A Highly Selective and Sensitive Chiral Derivatization Method for High- Performance Liquid Chromatographic Determination of the Stereoisomer Composition of Natural Products With Chiral Branched Alkyl Chains.

In the study of chiral biologically active compounds such as pheromones, the analysis of the stereoisomer composition is essential to gain more insight into their stereochemical diversity, which affects the pheromone communication channels and therefore the diversification of species. This mini-review summarizes the development of fluorescence derivatization reagents for high-performance liquid chromatographic (HPLC) determination of the absolute configuration and stereoisomer composition of natural products with a chiral branched alkyl chain. The diastereomeric separation of anteiso fatty acids bearing a branched methyl group up to the C-26 position was achieved by reversed-phase HPLC under very low column temperature conditions using (1S,2S)-2-(2,3-anthracenedicarboximido)cyclohexanol as a derivatization reagent, enabling fluorescent detection of these compounds at femtomole levels. This method was also applicable to chiral alcohols and amines with chiral branched methyl groups using similar reagents containing a carboxyl group. These reagents were successfully applied to determine the absolute configurations and stereoisomer composition of the chiral alkyl chain of natural compounds including some insect pheromones, miyakosyne A, and plakoside A. The combination of these reagents and two-dimensional HPLC constitutes a very powerful tool for the analysis of the stereoisomers of natural crude samples. Furthermore, the analysis of some natural bioactive substances using this method demonstrated that natural substances are not always optically pure, consisting instead of stereoisomer mixtures exhibiting stronger activity than optically pure enantiomers. These results cast doubts on the concept of biological homochirality and demonstrate that natural pheromones do not always show the highest activity among all stereoisomers.

Bulbimidazoles A-C, Antimicrobial and Cytotoxic Alkanoyl Imidazoles from a Marine Gammaproteobacterium Microbulbifer Species.

Three new alkanoyl imidazoles, designated bulbimidazoles A-C (1-3), were found from the culture extract of the gammaproteobacterium Microbulbifer sp. DC3-6 isolated from a stony coral of the genus Tubastraea. The absolute configuration of the anteiso-methyl substitution in 1 was established to be a mixture of (R)- and (S)-configurations in a ratio of 9:91 by applying the Ohrui-Akasaka method. Compounds 1-3 displayed unique broad-spectrum antimicrobial activity against Gram-positive and -negative bacteria and fungi with MICs ranging from 0.78 to 12.5 µg/mL. They also exhibited cytotoxicity toward P388 murine leukemia cells with IC50 in the micromolar range.

Nocarimidazoles C and D, antimicrobial alkanoylimidazoles from a coral-derived actinomycete Kocuria sp.: application of 1 J C,H coupling constants for the unequivocal determination of substituted imidazoles and stereochemical diversity of anteisoalkyl chains in microbial metabolites.

Chemical investigation of secondary metabolites from a marine-derived actinomycete strain of the genus Kocuria, isolated from a stony coral Mycedium sp., led to the identification of two new alkanoylimidazoles, nocarimidazoles C (1) and D (2) as well as three known congeners, nocarimidazoles A (3) and B (4) and bulbimidazole A (5). Structure analysis of 1 and 2 by NMR and MS revealed that both are 4-alkanoyl-5-aminoimidazoles with a 6-methyloctanoyl or decanoyl chain, respectively. Two possible positions of the amino group on the imidazole rings (C-2 and C-5) posed a challenge in the structure study, which was settled by the measurement of 1 J C,H coupling constants for comparison with those of synthetically prepared model imidazoles. The absolute configurations of the anteisoalkanoyl group present in 1, 4, and 5 were determined by low-temperature HPLC analysis of the degradation products labeled with a chiral anthracene reagent, which revealed that 1 is a mixture of the R- and S-enantiomers with a ratio of 73:27, 4 is the pure (S)-enantiomer, and 5 is the (S)-enantiomer with 98% ee. The present study illustrates the diversity in the stereochemistry of anteiso branching in bacterial metabolites. Compounds 1-4 were moderately antimicrobial against Gram-positive bacteria and fungi, with MIC ranges of 6.25-25 µg/mL.

Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males.

Animals exhibit a spectacular array of traits to attract mates. Understanding the evolutionary origins of sexual features and preferences is a fundamental problem in evolutionary biology, and the mechanisms remain highly controversial. In some species, females choose mates based on direct benefits conferred by the male to the female and her offspring. Thus, female preferences are thought to originate and coevolve with male traits. In contrast, sensory exploitation occurs when expression of a male trait takes advantage of preexisting sensory biases in females. Here, we document in Drosophila a previously unidentified example of sensory exploitation of males by other males through the use of the sex pheromone CH503. We use mass spectrometry, high-performance liquid chromatography, and behavioral analysis to demonstrate that an antiaphrodisiac produced by males of the melanogaster subgroup also is effective in distant Drosophila relatives that do not express the pheromone. We further show that species that produce the pheromone have become less sensitive to the compound, illustrating that sensory adaptation occurs after sensory exploitation. Our findings provide a mechanism for the origin of a sex pheromone and show that sensory exploitation changes male sexual behavior over evolutionary time.

Nocapyrones: α- and γ-pyrones from a marine-derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2-4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2-4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.

Sporangimicins A-D, acylated maltose derivatives from a rare actinomycete of the genus Pseudosporangium.

Sporangimicins A-D (1-4), four anomeric pairs of diacyl disaccharides that represent a new metabolite class, were discovered from the culture extract of an actinomycete Pseudosporangium sp. RD061809. Compounds 1-4 caused peak separation in the HPLC chromatogram and partial duplication of the NMR resonances by anomeric interconversion of a maltose core modified at the two sugar 6-positions with an isobutanoyl and a methyl-branched long-chain dienoyl groups. A highlight of the structure elucidation was application of Ohrui-Akasaka's method to a chromatographically inseparable mixture of 3 and 4, which proved the composition ratio of 3 and 4 to be 82:18 and the R/S ratio at the anteiso-methyl bearing chiral center in 3 to be 66:34. Compounds 1-4 showed antimicrobial activity against Gram-positive bacteria and modest cytotoxicity toward P388 murine leukemia cells.

Anatomical localization and stereoisomeric composition of Tribolium castaneum aggregation pheromones.

We report that the abdominal epidermis and associated tissues are the predominant sources of male-produced pheromones in the red flour beetle, Tribolium castaneum and, for the first time, describe the stereoisomeric composition of the natural blend of isomers of the aggregation pheromone 4,8-dimethyldecanal (DMD) in this important pest species. Quantitative analyses via gas chromatography-mass spectrometry showed that the average amount of DMD released daily by single feeding males of T. castaneum was 878 ± 72 ng (SE). Analysis of different body parts identified the abdominal epidermis as the major source of aggregation pheromone; the thorax was a minor source, while no DMD was detectable in the head. No internal organs or obvious male-specific glands were associated with pheromone deposition. Complete separation of all four stereoisomers of DMD was achieved following oxidation to the corresponding acid, derivatization with (1R, 2R)- and (1S, 2S)-2-(anthracene-2,3-dicarboximido)cyclohexanol to diastereomeric esters, and their separation on reversed-phase high-performance liquid chromatography at -54°C. Analysis of the hexane eluate from Porapak-Q-collected volatiles from feeding males revealed the presence of all four isomers (4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) at a ratio of approximately 4:4:1:1. A walking orientation bioassay in a wind tunnel with various blends of the four synthetic isomers further indicated that the attractive potency of the reconstituted natural blend of 4:4:1:1 was equivalent to that of the natural pheromone and greater than that of the 1:1 blend of (4R,8R)/(4R,8S) used in commercial lures.

Contact sex pheromone components of the seed beetle, Callosobruchus analis (F.).

Callosobruchus analis (Coleoptera: Chrysomelidae: Bruchinae), found throughout tropical Asia and Africa, is a pest of stored legumes. Previous work has shown that females of this species produce a contact sex pheromone that elicits copulatory behavior in males. Comparisons of copulatory activity between any two of four congeneric species suggest that the contact sex pheromones are species specific. In laboratory bioassays, male C. analis exhibited copulatory behavior to a female dummy to which a crude extract of virgin females was applied. The extract had been collected by a filter paper method and was purified by acid-base partition and chromatographic techniques. Gas chromatography-mass spectrometry (GC-MS) analyses of active fractions revealed that the active compounds were 2,6-dimethyloctane-1,8-dioic acid (1) and callosobruchusic acid, (E)-3,7-dimethyl-2-octene-1,8-dioic acid (2), previously identified as contact sex pheromones of Callosobruchus maculatus (F.) and C. chinensis (L.), respectively. The stereoisomeric and chemical compositions were determined by the 2D-HPLC-Ohrui-Akasaka method as (2S,6R)-1:(S)-2=1.8:1, which meant that both compounds in C. analis were stereochemically pure, unlike the case of C. maculatus and C. chinensis. An examination of the influence of synthetic pheromone compounds on male copulatory activity revealed that (2S,6R)-1 is the main component, and that (S)-2 has an additive effect. In the examination of the stereochemistry-activity relationship, no copulatory behavior was elicited by (2R,6S)-1, and, furthermore, the enantiomer significantly masked the pheromonal activity of (2S,6R)-1. Glass rod dummy assays also suggested the presence of synergists. These results could elucidate the specificity of mate recognition in C. analis.

Ansaetherone, a new radical scavenger from Streptomyces sp.

We identified a new radical scavenger, ansaetherone (C26 H33 NO7), from a culture of the Streptomyces sp. USF-4727 strain. In our previous study, it was shown that this strain produced four lipoxygenase inhibitors, tetrapetalones A, B, C and D. The chemical structure of ansaetherone was elucidated by the spectroscopic method, indicating that this compound was constructed with an aglycon and a sugar moiety. This chemical structure suggested that ansaetherone was related to the tetrapetalones. This finding provided information regarding tetrapetalone biosynthesis. Ansaetherone showed radical scavenging activity with an ED50 value of 300 microM in our assay.

Selectivity enhancement for trans-2-(2,3-anthracenedicarboximido)-cyclohexane-derived diastereomers in HPLC by using an ordered organic stationary phase.

2-(2,3-anthracenedicarboximido)cyclohexane derivatives (AC) have been known as the evolutionary diastereomerizing reagents for enantiomer discrimination in HPLC with ODS. However, a substantial separation of diastereomers can be observed only at lower temperatures, such as -40 degrees C. Therefore, in this work, poly(octadecyl acrylate)-grafted silica, ODAn was applied as an alternative stationary phase to ODS for the separation of AC-derived diastereomers. As a result, complete separation was achieved even under the conventional condition: for example, methanol as the mobile phase and 0 degrees C as the column temperature. An investigation on the temperature dependency of the selectivity demonstrated that ODAn shows a remarkable increase in selectivity at temperatures below 30 degrees C, which almost agreed with the peak-top temperature of the endothermic peak in a DSC thermogram for ODA35 immersed in a mobile phase. The better separation would be derived from a highly ordered structure of ODAn and a carbonyl-pi interaction with AC-derived diastereomers.

Comparative study on the use of ortho-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde and anthracene-2,3-dicarboxaldehyde reagents for alpha-amino acids followed by the enantiomer separation of the formed isoindolin-1-one derivatives using quinine-type chiral stationary phases.

Cinchona alkaloid based chiral stationary phases (CSPs) were evaluated and compared for the enantiomer separation of a set of alpha-amino acid derivatives as selectands (SA), using ortho-phthalaldehyde (OPA), naphthalene-2,3-dicarboxaldehyde (NDA) and anthracene-2,3-dicarboxaldehyde (ADA) as reagents in the presence of acetonitrile. Protocols have been developed for the derivatization of most common amino acids in the absence of the usual thiol components (2-mercaptoethanol, mercaptosulphonic acid, sodium sulfite) under acidic and neutral conditions providing the corresponding isoindolin-1-one (phthalimidine) derivatives. They are stable for hours at various reaction conditions compared to thiol or sulfide modified isoindoles resulted by the OPA-thiol reaction type. Among the derivatizing agents, ADA afforded the highest retention factors (k) and for the majority of the analytes also resolution (Rs) and enantioselectivity (alpha) values (i.e. for tryptophan k1 = 23, Rs = 4.93 and alpha = 1.43). Structure variation of the CSPs and selector (SO), respectively indicates that steric arrangement around the binding cleft plays a major role in the enantiodiscriminating events. To provide more detailed information about the derivatization reaction itself, the proposed mechanism for the formation of the OPA derivative (isoindolin-l-one) was further evaluated by deuterium labeling and LC-MS analysis.

Highly potent chiral labeling reagents for the discrimination of chiral alcohols.

Highly sensitive chiral labeling reagents, (1R,2R)- and (1S,2S)-2-(2,3-anthracenedicarboximido)cyclohexanecarboxylic acids [(1R,2R)- and (1S,2S)-A] and (1R,2R)- and (1S,2S)-2-(2,3-naphthalenedicarboximido)cyclohexanecarboxylic acids [(1R,2R)- and (1S,2S)-A'], were prepared. Reagent A has enabled us to discriminate the enantiomers of anteiso fatty alcohols up to C9 methyl branching by 1H NMR, and both reagents A and A' have allowed up to C16 methyl branching at the 10(-15) molar level by fluorescence detected reversed-phase HPLC.

Simple determination of L-ascorbic acid on TLC by visual detection using autocatalytic reaction.

The L-ascorbic acid concentration in beverages was measured after separation by silica gel thin layer chromatography (TLC) by visually determining the time in autocatalytic reaction for the L-ascorbic acid spot to turn the same yellow color of the background and disappear (the end time of the induction period) after spraying the slide with a 3,6-dihydroxyxanthane solution. There was a good linear relationship between the end time of the induction period and the concentration of L-ascorbic acid for concentrations in the range of 5.0 - 20 mM (r(2) = 0.9944). In addition, there was a good relationship expressed by a quadratic equation in the concentration range of 0.1 - 5.0 mM (r(2) = 0.9975). The relative standard deviations of the L-ascorbic acid values for 3 beverages (2.2 - 8.6 mM) were less than 5% (n = 5), and the recovery of 5.0 mM L-ascorbic acid from 4 beverages (0.7 - 7 mM) was 97 - 110%. A good correlation was also observed between the L-ascorbic acid values of 23 beverages (0 - 86 mM) determined by the proposed TLC method and the colorimetric method contained in a commercially available kit for L-ascorbic acid (r(2) = 0.9945).

Structure of an unsaturated fatty acid with unique vicinal dimethyl branches isolated from the Okinawan soft coral of the genus Sinularia.

A new unsaturated fatty acid with unique vicinal dimethyl branches was isolated from the Okinawan soft coral of the genus Sinularia. The structure of the compound was determined based on the results of spectroscopic analysis and chemical conversion. The absolute configuration was deduced by applying the Ohrui-Akasaka method.

Direct determination of the stereoisomeric composition of callosobruchusic acid, the copulation release pheromone of the azuki bean weevil, Callosobruchus chinensis L., by the 2D-Ohrui-Akasaka method.

The stereoisomeric composition of the copulation release pheromone of the azuki bean weevil, Callosobruchus chinensis L., was determined to be R:S=3.3-3.4:1 by the 2D-Ohrui-Akasaka method.

Chiral discrimination of primary amines by HPLC after labeling with a chiral derivatization reagent, trans-2-(2,3-anthracenedicarboximido)-cyclohexanecarbonyl chloride.

Enantiomeric discrimination of chiral primary amines was performed by both reversed-phase HPLC and normal-phase HPLC after labeling with a chiral fluorescent derivatization reagent, (1R,2R)- and (1S,2S)-trans-2-(2,3-anthracenedicarboximido)cyclohexanecarbonyl chloride. Use of HPLC permits separation of diastereomeric derivatives of amines up to C30 which have a primary amino group at the middle of the alkyl chain. The derivatives of primary amines having an anteiso alkyl chain, which has a chiral branched-methyl at the n-3 position of the alkyl chain, were also separated by HPLC, and it was also possible to separate niphatesine D by reversed-phase HPLC after derivatization.

Effect of ascorbic acid on the chemiluminescence of polyphenols.

The chemiluminescence of gallic acid by hydrogen peroxide had completely inhibited by the presence of ascorbate. After ascorbate had disappeared by oxidation, chemiluminescence returned. The concentration of gallic acid was virtually unchanged by presence of ascorbate, but started to decrease after the disappearance of ascorbate. This might be attributable to the rapid reduction of quinone, which was the first product of the chemiluminescence reactions, to gallic acid by ascorbate or the donation of proton to the phenoxy radical from ascorbate to stop the chemiluminescence reaction at the first stage. The effects of ascorbate on the chemiluminescence of other polyphenols depended on their oxidation rate.

Absolute stereochemistry of fungal beauveriolide III and ACAT inhibitory activity of four stereoisomers.

Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII. Furthermore, the four beauveriolide III isomers ((3S,4S)BeauIII (23a), (3R,4R)BeauIII (23b), (3R,4S)BeauIII (23c), and (3S,4R)BeauIII (23d)) were synthesized, and it was shown that all the spectral data for 23a were identical with those for natural 1b. Isomers 23a and 23d showed potent inhibitory activity of lipid droplet accumulation in macrophages, while the other two isomers caused weak inhibition. Thus, the 3S configuration of BeauIII is important for this activity. Furthermore, 23a and 23d showed rather specific inhibition against the ACAT1 isozyme.

Chiral discrimination of secondary alcohols by both 1H-NMR and HPLC after labeling with a chiral derivatization reagent, 2-(2,3-anthracenedicarboximide)cyclohexane carboxylic acid.

Enantiomeric discrimination of chiral secondary alcohols was performed by both reversed-phase HPLC and 1H-NMR after labeling with a chiral fluorescent derivatization reagent, 2-(2,3-anthracenedicarboximide)cyclohexanecarboxylic acid. It was possible to discriminate by HPLC the chirality of alcohols up to C30 having a chiral hydroxyl group at the middle of the straight alkyl chain, and, by 1H-NMR, alcohols up to C16. For alcohols having one straight alkyl and one isoalkyl group with the same carbon numbers at both sides of a hydroxyl group, it was possible to discriminate the chiralities of alcohols up to C19 by both 1H-NMR and HPLC. The 1H-NMR methods also made it possible to determine absolute configurations empirically.

Development of highly potent D-glucosamine-based chiral fluorescent labeling reagents and a microwave-assisted beta-selective glycosidation of a methyl glycoside reagent.

We synthesized new chiral fluorescence labeling reagents having a 2,3-anthracenedicarboximide group from D-glucosamine, and it was possible to introduce target alcohols at the anomeric positions of the reagents with beta-selectivity by glycosidations. Especially, it was possible to use methyl glycoside reagent as a glycosyl donor with a Lewis acid and microwave irradiation, and it gave selectively beta-glycoside while the reaction without microwave irradiation gave alpha- and beta-mixed glycosides. Those reagents showed very high chiral discrimination ability, and they made it possible to separate the eight stereoisomers of 4,8,12,16-tetramethylheptadecanol by HPLC after derivatizations.