Under these conditions, 6–16% of the MAB-N-sulfate formed could be trapped as an aminoazo dye-GSH adduct. Upon subsequent purification, the adduct was shown to be chromatographically and spectrally identical to 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (3-GS-MAB), a known biliary metabolite of DAB and a product of the reaction of the synthetic ultimate carcinogen, N-benzoyloxy-N-methyl-4-aminoazobenzene(N-BzO-MAB), with GSH. Neither 2′- nor 4′-GS-MAB, both products of the latter reaction, were detected in the sulfotransferase incubation mixture.GSH-S-transferases did not appear to be involved in the reaction of MAB-N-sulfate or N-BzO-MAB with GSH. The addition of triethyltin, a potent GSH-S-transferase inhibitor, had no effect on the yield of 3-GS-MAB in (N-HO-MAB sulfotransferase)-GSH incubations; and the addition of cytosol or purified GSH transferases A and B to a (N-BzO-MAB)-GSH reaction mixture did not increase the amount of 3-GS-MAB formed.GSH was shown to inhibit only partially the covalent binding of [³H]-MAB-N-sulfate to DNA and rRNA. At 10 and 100 mM GSH, the sulfotransferase-mediated binding of [³H]N-HO-MAB to both nucleic acids was reduced by 30% and 70%, respectively. The role of GSH in the detoxification of chemical carcinogens is discussed.