Microbial Allyl Rearrangement and Resolution of Acetates of Unsaturated Cyclic Terpene Alchols by Pseudomonas sp. NOF-5 Strain

Microbial hydrolysis of the acetates of unsaturated cyclic terpene alcohols by Pseudomonas sp. NOF-5 isolated from soil was investigated. (±)-trans-Carveyl acetate ((±)-trans-3) was enantio-selectively hydrolyzed with NOF-5 strain to give ( – )-trans-carveol (( – )-trans-2 of 86.6% optical purity). However, the hydrolysis of (±)-cis-3 was less enantioselective, while (±)-piperitylacetate ((±)-6, a cis and trans mixture) was hydrolyzed to give the ( – )-trans- and ( – )-cis-piperitols (( – )- trans-5 and ( – )-cis-5) in a poor optical yield. In this case, other tert-alcohols, ( + )-trans- and ( – )- ds-2-p-menthen-1-ols ((±)-trans-7 and ( – )-cis-7), were also produced. Furthermore, microbial and enzymic allyl rearrangements of ( + )-trans-6 and ( – )-trans-verbenylacetate (( – )-trans-11) were studied. Biological treatment of (+)-trans-6 and ( – )-trans-11 with NOF-5 or its esterase gave (+)-trans- and (-)-cis-1 and ( + )-cis-3-pinen-2-ol (( + )-cis-12), respectively.     

Synthesis of Optically Pure (1S, 4S, 5S)-2-Pinen-4-ol (cis-Verbenol) and Its Antipode,the Pheromone of Ips Bark Beetles

The enantiomers of cis-verbenol (4a and 4a′) were first synthesized in optically pure state. (1S, 4S, 5S)-2-Pinen-4-ol (4a′) was dextrorotatory in acetone or in methanol but it was levorotatory in chloroform; cis-verbenols are indistinguishable by a prefix (+) or (?). The designation of the Ips pheromone as (+)-cis-verbenol is therefore ambiguous and it should be called as (1S, 4S, 5S)-2-pinen-4-ol (4a′) or (S)-cis-verbenol.     

Condensed tannins. Optically active diastereoisomers of (+)-mollisacacidin by epimerization

1. (+)-Mollisacacidin [(+)-3′,4′,7-trihydroxy-2,3-trans-flavan-3,4-trans- diol] is converted by autoclaving into the optically active free phenolic 2,3-trans-3-4-cis (12% yield), 2,3-cis-3,4-trans (11%) and 2,3-cis-3,4-cis (2·8%) diastereoisomers through epimerization at C-2 and C-4. 2. The relative configurations of the epimeric forms were determined by nuclear-magnetic-resonance spectrometry and paper ionophoresis in comparison with synthetic reference compounds, and was confirmed by chemical interconversions. 3. From this a scheme of epimerization is inferred and their absolute configurations are assigned as (2R:3S:4S), (2S:3S:4R) and (2S:3S:4S) respectively from the known absolute configuration (2R:3S:4R) of (+)-mollisacacidin.     

Synthesis and Antimicrobial Activity of Chiral cis-Cycloheximide Isomers

Such (+)- and (?)-cis-cycloheximide isomers as isocyclohcximide (1a, 1b), α-epiisocycloheximide (2a, 2b) and neocycloheximide (3a, 3b) were synthesized by aldol condensation of (?)-(2R, 4R)- and (+)-(2S, 4S)-cis-2,4-dimethyl-1-cyclohexanone (5a, 5b). obtained by microbial resolution, with 4-(2-oxoethyl)-2,6-piperidinedione (7). The absolute configuration of the (?)-cis-ketone 5a was confirmed by chemical correlation with natural (2S, 4S, 6S, αR)-cycloheximide (4). The newly synthesized isomer, (?)-α-epiisocycloheximide (2b), showed strong antimicrobial activity against S. cerevisiae andP. oryzae close to that of natural cycloheximide (4).     

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.     

The absolute configuration of phaseic and dihydrophaseic acids

A sample of phaseic acid methyl ester (5 mg, isolated from tomato plants fed (±)-abscisic acid, was reduced to a mixture of the epimeric dihydrophaseates which were separated by TLC. The more polar epimer was identical with the dihydrophaseate isolated from beans by Walton et al. [14]. Comparison of the NMR and IR spectra (H-bonding) of the two epimers shows the secondary hydroxyl of the less polar epimer is cis to the oxymethylene group, which is cis to the tertiary hydroxyl group. The absolute configuration of this centre is known so the absolute configuration of phaseic acid can be deduced. Phaseic acid is (−)-3-methyl-5{8[1(R), 5(R)-dimethyl-8(S)-hydroxy-3-oxo-6-oxabicyclo-(3,2,1)-octane]} 2-cis-4-trans-pentadienoic acid and both it and the reduction products exist in chair conformations. The more polar epimer isolated by Walton et al. is (−)-3-methyl-5{8[3(S,8(S)-dihydroxy-1(R,5(R)-dimethyl-6-oxabicyclo-(3,2,1)-octane]}2-cis-4-trans-pentadienoic acid. It is suggested that the less polar epimer should be referred to as epi-dihydrophaseic acid.     

A Preparation of Cow's Late Colostrum Fraction Containing αs1-Casein Promoted the Proliferation of Cultured Rat Intestinal IEC-6 Epithelial Cells

The asymmetric epoxidation of (±)-methyl (2Z,4E)-1′,4′-dihydroxy-α-ionylideneacetates is described for the preparation of chiral abscisic acid. A conventional Shapless kinetic resolution of (±)-1′,4′-cis-dihydroxyacetate with diethyl l-tartarate and then two simple steps of conversion gave (S)-abscisic acid, which was also obtained by the combination of (±)-1′,4′-trans-dihydroxyacetate with diethyl d-tartarte. Finally, (S)-abscisic acid was obtained in a 25% overall yield from the racemic mixture.     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

(−)-(S,S)-12-hydroxy-13-octadec-cis-9-enolide,a 14-membered lactone from Crepis conyzaefolia seed oil

Crepis conyzaefolia (Gouan) Dalle Torre seed oil contains about 3% of (?)-(S,S)-12-hydroxy-13-octadec-cis-9-enolide (1), a lactone of (?)-threo-12,13-dihydroxyoleic acid. The absolute configuration of the acid has been established as D-12, L-13 (12-S, 13-S) and the lactone has the same absolute configuration.     

Active site residues controlling substrate specificity in 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42

Acidovorax (formerly Pseudomonas) sp. strain JS42 utilizes 2-nitrotoluene as sole carbon, nitrogen, and energy source. 2-Nitrotoluene 2,3-dioxygenase (2NTDO) catalyzes the initial step in 2-nitrotoluene degradation by converting 2-nitrotoluene to 3-methylcatechol. In this study, we identified specific amino acids at the active site that control specificity. The residue at position 350 was found to be critical in determining both the enantiospecificity of 2NTDO with naphthalene and the ability to oxidize the ring of mononitrotoluenes. Substitution of Ile350 by phenylalanine resulted in an enzyme that produced 97% (+)-(1R, 2S)-cis-naphthalene dihydrodiol, in contrast to the wild type, which produced 72% (+)-(1R, 2S)-cis-naphthalene dihydrodiol. This substitution also severely reduced the ability of the enzyme to produce methylcatechols from nitrotoluenes. Instead, the methyl group of each nitrotoluene isomer was preferentially oxidized to form the corresponding nitrobenzyl alcohol. Substitution of a valine at position 258 significantly changed the enantiospecificity of 2NTDO (54% (−)-(1S, 2R)-cis-naphthalene dihydrodiol formed from naphthalene) and the ability of the enzyme to oxidize the aromatic ring of nitrotoluenes. Based on active site modeling using the crystal structure of nitrobenzene 1,2 dioxygenase from Comamonas sp. JS765, Asn258 appears to contribute to substrate specificity through hydrogen bonding to the nitro group of nitrotoluenes.     

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers. Received 15 November 1996/ Accepted in revised form 09 March 1997     

Asymmetric Hydrolysis of Prochiral Diesters with Pig Liver Esterase. Preparation of Optically Active Intermediates for the Synthesis of (+)-Biotin and (+)-α-Methyl-3,4-dihydroxyphenylalanine

With pig liver esterase, 1,3-dibenzyl-4,5-cis-bis(alkyloxycarbonyl)-2-oxoimidazolidine (1) was asymmetrically hydrolyzed to (4S,5R)-1,3-dibenzyl-5-alkyloxycarbonyl-2-oxoimidazolidine-4-carboxylic acid (2). This acid 2 was reduced with lithium borohydride to (4S,5R)-1,3-dibenzyl-5-hydroxymethyl-2-oxoimidazolidine-4-carboxylic acid lactone (3), which is known to be converted to (+)-biotin (4). With the same esterase, diethyl 3,4-dimethoxyphenylmethyl-(methyl)malonate (5) was asymmetrically hydrolyzed to (R)-ethyl hydrogen 3,4-dimethoxy-phenylmethyl(methyl)malonate (6), which can be converted to (S)-α-methyl-3,4-dihydroxyphenyl-alanine(l-α-methyldopa) (9).     

Discovery of Four New Compounds from Macropanax membranifolius and Their Cytotoxic Activity

A phytochemical investigation of the methanolic extract of the Macropanax membranifolius C.B. Shang leaves led to the isolation of three new flavans, (2R,3R)-4′-O-methylcatechin 5-O-β-D-glucopyranoside ( 1 ), (2S,3S)-4′-O-methylcatechin 5-O-β-D-glucopyranoside ( 2 ), (2S,3R)-4′-O-methylcatechin 5-O-β-D-glucopyranoside ( 3 ), one new triterpene glycoside 3-O-β-D-xylopyranosyl-(1→6)-[β-D-xylopyranosyl-(1→2)]-β-D-glucopyranosyl-oleanolic acid 28-O-β-D-glucopyranoside ( 4 ), together with nine known compounds ( 5 - 13 ). Their chemical structures were elucidated based on HR-ESI-MS, NMR spectroscopic data. The absolute configurations of compounds 1 – 3 were established by electronic circular dichroism (ECD) spectra. At concentration of 20 μM, compounds 1 – 13 showed the percentages of dead cell in the range of 2.14 % to 33.61 % against KB, HepG2, HL60, P388, HT29, and MCF7 cancerous cell lines by SRB assay.     

Preparation of Optically Active 2-Aminobutanoic Acid via Optical Resolution by Replacing Crystallization

An attempt was made to use a simple procedure to obtain (R)- and (S)-2-aminobutanoic acids [(R)- and (S)-1] which are non-proteinogenic α-amino acids and are useful as chiral reagents in asymmetric syntheses. Compound (RS)-1 p-toluenesulfonate [(RS)-2], which is known to exist as a conglomerate, was optically resolved by replacing crystallization with (R)- and (S)-methionine p-toluenesulfonate [(R)- and (S)-3] as optically active co-solutes. When (S)-3 was employed as the co-solute, (R)-2 was preferentially crystallized from a supersaturated solution of (RS)-2 in 1-propanol, as was (S)-2 in the presence of (R)-3. (R)- and (S)-2 recrystallized from 1-propanol were treated with triethylamine in methanol to give (R)- and (S)-1 in optically pure forms.     

Configuration of polyisoprenoids affects the permeability and thermotropic properties of phospholipid/polyisoprenoid model membranes

The influence of α-cis- and α-trans-polyprenols on the structure and properties of model membranes was analyzed. The interaction of Ficaprenol-12 (α-cis-Prenol-12, α-Z-Prenol-12) and Alloprenol-12 (α-trans-Prenol-12, α-E-Prenol-12) with model membranes was compared using high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. l-α-Phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the fractions of polyprenol as non-incorporated (Prenol_NonInc) and incorporated (Prenol_Inc) into liposomes. Consequently, distribution coefficients, P′, describing the equilibrium of prenol content between phospholipid (EYPC) membrane and the aqueous phase gave different log P′ for α-cis- and α-trans-Prenol-12, indicating that the configuration of the α-terminal residue significantly alters the hydrophobicity of the polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluorescence experiments α-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose while the effect of α-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior of DPPC membranes in the presence of both prenol isomers were observed. α-trans-Pren-12 completely abolished the pretransition while in the case of α-cis-Pren-12 it was noticeably reduced. Furthermore, for both prenol isomers, the temperature of the main phase transition (T_m) was shifted by about 1 °C to lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed a new peak at 38.7 °C, which may suggest the concomitant presence of more that one phase within the membrane.Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant tissues suggest that cis/trans isomerization of the α-residue of polyisoprenoid molecule might comprise a putative mechanism responsible for modulation of the permeability of cellular membranes.     

Occurrence of Taurine-Pyruvate Aminotransferase in Bacterial Extract

Natural ( + )-(1R,2S,3S)-methyl cucurbate (1b) and the ( – )-δ-lactone of 3-epi-cucurbic acid (16) were synthesized from (+)-(1R,6S,7R)-bicyclo [4.3.0] non-3-en-7-ol (5). Asymmetric hydrolysis of the acetate (8) of ( ± )-5 with pancreatin gave optically pure the ( + )-(7R)-alcohol (5) and (–)-(7S)-acetate (8). An ozonolysis product of ( + )-5 was transformed to ( – )-16 and ( + )-(3S)-1b with inversion of the (7R)-hydroxyl group. Similarly, unnatural (–)-1b and (+)-16 were prepared from optically pure ( — )-5. The growth inhibitory activities of these synthesized chiral compounds toward lettuce seedlings were examined.     

Synthesis and Distribution of (─)-Mintsulfide,a Novel Sulfur-containing Sesquiterpene

(─)-Mintsulfide [(─)-1], a new sulfur-containing sesquiterpene found in peppermint oil, was synthesized from (─)-germacrene-d[(─)-3] by a photochemical reaction and was confirmed to be (1R)-cis-2,6-epithio-cis-8-isopropyl-l-methyl-5-methylene-cis-bicyclo-[5,3,0]decane. The existence of 1 was confirmed in 13 of 74 essential oils analyzed.     

Design and in Vitro Characterization of Tricyclic Benzodiazepine Derivatives as Potent and Selective Antileukemic Agents

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient, and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell lines when compared to our previously reported compounds, with IC₅₀ values in the range of 2.9–5.6 μM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC₅₀=6.1 μM) and MV-4-11 (IC₅₀=11.0 μM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC₅₀=31.8–55.0 μM) and very weak cytotoxic effect toward the Balb/3T3 reference cell lines. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also established the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4],12(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had K_i values of 10–19 μM. The obtained results showed that the tested compounds exhibited a selective antileukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.     

Lipase-Catalyzed Kinetic Resolution of (±)-cis-Flavan-4-ol and Its Acetate: Synthesis of Chiral 3-Hydroxyflavanones

Lipase-catalyzed kinetic resolution of (±)-cis-flavan-4-ol and its acetate led to enantiomerically enriched flavan-4-ol and its acetate. These chiral compounds were converted to (2R, 3R)- and (2S, 3S)-3-hydroxyflavanones.     

Preparation of Aroma Compounds by Microbial Transformation of Isophorone with Aspergillus niger

( ± )-cis-γ-Irone (1a), ( ± )-cis-dihydro-γ-irone (2a) and their trans- isomers (1b, 2b) were synthesized via 3,3-(Claisen) or 2,3-sigmatropic rearrangement of 1-hydroxymethyl-3,3,4- trimethyl-1-cyclo he xene (8) derivatives as each key step.