Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of D. melanogaster on media supplemented with [1-¹³C]-palmitate lead to a family of ¹³C-palmitate-labeled fatty acid amides in the fly heads. The [1-¹³C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.     

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.     

Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides

Caffeic acid has been widely recognized as a versatile pharmacophore for synthesis of new chemical entities, among which caffeic acid derived phenethyl esters and amides are the most extensively-investigated bioactive compounds with potential therapeutical applications. However, the natural biosynthetic routes for caffeic acid derived phenethyl esters or amides remain enigmatic, limiting their bio-based production. Herein, product-directed design of biosynthetic schemes allowed the development of thermodynamically favorable pathways for these compounds via acyltransferase (ATF) mediated trans-esterification. Production based screening identified a microbial O-ATF from Saccharomyces cerevisiae and a plant N-ATF from Capsicum annuum capable of forming caffeic acid derived esters and amides, respectively. Subsequent combinatorial incorporation of caffeic acid with various aromatic alcohol or amine biosynthetic pathways permitted the de novo bacterial production of a panel of caffeic acid derived phenethyl esters or amides in Escherichia coli for the first time. Particularly, host strain engineering via systematic knocking out endogenous caffeoyl-CoA degrading thioesterase and pathway optimization via titrating co-substrates enabled production enhancement of five caffeic acid derived phenethyl esters and amides, with titers ranging from 9.2 to 369.1 mg/L. This platform expanded the capabilities of bacterial production of high-value natural aromatic esters and amides from renewable carbon source via tailoring non-natural biosynthetic pathways.     

Distribution of unsaturated aliphatic acid amides in japanese Zanthoxylum species

Seven species and two varieties of Zanthoxylum in Japan were investigated for unsaturated aliphatic acid amides. In addition to the known amides α-sanshoöl, γ-sanshoöl and hydroxy-γ-sanshoöl, a new compound, hydroxy-α-sanshoöl, was isolated and established by chemical and spectroscopic evidence. The compounds, corresponding to hydroxyl derivatives of the amides in the barks, commonly existed in the pericarps of all collected materials. Japanese Zanthoxylum species were divided chemotaxonomically into two taxa. These taxa ditter from the two assigned on the basis of botanical classification.     

Microorganisms hydrolyse amide bonds; knowledge enabling read-across of biodegradability of fatty acid amides

To get insight in the biodegradation and potential read-across of fatty acid amides, N-[3-(dimethylamino)propyl] cocoamide and N-(1-ethylpiperazine) tall oil amide were used as model compounds. Two bacteria, Pseudomonas aeruginosa PK1 and Pseudomonas putida PK2 were isolated with N-[3-(dimethylamino)propyl] cocoamide and its hydrolysis product N,N-dimethyl-1,3-propanediamine, respectively. In mixed culture, both strains accomplished complete mineralization of N-[3-(dimethylamino)propyl] cocoamide. Aeromonas hydrophila PK3 was enriched with N-(1-ethylpiperazine) tall oil amide and subsequently isolated using agar plates containing dodecanoate. N-(2-Aminoethyl)piperazine, the hydrolysis product of N-(1-ethylpiperazine) tall oil amide, was not degraded. The aerobic biodegradation pathway for primary and secondary fatty acid amides of P. aeruginosa and A. hydrophila involved initial hydrolysis of the amide bond producing ammonium, or amines, where the fatty acids formed were immediately metabolized. Complete mineralization of secondary fatty acid amides depended on the biodegradability of the released amine. Tertiary fatty acid amides were not transformed by P. aeruginosa or A. hydrophila. These strains were able to utilize all tested primary and secondary fatty acid amides independent of the amine structure and fatty acid. Read-across of previous reported ready biodegradability results of primary and secondary fatty acid amides is justified based on the broad substrate specificity and the initial hydrolytic attack of the two isolates PK1 and PK3.     

Nitrogen-containing non-steroidal secondary metabolites of Solanum, Cyphomandra, Lycianthes and Margaranthus

Of the 49 species of Solanum studied, cuscohygrine has been detected in 25, solamine and related amines in 17 and solamine-derived amides in 16. Five species of Cyphomandra examined all contained both amines and amides. From roots of Margaranthus solanaceus cuscohygrine has been isolated which probably occurs, too, in roots of Lycianthes rantonnettii. The distribution of these compounds throughout the taxa could be of chemotaxonomic value.     

An efficient one-pot synthesis of polyphenolic amino acids and evaluation of their radical-scavenging activity

A simple and efficient procedure for the synthesis of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy phenylglycine amides by a strategy based on the multicomponent Ugi reaction is proposed. Hydroxybenzaldehyde derivatives were reacted with 4-methoxybenzylamine, cyclohexyl isocyanide and benzoic acid or 2-naphthylacetic acid to give Ugi adducts that were treated with trifluoroacetic acid yielding N-acyl hydroxyphenylglycine amides in good yields. The same procedure using as acid component protocatechuic acid or hydrocaffeic acid gave N-catechoyl 3,4-dihydroxyphenylglycine amides. The use of N-benzyloxycarbonylglycine as acid component allowed the preparation of a 3,4-dihydroxyphenylglycyl dipeptide derivative. Radical-scavenging activity studies of the polyphenolic amino acid derivatives showed a sharp increase in activity with the increase in number of hydroxyl or catechol groups present. Cyclic voltammetry experiments established a correlation between oxidation peak potentials and the radical-scavenging activity.     

Structure–activity relationship studies of manzamine A: Amidation of positions 6 and 8 of the β-carboline moiety

Twenty manzamine amides were synthesized and evaluated for in vitro antimalarial and antimicrobial activities. The amides of manzamine A (1) showed significantly reduced cytotoxicity against Vero cells, although were less active than 1. The structure–activity analysis showed that linear, short alkyl groups adjacent to the amide carbonyl at position 8 are favored for antimalarial activity, while bulky and cyclic groups at position 6 provided the most active amides. Most of the amides showed potent activity against Mycobacterium intracellulare. The antimicrobial activity profile for position 8 series was similar to that for antimalarial activity profile, in which linear, slightly short alkyl groups adjacent to the amide carbonyl showed improved activity. Two amides 14 and 21, which showed potent antimalarial activity in vitro against Plasmodium falciparum were further evaluated in vivo in Plasmodium berghei infected mice. Oral administration of 14 and 21 at the dose of 30 mg/kg (once daily for three days) caused parasitemia suppression of 24% and 62%, respectively, with no apparent toxicity.     

Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Spilanthol-related amides from Acmella ciliata

From the flower heads of Acmella ciliata amides closely related to spilanthol were isolated. They possess isobutylamine, 2-phenylethylamine and 2-methylbutylamine parts. In the acidic part deca-2E,6Z,8E-trienoic acid, known from spilanthol, and its 2,3-dihydro derivative were found. Two compounds bear ester groups, a novelty for unsaturated amides. The structures were elucidated by means of high field ¹H NMR and high resolution mass spectrometry.     

Amides and other constituents from acmella ciliata

Fourteen highly unsaturated amides were isolated from Acmella ciliata. Their structures were determined by means of high field ¹H NMR including 2D-NMR, high resolution mass spectrometry and GC-MS. Some considerations on the biosynthesis of the amides are made.     

Thermochemistry of N-C bonds hydrolysis in amides,peptides, N-acetyl amino acids and high-energy N-C bonds hydrolysis in N-acetyl imidazole and urea

• 1.1. The reaction enthalpies of hydrolysis of amides, peptides and N-acetyl amino acids were calculated for both ionized and un-ionized forms of reaction components.
• 2.2. The average values of reaction enthalpies of amides, peptides and N-acetyl amino acids hydrolysis were essentially different from each other for ionized forms of reaction components and were equal for un-ionized forms of reaction components in the error interval.
• 3.3. As an example of high-energy N-C bonds N-acetyl imidazol and urea were discussed. It was found that the reaction enthalpies of hydrolysis of above compounds were different from analogous thermodynamic values of hydrolysis of amides, peptides and N-acetyl amino acids for any forms of components.

     

Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells

Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 μM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive.     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

Metabolites ofStreptomyces noursei

Branched chain amino acids are degraded byStreptomyces noursei to the corresponding amides: valine being the precursor of isobutyramide, leucine of isovaleramide and isoleucine of anteisovaleramide. The formation of amides is pyridoxalphosphate dependent and is catalyzed by peroxidase. Furthermore it was found that branched chain amino acids were also degraded via the corresponding 2-oxoacids into acylCoA which can be dehydrogenated to unsaturated acylCoA or hydrolyzed to acids and CoASH.     

Penicillinamidohydrolase inEscherichia coli

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) fromEscherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. SomeN-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial Tates of the enzyme hydrolysis of different substrates were found when using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space.N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.     

A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K_m = 6.2 mM; k_cat = 5.76 s⁻¹) and glutaramic acid (K_m = 2.8 mM; k_cat = 2.23 s⁻¹). However, the substrates of known amidases such as short-chain (C₂ to C₄) aliphatic amides, long-chain (above C₁₆) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M_r of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.     

Further amides from Echinacea purpurea

The aerial parts of Echinacea purpurea afforded, in addition to known compounds, five further highly unsaturated amides and a derivative of linolen     

Pyrrolidine and piperidine amides from Achillea

The underground parts of three Achillea species afforded, in addition to known unsaturated aliphatic acid isobutyl amides, four new pyrrolidides an     

Amides of Ottonia corcovadensis

Five amides were isolated from the roots of Ottonia corcovadensis. Two are the known piperlonguminine and piperovatine. The other three are new and