Sesquiterpene lactones and diterpene carboxylic acids in Helianthus niveus subspecies Canescens

Three new heliangolides (niveusin-A, -B and -C) and three known diterpene carboxylic acids were isolated and characterized from a CHCl₃, extract of Helianthus niveus. The structures of the three new lactones were deduced by ¹H and ¹³C NMR as well as extensive decoupling experiments and derivatizations.     

New natural diterpene acids from Juniperus communis

Three new diterpene acids have been isolated from the leaves of Juniperus communis and their structures, elucidated by spectroscopic methods, were identified as 7-oxo-13-epi- pimara-8,15-dien-18-oic acid, 7α-hydroxysandaracopimaric acid and (14 S)-14,15-dihydroxylabda- 8(17),13(16)-dien-19-oic acid. Biflavonyls, fatty acids and diterpenoids with known structures were also isolated.     

Conjugated fatty acids from latex of Euphorbia lathyris

The fatty acids esterified with triterpene and diterpene alcohols from the latex of Euphorbia lathyris were analyzed. 77% of the fatty acids esterified with triterpenes were conjugated, the main components of which were decadienoic and decatrienoic acids. Of the acids esterified with diterpenes, 81% were decatrienoic and dodecatrienoic acids.     

Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC₅₀ 6.5 μM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC₅₀ 55, 60, and 50 μM, respectively). The acylated amino acids themselves, except for AA-Glu, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC₅₀ 20 and 27 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes.     

Diterpene carboxylic acids and a heliangolide from Helianthus angustifolius

Two diterpene carboxylic acids, one a new kaurenoid derivative and one the previously characterized labdane, ()-cis-ozic acid, as well as a     

Cyp3a11 is not essential for the formation of murine bile acids

Humans and mice differ substantially in their bile acid profiles as mice in addition to cholic acid (CA) predominantly synthesize 6β-hydroxylated muricholic acids (MCAs) whereas humans produces chenodeoxycholic acid (CDCA) and CA as primary bile acids. Identifying the gene performing 6β-hydroxylation would be useful for ‘humanizing’ the bile acid profile in mice for studies of the interaction between bile acids, gut microbiota, and host metabolism. We investigated the formation of MCAs in primary murine hepatocytes and found that αMCA is synthesized from CDCA and βMCA from UDCA. It is commonly assumed that the P450-enzyme CYP3A11 catalyzes 6β-hydroxylation of bile acids, thus we hypothesized that mice without the Cyp3a11 gene would lack MCAs. To test this hypothesis, we analyzed bile acid profiles in Cyp3a deficient mice, which lack 7 genes in the Cyp3a gene cluster including Cyp3a11, and compared them with wild-type littermate controls. Bile acid composition in liver, gallbladder, caecum and serum from Cyp3a knock out mice and wild-type littermate controls was analyzed with UPLC-MS/MS and revealed no major differences in bile acid composition. We conclude that Cyp3a11 is not necessary for 6β-hydroxylation and the formation of MCAs.     

Grindelane diterpenoids from Chrysothamnus nauseosus

Two new grindelic acid diterpenoids, 18-hydroxygrindelic acid and 18-succinyloxygrindelic acid, were isolated from Chrysothamnus nauseosus. The assigned structures rest on their spectroscopic properties as well as chemical interconversions.     

New fatty acids from Colombian Caribbean Sea sponges

The fatty acid composition of whole phospholipids from marine sponges collected from the Colombian Caribbean Sea was determined as part of our studies on the order Halichondrida: Halichondria magniconulosa, Halichondria lutea, Petromica ciocalyptoides, Axinyssa ambrosia, Didiscus oxeata and Dragmaxia undata. Structure elucidation was accomplished by means of gas chromatography retention parameters and GCMS. Eight new fatty acids were identified by their methyl esters and N-acylpyrrolidide derivatives (i.e. 5-methyl-6-octadecenoic, 16-methyl-11-heptadecenoic, 8-methyl-4-tetracosenoic, 8,17-dimethyl-5-octadecenoic, 23-methyl-8-tetracosenoic, 20-methyl-18-tetracosenoic, 4,17-tetracosadienoic and 22,27-dimethyl-5,9-octacosadienoic acids). These findings establish alternative fatty acid biosynthetic possibilities for these organisms.     

Bioconversion of 2-amino acids to 2-hydroxy acids by Clostridium butyricum

Analysis by gas chromatography-mass spectrometry (GC-MS) of 24-h cultures of Clostridium butyricum type strain in synthetic BMG medium supplemented with various 2-amino acids (10 mM) revealed the presence of the corresponding 2-hydroxy acids. C. butyricum was able to bioconvert l-valine, dl-norvaline, l-leucine, dl-norleucine, l-methionine and l-phenylalanine as well as unusual 2-amino acids, i.e., l-2-aminobutyric acid, l-2-amino-4-pentenoic acid, dl-2-aminooctanoic acid, and dl-2-amino-4-phenylbutanoic acid. l-Isoleucine and cycloleucine were not converted into their corresponding 2-hydroxy acids. The bioconversion rate was maximal with dl-norvaline (6.2%). Chiral GC analysis demonstrated that only d-2-hydroxy-4-methylpentanoic acid is formed from l-leucine, indicating that the bioconversion is stereospecific, with inversion of configuration. d-Leucine and d-methionine were also converted to the corresponding 2-hydroxy acids. This observation opens new aspects in the study of C. butyricum and raises questions about the amino acid metabolism by this species.     

Major resin acids of Pinus nigra needles

Labdane diterpene acids were found to be the major resin acid components in Pinus nigra needles of various seed sources. The major constituents have been identified as 4-epiimbricataloic acid, manoyl oxide 19-oic acid, 4-epicommunic acid, and 15-monomethyl pinifolate. A GC method was developed to analytically differentiate pinifolic acid from its monomethyl ester in an admixture of both compounds. A minor resin acid was identified as 18-acetoxy-8(17)-labden-15-oic acid. 10-Nonacosanol and isoabienol were identified as major constituents of the needle and cortex extractives, respectively.     

Polyunsaturated fatty acids of mosquitos reared with single dietary polyunsaturates

Stock adults of Culex pipiens and tarsalis reared in crude media had a third of their phospholipid fatty acids as polyunsaturates, mainly 18C but including prominent proportions of arachidonic (20:4n6) and eicosapentaenoic (20:5n3) acids. Adults reared with synthetic media devoid of polyunsaturated fatty acids and therefore unable to fly at emergence contained no more than trace amounts of any polyunsaturate. With synthetic media containing single polyunsaturates the following findings emerged. Of four polyunsaturates known to be highly effective essential fatty acids individually 20:4n6 or 20:5n3 appeared unchanged in tissue phospholipids in proportions reflecting dietary concentrations; dietary 22:4n6 or 22:6n3 (docosahexaenoic acid) appeared also as 20:4n6 or 20:5n3, respectively, retroconverted from the administered dietary fatty acids, which were detected only in traces. Two moderately effective dietary fatty acids, 18:3n6 (γ-linolenic) and 20:3n6 (homo-γ-linolenic), which support weak flight at emergence, appeared in tissue phospholipids respectively as 18:3n6 only, or as similar proportions of 18:3n6 and 20:3n6, this latter indicating shortening to the 18C analogue as well as accumulation of the dietary 20C acid. Six other polyunsaturates [18:2n6 (linoleic), 18:3n3 (linolenic) and their 20C and 22C analogues], all considered slightly effective as essential fatty acids although unable to support proper flight, appeared in tissue phospholipid in dose-related proportions as the 18C basal n6 or n3 family analogues, with only traces of the higher analogues when these were the dietary fatty acids provided, indicating sequential chain shortening within each series, n6 or n3, no interconversion of n6 and n3 members (also shown by all other data), and efficient accumulation of the resultant 18C polyunsaturates. These findings show no capability for de novo synthesis of polyunsaturated fatty acids, afford an insight into the metabolic interrelations of diet-derived polyunsaturates and indicate a primary importance for endogenous arachidonic and eicosapentaenoic acids in mosquito essential fatty acid physiology.     

Generation of fad2 transgenic mice that produce omega-6 fatty acids

Fatty acid desaturase-2 (FAD2) introduces a double bond in position Δ12 in oleic acid (18︰1) to form linoleic acid (18︰2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20︰4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.     

Biosynthesis and biotechnological application of non-canonical amino acids: Complex and unclear

Compared with the better-studied canonical amino acids, the distribution, metabolism and functions of natural non-canonical amino acids remain relatively obscure. Natural non-canonical amino acids have been mainly discovered in plants as secondary metabolites that perform diversified physiological functions. Due to their specific characteristics, a broader range of natural and artificial non-canonical amino acids have recently been applied in the development of functional materials and pharmaceutical products. With the rapid development of advanced methods in biotechnology, non-canonical amino acids can be incorporated into peptides, proteins and enzymes to improve the function and performance relative to their natural counterparts. Therefore, biotechnological application of non-canonical amino acids in artificial bio-macromolecules follows the central goal of synthetic biology to: create novel life forms and functions. However, many of the non-canonical amino acids are synthesized via chemo- or semi-synthetic methods, and few non-canonical amino acids can be synthesized using natural in vivo pathways. Therefore, further research is needed to clarify the metabolic pathways and key enzymes of the non-canonical amino acids. This will lead to the discovery of more candidate non-canonical amino acids, especially for those that are derived from microorganisms and are naturally bio-compatible with chassis strains for in vivo biosynthesis. In this review, we summarize representative natural and artificial non-canonical amino acids, their known information regarding associated metabolic pathways, their characteristics and their practical applications. Moreover, this review summarizes current barriers in developing in vivo pathways for the synthesis of non-canonical amino acids, as well as other considerations, future trends and potential applications of non-canonical amino acids in advanced biotechnology.     

Resin acids from two amazonian species of Hymenaea

The major diterpene acid constituents of two Amazonian species of Hymenaea have been isolated and identified. A new resin acid, guamáic acid, is described.     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Nitro-fatty acids as activators of hSIRT6 deacetylase activity

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 μm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 μm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.     

A role for the human peroxisomal half-transporter ABCD3 in the oxidation of dicarboxylic acids

Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. Free fatty acids (FFAs) can enter peroxisomes through passive diffusion or by means of ATP binding cassette (ABC) transporters, including HsABCD1 (ALDP, adrenoleukodystrophy protein), HsABCD2 (ALDRP) and HsABCD3 (PMP70). The physiological functions of the different peroxisomal half-ABCD transporters have not been fully determined yet, but there are clear indications that both HsABCD1 and HsABCD2 are required for the breakdown of fatty acids in peroxisomes. Here we report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired oxidation of oleic acid, cannot only be partially rescued by HsABCD1, HsABCD2, but also by HsABCD3, which indicates that each peroxisomal half-transporter can function as homodimer. Fatty acid oxidation measurements using various fatty acids revealed that although the substrate specificities of HsABCD1, HsABCD2 and HsABCD3 are overlapping, they have distinctive preferences. Indeed, most hydrophobic C24:0 and C26:0 fatty acids are preferentially transported by HsABCD1, C22:0 and C22:6 by HsABCD2 and most hydrophilic substrates like long-chain unsaturated-, long branched-chain- and long-chain dicarboxylic fatty acids by HsABCD3. All these fatty acids are most likely transported as CoA esters. We postulate a role for human ABCD3 in the oxidation of dicarboxylic acids and a role in buffering fatty acids that are overflowing from the mitochondrial β-oxidation system.     

Antileishmanial activity of diterpene acids in copaiba oil

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC₅₀) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC₅₀ values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.     

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.     

13C NMR spectral analysis of grindelane diterpenoid acids

The ¹³C NMR spectra of methyl grindelate and some of its derivatives were recorded and the signals assigned. Based on these assignments and on a comparison with previously reported data, information regarding the structures and stereochemistry of the products under study was obtained.