Biosynthesis of marine natural products: isolation and characterization of a myrcene synthase from cultured tissues of the marine red alga Ochtodes secundiramea

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg(+2) as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (+/-)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.     

1-N-methyl-(6E)-(2-methylpropylidene)-(3Z)-3-(phenylmethylene)-2,5-piperazinedione,a metabolite from Streptomyces albus

The structure and stereochemistry of a new piperazinedione, isolated from the cells of Streptomyces albus, are assigned on the basis of spectroscopic data, including comparison with related 2,5-piperazinediones.     

Synthesis of (S)-13-Hydroxy (2E,4E,8E)- and (2E,4E,8Z)-Tetradecatrienoic Acids

The syntheses of (S)-13-hydroxy-(2E,4E,8E)-tetradecatrienoic acid (1) and (2E,4E,8Z)-tetradecatrienoic acid (2) were carried out by using the Wittig reaction as the key step. The asymmetric center at C-13 and the double bond between C-8 and C-9 for natural compound 1 were reconfirmed as being of (S) configuration and E, respectively.

The relationship between the structure of the unsaturated hydroxy fatty acids and their inhibitory effect on the growth of lettuce was investigated.     

Discovery of 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid [12-(S)-HETE] in the tropical red alga Platysiphonia miniata

The potent mammalian immunohormone, 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid (12-(S)-HETE), is a 12-lipoxygenase metabolite of arachidonic acid that is widely distributed in animal tissues. In humans, it is produced and secreted by platelet cells and elicits both chemotactic and degranulatory responses in target neutrophils. As widely as 12-lipoxygenase activity and one of its major products, 12-(S)-HETE, have been found in animal tissues, it has never been found in plants. Herein, we report the first isolation of the 12-lipoxygenase product, 12-(S)-HETE, from a plant, the tropical marine alga Platysiphonia miniata (C. Agardh) B?rgesen.     

Cytotoxicities, cell cycle and caspase evaluations of 1,6-diaryl-3(Z)-hexen-1,5-diynes, 2-(6-aryl-3(Z)-hexen-1,5-diynyl)anilines and their derivatives

Compounds 3, 6-7, 9-10, 15-17, and 20-21 showed growth inhibition effects on a full panel of 60 human cancer cell lines, and most of the average IC50 values of the indicated analogues were from < 0.01 to 96.6 microM, in which analogues 16 and 17 revealed the highest cytotoxic activity with the cancer cell lines at 10(-7) M concentration range. During the cell cycle analysis, a moderate to high apoptotic progress induction was shown by 3, 9, 16-17, and 20 compared with the control, which 2-(6-(2-thienyl)-3(Z)-hexen-1,5-diynyl)aniline 16 showed the highest apoptotic effect. Structures 16-17 displayed a significant G2/M phase arrest in the cell growth cycle compared with other derivatives, which the proportions of the G2/M phase cells were accumulated to 71.5% and 82.6%, respectively. Moreover, the colorimetric assay of 16-17 also provided advanced evidence to the relationship between the compounds and the caspase-3 enzyme, which was one of the major promoters of apoptotic effect.     

Production of highly optically active (R)-3-chloro-1,2-propanediol using a bacterium assimilating the (S)-isomer

A bacterium that assimilates (S)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was isolated from soil by enrichment culture. The bacterium was identified as Pseudomonas sp. by taxonomic studies. The strain grew in a medium containing racemic MCH as a source of carbon and degraded (S)-MCH stereoselectively, liberating chloride ions. The residual isomer was the (R)-form [99.5% enantiomeric excess (ee)], which was obtained from the racemate in a final yield of 36% by using this strain. Subsequently, highly optically active (R)-glycidol (GLD) (99.3% ee) was prepared from the (R)-MCH obtained by reaction in alkaline solution. The cell-free extracts of the cells had both dehalogenating and epoxide-opening activities, which converted various halohydrins to the corresponding epoxides and epoxides to the corresponding diols, respectively.Correspondence to: T. Suzuki     

Construction of a two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate ethyl ester

Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP⁺. An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.     

The effect of a liquid seaweed extract from Ascophyllum nodosum (Fucales,Phaeophyta) on the two-spotted red spider mite Tetranychus urticae

This paper presents evidence from an initial trial in favor of claims that the liquid seaweed extract of Ascophyllum nodosum, marketed in the UK as Maxicrop, can reduce numbers of the two-spotted red spider mite Tetranychus urticae on crop plants. Seven applications of Maxicrop Triple over 20 days significantly suppressed the population build-up of two-spotted red spider mites on treated strawberry plants (Fragaria sp. var. Redgauntlet) raised under high polythene tunnels as compared to untreated controls.     

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discovery of (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796): a gamma-secretase inhibitor with Abeta lowering activity in a transgenic mouse model of Alzheimer's disease

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.     

Formation of 3-(2'-deoxyribofuranosyl) and 9-(2'-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.     

Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol

(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 μmol/min/mg, ten times of that of the wild-type. Its k _cat/K _m increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.

     

Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.     

Biosynthetic Origin of [R-(Z)]-4-Amino-3-chloro-2-pentenedioic Acid in Streptomyces viridogenes

The biosynthesis of the chlorinated amino acid [R-(Z)]-4-amino-3-chloro-2-pentenedioic acid (ACPA) was investigated. Feeding studies with Streptomyces viridogenes were conducted in resting cells. Substantial incorporation from [¹⁵N]- and [¹³C]-enriched glutamate and proline indicated that the biosynthetic origin of ACPA is one of these amino acids. Experiments with deuterated glutamate and proline imply that chlorination does not occur via a radical mechanism, but rather suggest that a FADH₂-dependent halogenase is involved.     

A rearranged chamigrene derivative and its potential biogenetic precursor from a new species of the marine red algal genus Laurencia (rhodomelaceae)

The structures of a novel rearranged sesquiterpenoid and a biogenetically-related chamigrene derivative have been determined by combined spectral and chemical methods. These sesquiterpenoids were components of an undescribed Laurencia species, and each was toxic toward the damselfish Pomacentrus coeruleus.     

Transformation of 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one in a polar metabolite in HepG2 hepatoma cells

Incubation of 3 beta-(2-hydroxy-2[3H]-ethoxy)-5 alpha-cholest-8(14)-en-15-one with Hep G2 cells led to the accumulation of a radioactive polar product in the culture medium, which was identified as 3 beta-(2-hydroxyethoxy)-15-keto-5 alpha-cholest-8(14)-ene-24-oic acid. Its structure was confirmed by a chemical counter synthesis. The labeled ketosterol was rapidly (tau 1/2 = 6 min) and reversibly bound by Hep G2 cells. The intracellular concentration of 15-ketosterol decreased during incubation mainly due to the formation of a polar metabolite, secreted to the medium. The level of cholesterol biosynthesis was 22 +/- 5% of the control value in Hep G2 cells at a 15-ketocholesterol concentration in the medium of 30 microM. However, further incubation for 3 h in the medium without the ketosterol led to restoration of the level of biosynthesis to 85 +/- 11% of the control value. These results suggest that inhibition of the cholesterol biosynthesis by 15-ketocholesterol in Hep G2 cells depends on the intracellular concentration of the inhibitor, which, in turn, is determined by the rate of its conversion into the polar metabolite. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-analogues through a common synthetic route

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-³H₈]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.     

Discovery of ((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone, PF-4254196, a CCR2 antagonist with an improved cardiovascular profile

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45 mg BID and a CV-TI = 3800-fold.     

Asymmetric synthesis and teratogenic activity of (R)- and (S)-2-ethylhexanoic acid, a metabolite of the plasticizer di-(2-ethylhexyl)phthalate

The stereoselectivity of the teratogenic activity of 2-ethylhexanoic acid (EHXA), a metabolite of the widely-used plasticizer di-(2-ethylhexyl)phthalate, was investigated. The enantiomers of EHXA were prepared via asymmetric synthesis with the aid of the chiral auxiliaries (R)- and (S)-1-amino-2-(methoxymethyl)pyrrolidine (RAMP, SAMP). The aqueous solutions of the sodium salts of (R)- and (S)-EHXA and the racemic EHXA [+/- )-EHXA) were injected each morning and evening of day 7 and 8 of gestation in the NMRI mouse (500 mg/kg, i.p.), a period highly sensitive in regard to the production of neural tube defects (exencephaly) by branched-chain carboxylic acids. (S)-EHXA did not yield any teratogenic or embryotoxic response in this model, while (R)-EHXA was highly teratogenic (59% of living fetuses exhibited exencephaly) and embryotoxic (as indicated by embryolethality and fetal weight retardation); the exencephaly rate induced by (+/- )-EHXA was between those of the two enantiomeres (32%). It is therefore likely that stereoselective interactions of the enantiomers of EHXA with chiral molecules in the embryo are decisive in regard to the teratogenic response. This first example of the stereoselectivity of the teratological activity of an environmental pollutant suggests that the safety of man-made chemicals can be improved by the use of pure enantiomers instead of racemates.