Studies on the synthesis of β-keto esters derived from dipeptides: search for a low-epimerizing method

-Keto esters derived from dipeptides areprepared by application of common methodologiesemployed for the synthesis of amino acid-derived-keto esters; however, epimerization of theC-terminal residue occurred to different extentsdepending on the method. In imidazolide activateddipeptides, this epimerization is due to the CDIactivation step and to the configurational instabilityof the intermediate imidazolides in different reactionmedia. Regarding yield and diastereomeric purity, themethod of choice proved to be the reaction ofdipeptide-derived imidazolide with the potassium saltof malonic half esters in the presence of MgCl₂.     

Amino acid esters of 9-(2′,3′-dihydroxypropyl-1′)-adenine are the specific inhibitors of protein synthesis on ribosomes

Peptide acceptor properties of phenylalanine and glycine esters of 9-(2,3-dihydroxypropyl-1)-adenine and 1-(2,3-dihydroxypropyl-1)-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[¹⁴C] Phe-tRNA in a ribosomal system charged with poly U. However, peptidyl esters of 9-(2,3-dihydroxypropyl-1)-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens.Abbreviations AcPhe acetyl-l-phenylalanine - HP-Ade 9-(2,3-dihydroxypropyl-1)-adenine - Phe-HP-Ade and Gly-HP-Ade l-phenylalanine and glycine esters of HP-Ade - Phe-HP-TUra l-phenylalanine ester 1-(2,3-dihydroxypropyl-1)-4-thiouracyl - AcPhePhe-HP-Ade and AcPheGly-HP-Ade acetyl-l-diphenylalanine and acetyl-l-phenylalanylglycine esters of HP-Ade respectively - AcPhe-puromycin acetyl-l-phenylalanyl-puromycin     

Mixed anhydrides (phosphoric-carboxyl) are also formed in the esterification of 5′-amp with n-acetylaminoacyl imidazolides: Implications regarding the origin of protein synthesis

Procedures for the formation of aminoacyl esters of monoribonucleotides with aminoacyl imidazolides were first reported by Gottikhet al. and summarized in 1970. This reaction has been widely used by us and numbers of other workers as a convenient means of preparing aminoacyl esters of nucleotides. We have previously reported that, under conditions of excess imidazolide, large amounts of bis 2, 3 esters are formed in addition to the monoesters, (Laceyet al., 1991). However, to our knowledge, no one has reported that in addition to the esters, relatively large amounts of the mixed anhydride, with the amino acid carboxyl attached to the phosphate, are also formed at short reaction times. We report here on the relative amounts of anhydride and esters formed in this reaction of racemic mixtures of eleven N-acetyl amino acid imidazolides with 5-AMP and discuss the relevance of the findings to the origin of protein synthesis.     

Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors

Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - PBS phosphate buffered saline - PDBu 4-phorbol-12,13-dibutyrate - PDGF platelet-derived growth factor - PK-C protein kinase-C - TGF- transforming growth factor- - TPA 12-0-tetradecanoylphorbol-13-acetate     

Esterolytic and proteolytic enzymes of the rat adenohypophysis

Summary Characteristics of the hydrolysis of histochemical substrates 5-bromoindoxyl acetate, naphthyl acetate, proprionate, butyrate, caprylate, laurate, myristate, and palmitate, acetyl and butyryl thiocholhie, chloroacetyl and trifluoroacetyl -naphthylamide, benzoyl-arginine -naphthylamide and proteinase substrates human hemoglobin and glycyl-phenylalanine amide by the rat pituitary tissue homogenate and DEAE-cellulose chromatography fractions were determined.In DEAE-cellulose chromatography fractions four separate activities were found splitting short and long chain carboxylic esters. The activity hydrolysing most rapidly 5-bromoindoxyl acetate was resistant to E 600 and was identified as C esterase. Three of the remaining esterase activities were sensitive to E 600 and two of them hydrolysed more rapidly short-chain fatty acid esters while one preferred long-chain fatty acid esters as substrate.One peak of activity was identified as nonspecific cholinesterase on the basis of inhibition studies and hydrolysis of thiocholine substrates. Chloroacetyl -naphthylamide was hydrolysed minimally. Hydrolysis of trifluoroacetyl -naphthylamide was ascribed to E 600 resistant enzyme with pH-optimum at 8.3 hydrolysing also the thiocholine substrates and slowly long-chain fatty acid esters.Five different proteinases hydrolysing human hemoglobin were separated, three of them with pH-optima on the acid and two on the alkaline side of pH. The activities hydrolysing benzoyl-arginine naphthylamide were cysteine activated and had pH-optima around 5.3. None of the peaks of the proteinase activities appeared to coinside with the hydrolysis peaks of any of the histochemical ester substrates in the DEAE fractions.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.     

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

Synopsis The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper.Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the whorls observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative.Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 721. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K₄[Fe(CN)₆] containing OsO₄ in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).     

Some investigations of the mechanism of the so-called “methylation” reactions used in mucosubstance histochemistry

Summary In 1968, Vilter suggested that acid methylating reagents such as warm methanolic hydrochloric acid convert the anionic groups of acid mucosaccharides to a lactone form instead of esterifying them as had been commonly assumed hitherto. The three new histochemical methods described in this paper for distinguishing lactones from esters have been used to show that Vilter's hypothesis has some substance. Among other things, methylated mucosaccharides do not regain their affinity for Azure A after treatment with first, either aqueous sodium borohydride at 0 ° or aqueous, neutral solutions of hydroxylamine at room temperature and, second, a saponifying reagent. In vitro the borohydride and hydroxylamine reagents react with lactones but not with esters. Thus, it is argued, if esters are present in methylated mucosaccharides, they would not react with either reagent and consequently the original anionic groups would be regenerated by the saponifying reagent and thence available for staining by Azure A.     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

Energy-rich glyceric acid oxygen esters: Implications for the origin of glycolysis

The apparent Gibbs free energy change of hydrolysis (G°', pH 7) of the 2- and 3-O-glyceroyl esters of 2- and 3-O-L-glyceroyl-L-glyceric acid methyl ester were measured at 25 °C. The 2- and 3-glyceroyl esters were found to be energy-rich with G°' values of –9.1 kcal mol^–1 and –7.8 kcal mol, respectively. This result indicates that the analogous 2- and 3-glyceroyl esters of polyglyceric acid are also energy-rich and, therefore, could have acted as an energy source for primitive phosphoanhydride synthesis.     

Enzymic alcoholysis of blackcurrant oil

Alcoholysis of blackcurrant oil mediated by Pseudomonas fluorescens lipase performed at 30°C in ethanol (96%, v/v) used both as a solvent and as a reactant. After 16 h, 95% of triacylglycerols present in the oil was converted into a mixture consisting of fatty acid ethyl esters, free fatty acids, monoacylglycerols and diacylglycerols. The highest amount of fatty acid ethyl esters (52%) was achieved after 8 h.     

Synthesis of flavor and fragrance esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral -substituted or -substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.     

Synthesis ofα-triazolylα-amino acid derivatives

Summary We report the synthesis of-triazolyl-amino esters by 1,3 dipolar cycloaddition of acetylenic compounds and-azido-amino esters.     

Nicotinic and muscarinic agonists,phorbol esters,and agents which raise cyclic AMP levels phosphorylate distinct groups of proteins in the superior cervical ganglion

The phosphorylation of proteins in the superior cervical ganglion of the rat was investigated. Ganglia were incubated with³²P_i, and the³²P-labeled proteins in the ganglion were separated by two-dimensional electrophoresis and visualized by autoradiography. Approximately 40 distinct phosphoproteins could be visualized by these methods. The most heavily labeled ganglionic protein was an acidic protein with an M_r of approximately 83,000. Tyrosine hydroxylase was identified as a doublet of two closely-migrating radioactive spots. Treatment of intact ganglia with depolarizing agents, nicotinic and muscarinic agonists, phorbol esters, and agents that increase the content of cyclic adenosine 35-monophosphate in the ganglion stimulated the incorporation of³²P_i into distinct but overlapping groups of phosphoproteins. All of these agents increased the phosphorylation of tyrosine hydroxylase. In contrast, only phorbol esters and muscarcinic agonists increased the phosphorylation of the 83,000 ganglionic phosphoprotein. Our data are consistent with the idea that the various classes of agonists may activate distinct protein kinases in the ganglion.     

Lipid composition of positively buoyant eggs of reef building corals

Lipid composition of the eggs of three reef building corals, Acropora millepora, A. tenuis and Montipora digitata, were determined. Sixty to 70% of the egg dry weight was lipid, which consisted of wax esters (69.5–81.8%), triacylglycerols (1.1–8.4%) and polar lipids c/mainly phospholipids (11.9–13.2%). Montipora digitata also contained some polar lipids typical of the thylakoid membrane in chloroplasts, probably due to the presence of symbiotic zooxanthellae in the eggs. The wax esters appeared to be the major contributor to positive buoyancy of the eggs, and specific gravity of wax esters in A. millepora was estimated to be 0.92. Among the fatty acids of the wax esters, 34.9–51.3% was hexadecanoic acid (16:0) while the major fatty acids in polar lipids were octadecenoic acid (18:1), hexadecanoic acid (16:0), eicosapentaenoic acid (20:5) and eicosatetraenoic acid (20:4). The wax ester appears to be the main component of the 4.5 6.0 m diameter lipid droplets which fill most of the central mass of the coral eggs.     

Stereochemical aspects of synthetic and naturally occurring 2-hydroxy fatty acids. Their absolute configurations and assays of optical purity

d- and l-Cerebronic acid⁵, ⁶ methyl esters were obtained from a dl-mixture by converting the racemic ester to the l-acetylmandelates and separating the diastereomers by thin-layer chromatography; the diasteromers were then decomposed with mild acid methanolysis. The optical purities of the d- and l-enantiomers were determined as described below and found to be 82.5% and 87.1%, respectively. Absolute configurations of the enantiomers were confirmed by their ORD spectra; specific rotations at the peak of the anomalous curves at 223 nm of the d- and l-isomers were ?989 ° and +1120 °, respectively. Considering their optical purities, the actual maximum rotations of these enantiomers were calculated as ?1520 ° and +1509 °, respectively. The conditions for the reaction of l-acetylmandelyl chloride and 2-hydroxy fatty acid esters were modified to be suitable for the optical assay of 2-hydroxy fatty acid methyl esters. The method was also shown to be applicable to the assay of 3-hydroxy isomers and possibly to other positional isomers. The absolute configuration of 2-hydroxy fatty acid methyl esters obtained from calf brain cerebrosides and from yeast cerebrins were determined to be d(?), confirming previous assignments. Methyl cerebronate obtained from its less soluble strychnine salt was found to be the l(+)-enantiomer, contrary to a previous assignment. Methyl d-cerebronate was hydrolyzed with tetrahydrofuran-HCl to obtain D-cerebronic acid without racemization.     

The enzyme lecithin‐cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH‐SY5Y cells

Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)‐hydroxycholesterol, 24(S)OH‐C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin‐cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH‐C. Proteoliposomes containing apolipoprotein A‐I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A‐I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH‐C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH‐C esterification. When highly neurotoxic 24(S)OH‐C was treated with enzyme and proteoliposomes before incubation with differentiated SH‐SY5Y cells, the neuron survival improved. The esters of 24(S)OH‐C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH‐C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH‐C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol‐induced neurotoxic injuries to neurons in culture.

     

Asymmetric imine-ene reactions: Diastereofacial selective reactions with chiral glyoxylate-derivedα-imino esters and asymmetric catalysis of enantiofacial selective reactions with prochiralα-imino esters

Summary The diastereofacial selective imine-ene reactions with-imino esters, prepared from (–)-8-phenylmenthyl glyoxylate, are shown to provide an efficient entry to the asymmetric synthesis of-amino acids. The feasibility study of the asymmetric catalysis is also reported on the enantiofacial selective ene reactions with prochiral-imino esters.