Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.     

Long-chain N-acyl amino acid synthases are linked to the putative PEP-CTERM/exosortase protein-sorting system in Gram-negative bacteria

Clones that encode the biosynthesis of long-chain N-acyl amino acids are frequently recovered from activity-based screens of soil metagenomic libraries. Members of a diverse set of enzymes referred to as N-acyl amino acid synthases are responsible for the production of all metagenome-derived N-acyl amino acids characterized to date. Based on the frequency at which N-acyl amino acid synthase genes have been identified from metagenomic samples, related genes are expected to be common throughout the global bacterial metagenome. Homologs of metagenome-derived N-acyl amino acid synthase genes are scarce, however, within the sequenced genomes of cultured bacterial species. Toward the goal of understanding the role(s) played by N-acyl amino acids in environmental bacteria, we looked for conserved genetic features that are positionally linked to metagenome-derived N-acyl amino acid synthase genes. This analysis revealed that N-acyl amino acid synthase genes are frequently found adjacent to genes predicted to encode PEP-CTERM motif-containing proteins and, in some cases, other conserved elements of the PEP-CTERM/exosortase system. Although relatively little is known about the PEP-CTERM/exosortase system, its core components are believed to represent the putative Gram-negative equivalent of the LPXTG/sortase protein-sorting system of Gram-positive bacteria. During the course of this investigation, we were able to provide evidence that an uncharacterized family of hypothetical acyltransferases, which had previously been linked to the PEP-CTERM/exosortase system by bioinformatics, is a new family of N-acyl amino acid synthases that is widely distributed among the PEP-CTERM/exosortase system-containing Proteobacteria.     

Differential scanning calorimetry of chain-melting phase transitions of N-acylphosphatidylethanolamines.

Phosphatidylethanolamines in which the polar headgroup is N-acylated by a long-chain fatty acid (N-acyl PEs) are present in many plasma membranes under normal conditions, and their content increases dramatically in response to membrane stress in a variety of organisms. The thermotropic phase behavior of a homologous series of saturated N-acyl PEs, in which the length of the N-acyl chain is equal to that of the O-acyl chains attached at the glycerol backbone, has been investigated by differential scanning calorimetry (DSC). All fully hydrated N-acyl PEs with even chain lengths from C-12 to C-18 exhibit sharp endothermic chain-melting phase transitions in the absence of salt and in 1 M NaCl. Cooperative chain-melting is demonstrated directly by the temperature dependence of the electron spin resonance spectra from probe phospholipids bearing a spin label group in the acyl chain. The calorimetric transition enthalpy and the transition entropy obtained from DSC depend approximately linearly on the chain length with incremental values per CH2 group that exceed those of normal diacyl phosphatidylethanolamines, but to an extent that underrepresents the additional N-acyl chain. A thermodynamic model is constructed for the chain-length dependences and end effects of the calorimetric quantities, which includes a deficit proportional to the difference in O-acyl and N-acyl chain lengths for nonmatched chains, as is found and justified structurally for mixed-chain diacyl phospholipids. From data on the chain-length dependence of N-acyl diC16PEs, it is then deduced that the N-acyl chains are less well packed than the O-acyl chains and, from the data on the matched-chain N-acyl PEs, that the O-acyl chain packing is similar to that in normal diacyl PEs. The gel-to-fluid phase transition temperatures of the N-acyl PEs in the absence of salt are practically the same as those of the normal diacyl PEs of the corresponding chain lengths, although the transition enthalpies and entropies are appreciably greater, indicating entropy-enthalpy compensation. In 1 M NaCl, the transition temperatures are 3-4.5 degrees higher than in the absence of salt, representing the contribution of the electrostatic surface potential of the N-acyl PEs.     

Establishing the potency of N-acyl amino acids versus conventional fatty acids as thermogenic uncouplers in cells and mitochondria from different tissues

The possibility that N-acyl amino acids could function as brown or brite/beige adipose tissue-derived lipokines that could induce UCP1-independent thermogenesis by uncoupling mitochondrial respiration in several peripheral tissues is of significant physiological interest. To quantify the potency of N-acyl amino acids versus conventional fatty acids as thermogenic inducers, we have examined the affinity and efficacy of two pairs of such compounds: oleate versus N-oleoyl-leucine and arachidonate versus N-arachidonoyl-glycine in cells and mitochondria from different tissues. We found that in cultures of the muscle-derived L6 cell line, as well as in primary cultures of murine white, brite/beige and brown adipocytes, the N-acyl amino acids were proficient uncouplers but that they did not systematically display higher affinity or potency than the conventional fatty acids, and they were not as efficient uncouplers as classical protonophores (FCCP). Higher concentrations of the N-acyl amino acids (as well as of conventional fatty acids) were associated with signs of deleterious effects on the cells. In liver mitochondria, we found that the N-acyl amino acids uncoupled similarly to conventional fatty acids, thus apparently via activation of the adenine nucleotide transporter-2. In brown adipose tissue mitochondria, the N-acyl amino acids were able to activate UCP1, again similarly to conventional fatty acids. We thus conclude that the formation of the acyl-amino acid derivatives does not confer upon the corresponding fatty acids an enhanced ability to induce thermogenesis in peripheral tissues, and it is therefore unlikely that the N-acyl amino acids are of specific physiological relevance as UCP1-independent thermogenic compounds.     

N-acylphosphatidylethanolamines: effect of the N-acyl chain length on its orientation

N-Acylphosphatidylethanolamines, or NAPEs, are found in tissues involved in degenerating processes, such as dehydrated endosperm of seeds, erythrocyte membranes, or cell injury. To determine the conformation and orientation of the acyl chains of these phospholipids, NAPEs with deuterated N-acyl chains of 6 and 16 carbon atoms were synthesized and studied by transmission and attenuated total reflectance (ATR) infrared spectroscopy. For N-C16d-DPPE, the ATR measurements show that the N-acyl chain has the same orientation as the two acyl chains attached to the glycerol moiety, while the N-acyl chain of N-C6d-DPPE is randomly oriented. These results demonstrate that for N-C16d-DPPE, the N-acyl chain is embedded into the hydrophobic core of the bilayer, while for the short chain derivative the N-acyl chain remains in the lipid headgroup region. The analysis of the carbonyl stretching band and of the amide I band suggests that, for the long N-acyl chain lipid, the ester C=O and the N-H groups are linked by intermolecular hydrogen bonds.     

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.     

Glycerophospho-(N-acyl)-ethanolamine lipids in degenerating BHK cells.

The phospholipids, which accompany semilysobisphosphatidic acid from degenerating BHK cells, were identified as a mixture of glycerophospho-(N-acyl)-ethanolamine lipids. The identification was based on infrared spectroscopy, thin-layer chromatography and ethanolamine content of the intact lipids or their partial degradation products. Sequential treatments with mild acid and alkali revealed the presence of three different derivatives: the most abundant of these was the O-(1-alkenyl) ether derivative (plasmenyl-(N-acyl)-ethanolamine), which represented 55-60% of the total glycerophospho-(N-acyl)-ethanolamine lipids; the O-alkyl derivative (plasmanyl-(N-acyl)-ethanolamine) and the di-O-acyl derivative (phosphatidyl-(N-acyl)-ethanolamine) each represented about 20% of the total.     

Study of the structure of N-acyldipalmitoylphosphatidylethanolamines in aqueous dispersion by infrared and Raman spectroscopies

The effect of the headgroup chain length on the structure and on the thermotropic behavior of N-acyldipalmitoylphosphatidylethanolamines (N-acyl-DPPEs) has been studied by infrared and Raman spectroscopies. The results show that the N-acyl-DPPEs can be divided in two classes depending on the N-acyl chain length. When the N-acyl chain contains 10 carbon atoms or more, it penetrates into the bilayer while it remains at the level of the glycerol backbone for shorter N-acyl chains. For both classes of N-acyl-DPPEs, the rotation of the lipid chains in the liquid-crystalline phase is hindered by the presence of the N-acyl group. In addition, the disruption of the hydrogen bonds between the amino and phosphate groups by N-acylation of the amino group results in an increase of the hydration of the phosphate group compared to that in DPPE. The hydration occurred at both the phosphate and amide group levels; the phosphate group is more hydrated for phospholipids with long N-acyl chains while in the case of short-chain derivatives both the phosphate and amide groups are hydrated. This higher degree of hydration coupled with the immobilization of the lipid molecule may contribute to the bilayer stabilizer role of N-acyl-PEs since hydration is an important factor in bilayer stability.     

Derivatised lipids in membranes. Physico-chemical aspects of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and N-acyl ethanolamines

The physical properties of N-biotinyl phosphatidylethanolamines, N-acyl phosphatidylethanolamines and of N-acyl ethanolamines, in aqueous dispersions, are reviewed. Emphasis is placed on the calorimetric (i.e. chain melting) properties, the thermotropic phase behaviour, certain aspects of the structure and dynamics, and the miscibility with other membrane lipids. In the case of N-biotinyl phosphatidylethanolamines, the specific binding of avidin, and in the case of N-acyl ethanolamines, the function of the third chain, is also considered. All of these properties are relevant to the role of these rather unusual lipids in membranes.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.     

菜籽蛋白酶解液制备N酰化肽的研究(Ⅰ)——酰化肽制备工艺的优化

对N 酰化肽的合成条件进行了研究 ,得出较佳的工艺条件为 :温度 1 0 2 5℃ ,酶解液与脂肪酰氯的摩尔比为0 .7:1 ,pH值为 9.0。温度对氨基氨的转化率影响不大 ,在实际生产中可选择室温条件。pH值对反应的影响较大 ,pH9时比 pH8时转化率提高近 50 %。氨基氮与酰氯的配比为 0 .7时 ,能使 90 %的氨基氮转化为酰化肽。     

Base- and acid-catalyzed interconversions of O-acyl- and N-acyl-ethanolamines: a cautionary note for lipid analyses

The isolation and quantification of ethanolamine containing lipids from animal tissues may expose neutral lipid extracts to acidic or basic conditions during chromatographic separations or derivatization chemistry. While investigating the acid- and base-catalyzed production of anandamide in chromatographic fractions of rat brain extracts not containing anandamide, we observed that O,N-acyl migrations are facile. O,N-acyl migrations are well documented in synthetic organic chemistry literature, but are not well described or recognized with regard to methods in lipid isolation or lipid enzyme studies. We report here the synthesis and characterization of O- and N-acyl (palmitoyl- or arachidonoyl-)ethanolamines. Their rearrangements in base and acid are quantified by liquid chromatography;-electrospray ionization mass spectrometry. The rearrangements proceed through a cyclic intermediate that is also formed during chemical reactions commonly used for derivatization of acylethanolamines for gas chromatography-mass spectrometry.The isolation and characterization of N- or O-acylethanolamines and their enzymatic formation requires awareness and consideration of the proclivity of these compounds to chemically rearrange.     

Synthesis and biological activity of N-acyl O-indolylalkyl ethanolamines

The plant-growth regulators, indole-3-carboxylic acids, were introduced into N-acyl ethanolamines, and a series of N-acyl O-indolylalkyl ethanolamines were prepared. Their biological activities to regulate rape hypocotyl elongation, cucumber cotyledon expansion and common wheat coleoptile growth were tested. The results indicate that the title compounds inhibited rape hypocotyl elongation, especially the indole-3-propionic acid derivatives, whose bioactivity was better than that of indole-3-acetic acid.     

Rapid and selective reduction of amide group by borane-amine complexes in acyl protected nucleosides

Borane-amine complexes provide an unusually fast and selective reduction of a deoxynucleoside N-acyl group to a corresponding N-alkyl group. Three different nucleosides (dG, dA, and dC) each having one of three N-protecting groups (benzoyl, isobutyryl, or acetyl) were used to prepare N-alkylated nucleosides in good yields under mild conditions. Deoxyribose O-acyl protecting groups remain intact at the conditions of N-acyl group reduction.     

Evaluation of endogenous fatty acid amides and their synthetic analogues as potential anti-inflammatory leads

A series of endogenous fatty acid amides and their analogues (1-78) were prepared, and their inhibitory effects on pro-inflammatory mediators (NO, IL-1β, IL-6, and TNF-α) in LPS-activated RAW264.7 cells were evaluated. Their inhibitory activity on the pro-inflammatory chemokine MDC in IFN-γ-activated HaCaT cells was also examined. The results showed that the activity is strongly dependent on the nature of the fatty acid part of the molecules. As expected, the amides derived from enone fatty acids showed significant activity and were more active than those derived from other types of fatty acids. A variation of the amine headgroup also altered bioactivity profile remarkably, possibly by modulating cell permeability. Regarding the amine part of the molecules, N-acyl dopamines exhibited the most potent activity (IC(50) ～2 μM). This is the first report of the inhibitory activity of endogenous fatty acid amides and their analogues on the production of nitric oxide, cytokines (IL-1β, IL-6, and TNF-α) and the chemokine MDC. This study suggests that the enone fatty acid-derived amides (such as N-acyl ethanolamines and N-acyl amino acids) and N-acyl dopamines may be potential anti-inflammatory leads.     

Variability in the carbazole assay for N-desulfonated/N-acylated heparin derivatives

The carbazole assay is commonly employed to quantify heparin and other uronic acid-containing polysaccharides. Heparin-derived standard curves are often employed to quantify solutions of various natural and unnatural heparin structures that have different levels of sulfate substitution, different levels of N-sulfo and N-acetyl groups, and other structural changes as a consequence of reducing molecular weight. Recent studies in our laboratory have focused on chemically modified heparin derivatives comprised of structurally diverse N-acyl moieties substituted into heparin in place of N-sulfo groups. We report here that although differing degrees of 2-N-sulfo-, 2-N-acetyl- or 2-amino-d-glucosamine residues within heparin do not affect signal intensity in the carbazole assay, replacing N-sulfo groups in heparin with structurally diverse N-acyl moieties affords products that display significant variation in the assay. The structure of different N-acyl groups, and to a lesser extent the degree of N-acylation by individual N-acyl groups, is shown to variably alter signal intensity in the carbazole assay even though content and structure of uronic acid residues is unaltered.     

Synthetic libraries of tyrosine-derived bacterial metabolites

The preparation of a collection of 131 small molecules, reminiscent of families of long chain N-acyl tyrosines, enamides and enol esters that have been isolated from heterologous expression of environmental DNA (eDNA) in Escherichia coli, is reported. The synthetic libraries of N-acyl tyrosines and their 3-keto counterparts were prepared via solid-phase routes, whereas the enamides and enol esters were synthesized in solution-phase.     

菜籽蛋白酶解液制备N-酰化肽的研究(Ⅱ)——蛋白水解率对N-酰化肽性能的影响

用不同水解率的水解液合成了N 酰化肽 ,对比了它们的表面活性 ,由碱性蛋白酶水解菜籽蛋白 ,当水解率为 30 .6%时 ,所合成的N 酰化肽性能比水解率为 1 5.7%、2 0 .3%时要好 ,用SephadexG 1 5分析了水解液中肽分子量的分布情况 ,发现较佳水解率的水解液中 80 %的肽分子量小于 1 50 0Da。