Synthesis of l-muscone by asymmetric methylation via enol esters

An effective synthetic route of l-muscone (1) by asymmetric methylation, followed by enolate-trapping to generate enol esters as intermediates, was described. Interestingly, the enol esters can be used as substrates for enzymatic optical resolution to improve optical purity. Additionally, several excellent new chiral ligands were discovered for asymmetric methylation of (E)-cyclopentadec-2-enone to produce l-muscone with high optical purity.     

Esterification of acid chlorides with thallium and potassium salts of 19-norethisterone: Formation of 17-enol esters

During the esterification of acid chlorides with thallium and potassium salts of 19-norethisterone, 17-enol esters were isolated. The enol esters are formed $\text{Via}$ the intermediate, 4-estrene-3, 17-dione.     

Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwanensis.

The polar glycolipids were isolated from the thermophilic bacteria Meiothermus taiwanensis ATCC BAA-400 by ethanol extraction and purified by Sephadex LH-20 and silica gel column chromatography. The fatty acid composition of O-acyl groups in the glycolipids was obtained by gas chromatography mass spectroscopy analysis on their methyl esters derived from methanolysis and was made mainly of C(15:0) (34.0%) and C(17:0) (42.3%) fatty acids, with the majority as branched fatty acids (over 80%). Removal of O-acyl groups under mild basic conditions provided two glycolipids, which differ only in N-acyl substitution on a hexosamine. Electrospray mass spectroscopy analysis revealed that one has a C(17:0) N-acyl group and the other hydroxy C(17:0) in a ratio of about 1 : 3.5. Furthermore, complete de-lipidation with strong base followed by selective N-acetylation resulted in a homogeneous tetraglycosyl glycerol. The linkages and configurations of the carbohydrate moiety were then elucidated by MS and various NMR analyses. Thus, the major glycolipid from M. taiwanensis ATCC BAA-400 was determined with the following structure: alpha-Galp(1-6)-beta-Galp(1-6)-beta-GalNAcyl(1,2)-alpha-Glc(1,1)-Gro diester, where N-acyl is C(17:0) or hydroxy C(17:0) fatty acid and the glycerol esters were mainly iso- and anteisobranched C(15:0) and C(17:0).     

The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C6H5CONHCH2CONHCH2CO2CRR1CO2H (2 approximately) and seven esters of N-pivaloylglycine ((CH3)3CCONHCH2CRR1CO2H (3 approximately)) by bovine pancreatic carboxypeptidase A (Peptidyl-L-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25 degrees C and ionic strength 0.5. All N-hippurylglycine esters (2: R=H, R1=H, C2H5, 4-ClC6H4, C6H5CH2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics (3 approximately: R=H, R1=H, C2H5 C6H5), substrate activation (3 approximately: R=H, R1=4-ClC6H4; R=R1=CH3) or substrate inhibition (3 approximately: R=H, R1=(CH3)2CHCH2, C6H5CH2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters (1 approximately). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 approximately, 2 approximately and 3 approximately and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.     

Phosphatidyl-(N-acyl)-ethanolamine. A lipid component of mammalian epidermis.

A lipid present in the granular cells of mammalian epidermis was identified as phosphatidyl-(N-acyl)-ethanolamine. The structure was deduced from the ratio of phosphorus : nitrogen : glycerol : fatty acid esters : total fatty acid (1 : 0.94 : 0.97 : 2.1 : 2.9), from analyses of the products of alkaline and acid hydrolyses and from its infrared spectrum. Conclusive evidence was obtained by a direct comparison of the chromatographic properties, degradation products and infrared spectrum of the isolated lipid with those of synthetic 1,2-dipalmitoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanolamine. The fatty acids attached to the ethanolamine were predominantly saturated (69% of total) and hexadecanoic acid was the major component (41% of total). Phosphatidyl-(N-acyl)-ethanolamine was hydrolysed by a phospholipase C (Bacillus cereus) to diacylglycerol, inorganic phosphorus and N-acylethanolamine. Evidence for the presence of N-acylethanolamine in granular cells and in stratum corneum suggested that an epidermal phospholipase C may be involved in the catabolism of phosphatidyl-(N-acyl)-ethanolamine.     

Preparation of some long-chain N-acyl derivatives of essential amino acids for nutritional studies

Long-chain N-acyl derivatives of methionine, tryptophan, threonine, and lysine (N6) have been obtained by the reaction of succinimidyl esters of fatty acids with the unprotected amino acids. Their physical properties have been characterized.     

p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins

A new biotin-containing reagent, p-diazobenzoyl biocytin (DBB), has been developed for labeling tyrosines and histidines in proteins. The reagent has used to label these residues in both model proteins and erythrocyte membrane proteins on dot blots and blot transfers. In some cases, sub-nanogram levels of individual proteins could be detected. The utility of DBB as a versatile alternative to biotin-containing N-hydroxysuccinimide esters for the general labeling of proteins is discussed.     

Lipids of a T Strain of Mycoplasma

Cholesterol, free fatty acids, and phosphatidic acid are the predominant lipids of a T strain of Mycoplasma. The remaining neutral lipids are composed of cholesteryl esters, triglycerides, and diglycerides. Three glucose-containing glycolipids are present in trace amounts. In addition to phosphatidic acid, the phospholipids are comprised of phosphatidyl glycerol, diphosphatidyl glycerol, and phosphatidyl ethanolamine. Another polar lipid was found to be ninhydrin-positive and phosphate-free. It appears to be a diamino hydroxy compound containing adjacent fatty acid ester and N-acyl groups.     

Non-natural phenolic amino acids Synthesis and application in peptide chemistry

Summary Several non-natural phenolic amino acids have been synthesized.t-Butylated tyrosine and thyroxine derivatives, on one-electron oxidation, give persistent radicals which can be used as positional and/or spin labels for amino acids. Two-electron oxidation ofN-protected tyrosines leads to spirolactones, useful active esters for peptide coupling.     

Hydrophobic derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate: a new type of D-glucosamine-6-phosphate synthase inhibitors with antifungal action

Several N-acyl and ester derivatives of 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) have been synthesised and tested as inhibitors of fungal enzymes involved in early steps of chitin biosynthesis and for antifungal activity. All the tested derivatives were found to be much poorer inhibitors of the enzyme, D-glucosamine-6-phosphate (GlcN-6-P) synthase, than the parent compound but some of them exhibited much better antifungal activity. MIC values for the investigated compounds ranged between 10 mg mL(-1), found for ADGP and 0.3 mg mL(-1) for the most active derivative, namely ADGP dimethyl ester. Increased affinity of ADGP derivatives to the artificial immobilised cell membrane was correlated with their enhanced ability to be taken up by fungal cells by free diffusion. It was found that some of the examined derivatives behaved as 'pro-drugs' and after internalisation were converted into ADGP in the cell-free extract. This conversion was relatively rapid for ADGP esters but very slow for N-acyl derivatives. Results of our studies demonstrate a possibility of design and preparation of GlcN-6-P synthase inhibitors exhibiting antifungal activity.     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

Determination of ganglioside non-hydroxy fatty acid and long chain base by analysis of perbenzoylated derivatives

The non-hydroxy fatty acid and long chain base compositions from as little as 2.7 nmol of ganglioside were ascertained from perbenzoylated ganglioside derivatives. Non-hydroxy fatty acids were determined by mild alkaline methanolysis of the derivatives, followed by gas-liquid chromatography (GLC) of the methyl esters. N-acyl and N-benzoyl "gangliosides" that were generated by the methanolysis were hydrolyzed by a standard procedure that utilized aqueous acetonitrile-HCl, followed by high performance liquid chromatography (HPLC) determination of the biphenylcarbonyl derivatives with ultraviolet (UV) detection at 280 nm. A critical aspect of this procedure is a modified workup for the isolation of the biphenylcarbonyl derivatives which eliminates by-products that otherwise interfere with their separation by HPLC, especially when high sensitivity is required.     

Enantioselectivity-promoting factor in enzyme-mediated asymmetric hydrolysis of enol esters

Enantioselectivity-promoting factor enhances enantioselectivity of protonation in lipase AP-catalyzed asymmetric hydrolysis of enol esters. The factor was partially purified by chromatography using Phenyl-TOYOPEARL 650M and Sephacryl S-200HR. The hydrolysis of 2-benzyl-l-cyclohexenyl acetate by PLE in the presence of the purified factor produced (R)-2-benzylcyclo-hexanone in 92% ee, while the reaction without the factor gave the racemate.     

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.     

Biosynthetic labelling of membrane lipids of eukaryotic cells in tissue culture by a novel type of fluorescent fatty acids.

W-Anthryl labelled fatty acids with hydrocarbon chains of different lengths (C8, C11, C15) and different degrees of unsaturation have been incorporated into the membrane lipids of three different cell lines in tissue culture by addition of these 3H-labelled precursor fatty acids to the growth medium. The cell lines were baby hamster kidney cells (BHK 21), Chang liver cells and the RN6 cell line derived from a chemically induced Schwannoma tumor cell clone. Cell growth was normal. The quantitative analysis on the basis of radioactivity determinations demonstrated that the fluorescent-labelled fatty acids were introduced into the neutral lipid fraction (triglycerides, diglycerides, and cholesterol esters, all present in small amounts), but mainly into the phospholipid classes phosphatidylcholine, -ethanolamine and -serine, and to a lesser extent, as N-acyl component of sphingolipids (sphingomyelins, ceramides, mono- and diglycosylceramides). Cell fractionation studies indicated that the membranes of all subcellular particles were labelled with the fluorescent probes in their lipid moieties. These w-anthryl fatty acids are the first type of fluorescent lipid precursors which can be incorporated biosynthetically in vivo into membrane lipids of eukaryotic cells. The effective incorporation of the bulky fluorescent anthryl group in the terminal position of fatty acids of different chain lengths into the complex membrane lipids of the cell gives proff of 1) their uninhibited membrane transport, 2) their activation by the acyl-CoA synthetase and 3) their substrate properties for the O- acyl and N-acyl transferases in phospho- and sphingolipid biosynthesis.     

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.     

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.     

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.     

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.     

A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization

It has recently been shown that fatty acid vinyl esters serve as effective acylating agents for the synthesis of esters by enzymatic transesterification in high yields. To enhance the usefulness of this system at low temperatures, we have searched for the gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters and obtained it from the genomic library of Acinetobacter sp. strain no. 6, a psychrotroph isolated from Siberian soil. The gene (termed aelh, 777 bp) encoded a protein of 258 amino acids, and sequence analysis revealed that the enzyme shows a high sequence similarity to β-ketoadipate enol-lactone hydrolase involved in the β-ketoadipate pathway for the bacterial catabolism of benzoic acid. The aelh gene was expressed in the E. coli C600 cells under the control of lac promoter and the expression product was purified to homogeneity and characterized. It was a monomeric esterase preferentially catalyzing the hydrolysis of enol esters, such as fatty acid vinyl esters with a short-chain acyl group. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, a specific inhibitor for serine hydrolases. The enzyme could also catalyze transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4 °C. These results indicate the usefulness of an esterase (termed AELH) for the enzymatic synthesis of esters by transesterification using vinyl esters as an acyl donor.