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.     

Identification of reactive toxicants: Structure–activity relationships for amides

A diverse series of amides were evaluated for aquatic toxicity (IGC₅₀) assessed in the Tetrahymena pyriformis population growth impairment assay and for reactivity (EC₅₀) with the model soft nucleophile thiol in the form of the cysteine residue of the tripeptide glutathione. All alkylamides along with some halo-substituted amides are well predicted by the simple hydrophobicity (log K _ow)–electrophilicity (E _lumo) response-surface model [log(IGC⁻¹ ₅₀) = 0.45(log K _ow) − 0.342(E _lumo) − 1.11]. However, 2-halo amides with the halogen at the end of the molecule and α,β-unsaturated primary amides are among those derivatives identified as being more toxic than predicted by the model. Amides, which exhibit excess toxicity, were capable of forming covalent bonds through an S_N2 displacement or a Michael addition. Moreover, only those amides exhibiting excess toxicity were reactive with thiol, suggesting that the reactivity with model nucleophiles such as the thiol group may provide a means of accurately defining reactive toxicants.     

Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots

From the fresh roots of Heliopsis longipes three new minor alkamides: longipinamide A (N-isobutyl-8,10-diynoic-3Z-undecenamide), longipenamide A (N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide) and longipenamide B (N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide); three known alkamides: affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide), N-isobutyl-2E,6Z-decadienamide and N-isobutyl-2E-decenamide; and 11 other known compounds were isolated. The structures of the three new minor alkamides were established by 1D and 2D NMR spectroscopy including ¹H, ¹³C, DEPT, COSY, HSQC, and HMBC experiments, as well as by EI and FAB⁺ mass spectrometry. To our knowledge, this is the first report of the isolation of linear dihydroxyalkamides as natural products.     

Hydroxycinnamic acid spermidine amides from pollen of corylus avellana L.

Two hydroxyannamic acid amides from the pollen of Corylus avellana L. have been identified as (E)-caffeoyl-(E)-feruloylspermidine and di-(E)-feruloylspermidine on the basis of ¹H NMR, ¹³C NMR and mass spectral data.     

Synthesis and structural characterization of solvated calcium amides containing bulky silylamide ligands

A series of solvated calcium bis(amides) with the general formula Ca[N(R)(SiMe₃)]₂(solv)_x (R = -SiMe₂t-Bu, -SiPh₂t-Bu, and -SiPh₃) was prepared from the reaction of CaI₂ with 2 equiv of the corresponding potassium amide. Solvent ligands used in this study include THF, pyridine (py), hexamethylphosphoranide (HMPA), and 4-dimethylaminopyridine (DMAP). The coordinated THF in Ca[N(SiMe₂t-Bu)(SiMe₃)]₂(THF)₂ could be replaced by stronger donating ligands. When -N[(SiPh₃)(SiMe₃)] was used as the amide ligand again a bis(THF) adduct was formed. Quite interestingly, when the more sterically-demanding ligand -N[(SiPh₂t-Bu)(SiMe₃)] ligand was used only one THF molecule could coordinate to Ca, leading to a rare example of a tri-coordinate Ca bis(amide). One of the starting amides, [KN(SiMe₂t-Bu)(SiMe₃)]₂, was isolated and found to adopt a dimeric structure in the solid state. All complexes were characterized with ¹H NMR, ¹³C NMR, elemental analyses, and X-ray crystallography when appropriate.     

Structural and synthetic studies on the retrofractamides—amide constituents of Piper retrofractum

Two new unsaturated amides, retrofractamides A and C, were isolated from the total above-ground parts of Piper retrofractum. Retrofractamide A was shown to be N-isobutyl-9(3′,4′-methylenedioxyphenyl)2E,4E,8E-nonatrienamide from spectroscopic and chemical investigations. The structure 6 for retrofractamide C was suggested from spectroscopic and chemical studies and was confirmed by a total stereoselective synthesis. The presence of sesamin and 3,4,5-trimethoxydihydrocinnamic acid as well as two higher homologues of retrofractamide A, viz. pipericide (retrofractamide B) and retrofractamide D was demonstrated. The synthesis of pipericide was also achieved.     

Phenolic amides from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities

Three new phenolic amides, tabamides A–C (1–3), together with three known phenolic amides (4–6), were isolated from the leaves of Nicotiana tabacum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1–6 were also tested for their anti-tobacco mosaic virus (anti-TMV) activity. The results showed that compound 1 exhibited high anti-TMV activity with inhibition rate of 38.6%, which is higher than that of positive control (ningnanmycin). The other compounds also showed potential anti-TMV activity with inhibition rates in the range of 15.3–26.5%, respectively.     

Produits neutres et alcaloides de Myrtopsis macrocarpa,M. myrtoidea,M. novae-caledoniae et M. sellingii

Stems and leaves of Myrtopsis macrocarpa, M. myrtoidea, M. novae-caledoniae and M. sellingii yielded terpenes, sterols, coumarins, alkaloids (furoquinolines and quinolones) and amides. A new quinolone (8-methoxy flindersine) occurs in Myrtopsis macrocarpa, a new amide (N-benzoyltryptamine) in M. myrtoidea, two new coumarins (myrsellin and myrsellinol) and a new dihydrofuroquinoline (myrtopsine) in M. sellingii. Structures of the new compounds are proposed from chemical and spectroscopic evidence.     

Synthesis,antimycobacterial and cytotoxic activity of α,β-unsaturated amides and 2,4-disubstituted oxazoline derivatives

The synthesis of six α,β,-unsaturated amides and six 2,4-disubstituted oxazolines derivatives and their evaluation against two Mycobacterium tuberculosis strains (sensitive H37Rv and a resistant clinical isolate) is reported. 2,4-Disubstituted oxazolines (S)-3b,d,e were the most active in the sensitive strain with a MIC of 14.2, 13.6 and 10.8 μM, respectively, and the compounds (S)-3d,f were the most active against resistant strain with a MIC of 6.8 and 7.4 μM. The ex-vivo evaluation of hepatotoxicity on precision-cut rat liver slices was also tested for the α,β-unsaturated amides (S)-2b and (S)-2d,f and for the oxazolines (S)-3b and (S)-3d,f at different concentrations (5, 15 and 30 μg/mL). The results indicate that these compounds possess promising antimycobacterial activity and at the same time are not hepatotoxic. These findings open the possibility for development of new drugs against tuberculosis.     

Transposable Element Insertion and Epigenetic Modification Cause the Multiallelic Variation in the Expression of FAE1 in Sinapis alba

Naturally occurring heritable variation provides a fundamental resource to reveal the genetic and molecular bases of traits in forward genetic studies. Here, we report the molecular basis of the differences in the four alleles E¹, E², E³, and e of the FATTY ACID ELONGATION1 (FAE1) gene controlling high, medium, low, and zero erucic content in yellow mustard (Sinapis alba). E¹ represents a fully functional allele with a coding DNA sequence (CDS) of 1521 bp and a promoter adjacent to the CDS. The null allele e resulted from an insertional disruption in the CDS by Sal-PIF, a 3100-bp PIF/Harbinger-like DNA transposon, whereas E² and E³ originated from the insertion of Sal-T1, a 4863-bp Copia-like retrotransposon, in the 5′ untranslated region. E³ was identical to E² but showed cytosine methylation in the promoter region and was thus an epiallele having a further reduction in expression. The coding regions of E² and E³ also contained five single-nucleotide polymorphisms (SNPs) not present in E¹, but expression studies in Saccharomyces cerevisiae indicated that these SNPs did not affect enzyme functionality. These results demonstrate a comprehensive molecular framework for the interplay of transposon insertion, SNP/indel mutation, and epigenetic modification influencing the broad range of natural genetic variation in plants.     

Pungent Alkamides from Spilanthes acmella L. var. oleracea Clarke

A main pungent amide, spilanthol (1), and three alkamides, (2E)-N-(2-methylbutyl)-2-undecene-8,10-diynamide (2), (2E,7Z)-N-isobutyl-2,7-tridecadiene-10,12-diynamide (3), and (7Z)-N-isobutyl-7-tridecene-10,12-diynamide (4) were isolated from the flower heads of Spilanthes acmella L. var. oleracea Clarke. Their structures were established by spectroscopic methods. Compounds 2 and 4 were new and 3 was found for the first time in Spilanthes species. Chemotaxonomic aspects are discussed.     

Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 ? E2 Conformational Changes

This work investigates the role of charge of the phosphorylated aspartate, Asp³⁶⁹, of Na⁺,K⁺-ATPase on E₁ ↔ E₂ conformational changes. Wild type (porcine α₁/His₁₀-β₁), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5′-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S. J. (1980) J. Bioenerg. Biomembr. 12, 111–136). One central finding is that the D369N/D369A mutants are strongly stabilized in E₂ compared with wild type and D369E or T212A mutants. Stabilization of E₂(Rb) is detected by a reduced K_0.5Rb for the Rb⁺-induced E₁ ↔ E₂(2Rb) transition. The mechanism involves a greatly reduced rate of E₂(2Rb) → E₁Na with no effect on E₁ → E₂(2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E₂ but affects the D369N mutant only weakly. Thus, this “Bohr” effect of pH on E₁ ↔ E₂ is due largely to protonation of Asp³⁶⁹. Two novel effects of phosphate and vanadate were observed with the D369N/D369A mutants as follows. (a) E₁ → E₂·P is induced by phosphate without Mg²⁺ ions by contrast with wild type, which requires Mg²⁺. (b) Both phosphate and vanadate induce rapid E₁ → E₂ transitions compared with slow rates for the wild type. With reference to crystal structures of Ca²⁺-ATPase and Na⁺,K⁺-ATPase, negatively charged Asp³⁶⁹ favors disengagement of the A domain from N and P domains (E₁), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E₂). Changes in charge interactions of Asp³⁶⁹ may play an important role in triggering E₁P(3Na) ↔ E₂P and E₂(2K) → E₁Na transitions in native Na⁺,K⁺-ATPase.     

Molecular modeling of the interaction of 17(20)Z- and 17(20)E-pregna-5,17(20)-dien-21-oyl amides with the nuclear receptor LXRβ

Eight isomeric 17(20)Z- and 17(20)E-pregna-5,17(20)-dien-21-oyl amides, conformationally rigid oxysterol analogues, differing in the structure of the amide moiety have been analyzed. Analysis of low energy conformers revealed that all 17(20)E-isomers had three main energy minima (corresponding to the values of the dihedral angle θ_20,21 (C17=C20-C21=O) about ～0°, ～120°, and ～240°); the most occupied minimum corresponded to θ_20,21 about ～0°. 17(20) Z-Isomers had either one or two pools of stable low energy conformations. Molecular docking of these compounds to the ligand-binding site of the nuclear receptor LXRβ (a potential target) demonstrated high probability of binding of E-isomers but not Z-isomers with this target. Results of the molecular modeling were confirmed by an experiment in which stimulation of triglyceride biosynthesis in Hep G2 cells in the presence of 17(20)E-3β-hydroxypregna-5,17(20)-dien-21-oyl (hydroxyethyl)amide was demonstrated.     

Head-tail connector of bacteriophage lambda

The head-tail connector of phage λ, a protein knob inside the head shell to which the tail attaches, is composed primarily of head protein gpB ⁴ and its cleaved form gpB¹. All of the gpB and gpB¹ in the virion is located in the connector. gpFII, the protein that is thought to form the site on the head to which the tail binds, is also located in the connector. Head proteins gpE, gpD, X1 and X2 are not components of the connector. These assignments were made by disrupting virions with guanidine hydrochloride, in such a way that heads and tails separate with the connectors attached to the tails, and determining which head proteins co-purify with the tails.We find that lysates from a λE^? infection contain a high proportion of tails with connectors attached. (Gene E codes for the major component of the head shell.) Connectors are also present on tails from a λE^?C^? infection, arguing that gpE, gpC, and their processed forms, X1 and X2, are all unnecessary for assembly of biologically competent connectors. The gpB in the connectors on E^? and E^?C^? tails is in the uncleaved form. Connectors are not seen on tails from infections by λE^?B^?, λE^?FII^?, or λE^? in a groE^? host.     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca²⁺ at the transport sites (E2PCa₂) of sarcoplasmic reticulum Ca²⁺-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca²⁺ deocclusion/release to the lumen; E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. Stabilization was achieved by elongation of the Glu⁴⁰-Ser⁴⁸ loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly⁴⁶/Lys⁴⁷ and by binding of beryllium fluoride (BeF_x) to the phosphorylation site of the Ca²⁺-bound ATPase (E1Ca₂). The complex E2Ca₂·BeF₃⁻ was also produced by lumenal Ca²⁺ binding to E2·BeF₃⁻ (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 °C. Only BeF_x, but not AlF_x or MgF_x, produced the E2PCa₂ structural analog. Complex formation required binding of Mg²⁺, Mn²⁺, or Ca²⁺ at the catalytic Mg²⁺ site. Results reveal that the phosphorylation product E1PCa₂ and the E2P ground state (but not the transition states) become competent to produce the E2PCa₂ transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca²⁺ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu⁴⁰-Ser⁴⁸ linker and of Mg²⁺ at the catalytic site in these processes.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

A mosquito larvicide in Spilanthes mauritiana

The methanol extract of fresh vegetative aerial parts of Spilanthes mauritiana afforded, after repeated chromatographic separations and mosquito larvicidal bioassays, a potent mosquito larvicide N-isobutyl-2E,4E,8^E,10Z-dodeca-2,4,8,10-tetraenamide.The structure of the compound followed from spectroscopic considerations. It gave 100% mortality against third instar larvae of Aedes aegypti at 10⁻⁵ mg/ml.     

Prenyltransferases from the flavedo of Citrus sinensis

A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2E,6E)- and (2Z,6E)-farnesyl pyrophosphate. The two C₁₅-products are predominant. The E- and Z-synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z-synthetase were obtained. Mg^{2 +} is required for full activity. Mn^{2 +} or Co^{2 +} can replace Mg^{2 +}. The ratio of E/Z-products formed is different for each cation. Mg^{2 +} complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E- and Z-products.     

Isobutyl Amides from Pepper (Piper nigrum L.)

Four isobutyl amides were isolated from the fruits of white pepper (Piper nigrum L.) and identified to be N-isobutyl-13-(3,4-methylenedioxyphenyl)-2E,4E,12E-tridecatrienamide (3, guineensine), N-isobutyl-2E,4E,8Z-eicosatrienamide (5), N-isobutyl-2E,4E-octadecadienamide (6) and N-isobutyl-2E,4E-decadienamide (7, pellitorine).