Bright oligothiophene N-succinimidyl esters for efficient fluorescent labeling of proteins and oligonucleotides

The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported.     

Carboxylic ester hydrolases of rat pancreatic juice

An attempt was made to establish the number and characteristics of the enzymes in pancreatic juice that hydrolyze nitrogen- and phosphorus-free esters of fatty acids. For this purpose model compounds were hydrolyzed by lyophilized rat pancreatic juice under conditions that accelerated or inhibited the reactions. Although it is not established with certainty, it is suggested that three enzymes are responsible for the hydrolysis of fatty acid esters. The first enzyme is glycerol-ester hydrolase (EC 3.1.1.3) or lipase. This enzyme hydrolyzes water-insoluble esters of primary alcohols. The reaction occurs at an oil/water interface and is inhibited by bile salts at pH 8. The enzyme is relatively stable at pH 9, but unstable at pH 4. It has a broad pH optimum between 7.5 and 9.5. The second enzyme hydrolyzes esters of secondary alcohols and of other alcohols as well. It has an absolute requirement for bile salts and has a pH optimum at about 8. The enzyme is unstable in pancreatic juice when maintained at pH 9, probably due to the action of trypsin. It may be identical with sterol-ester hydrolase (EC 3.1.1.13). The third enzyme hydrolyzes water-soluble esters. It too has an absolute requirement for bile salts, although a smaller amount is necessary for maximum activity. This enzyme also is unstable at pH 9, but can be differentiated from the preceding enzyme by its stability at pH 4 and its pH optimum of 9.0. Carboxylic-ester hydrolase (EC 3.1.1.1) is not found in pancreatic juice, although it is present in pancreatic tissue.     

Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase

Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.     

Acyl unsaturation and cholesteryl ester miscibility in surfaces. Formation of lecithin-cholesteryl ester complexes

The surface behavior of a series of cholesteryl esters was studied in mixtures with a model phospholipid, 1-palmitoyl-2-oleoyl phosphatidylcholine. The cholesteryl esters were representative of the predominant forms occurring naturally and qualitative similarities in their phase behavior permits generalization of their surface properties. Quantitative differences, however, show that the availability of cholesteryl esters in all surface states is dependent on the structure of the acyl moiety. All except cholesteryl stearate were surface-active and formed preferred packing arrays, i.e., complexes, with the lecithin at compositions grouped around cholesteryl ester mol fractions of 0.015. Exceptions were cholesteryl arachidonate and docosahexaenoate, with complex stoichiometries of 0.034 and 0.032, respectively. Lecithin had the same apparent area in all complexes, 56.5 +/- 1.04 A2, which was larger than that of uncomplexed lecithin, 53.3 +/- 0.7 A2. This implies that the conformation or orientation of the two polyunsaturated species in complexes is markedly different than the others studied. The areas and hydrations of all uncomplexed cholesteryl esters were similar. Because mixing of complex with uncomplexed cholesteryl ester deviated positively from ideality, the apparent molecular areas of the uncomplexed cholesteryl esters ranged from 161 +/- 22 (complex-rich) to 107 +/- 15 A2 (cholesteryl ester-rich). The similarity of the monolayer phase complex stoichiometries and the bilayer miscibilities of cholesteryl oleate suggests a correspondence between states. If so, the availability of cholesteryl arachidonate or docosahexaenoate in bilayers should be approximately twice that of other naturally occurring cholesteryl esters.     

Use of multiple carboxylates to increase intracellular retention of fluorescent probes following release from cell penetrating fluorogenic conjugates

Fluorogenic reporter systems for use inside cells require that the fluorophore be retained inside the cell following activation to ensure accumulation of an observable signal. In the process of developing ester-based nucleic acid-triggered probe activation systems for use in cells, we found that simple O-alkylated fluorescein esters coupled to cell-penetrating peptides led to very poor signals, presumably because the released fluorophore was too membrane permeable and rapidly exited the cell. To circumvent this problem, we have examined the effect of adding one or two carboxylates to the fluorescein to reduce its membrane permeability. N-maleimido d-valine and alpha-methyl-beta- L-alanine esters of fluorescein, in which the second phenolic hydroxyl group was derivatized with a carboxymethyl group and then further conjugated with glutamate, were linked to the cell-penetrating peptide Arg9Cys through conjugate addition of the thiol group to the maleimido group. HeLa cells were incubated with these conjugates, washed, and then further incubated for various times prior to analysis by flow cytometry. Quantitative analysis of the data by a simplified kinetic scheme showed that the fluorescein with two appended carboxylic acid groups effluxed with a rate constant of about 0.00113 min (-1), corresponding to a half-life of 8.8 h. The dicarboxylated fluorescein effluxed about 6.1 times more slowly than the fluorescein with a single carboxylic acid group and led to a fairly stable signal. The analysis also showed that the d-Val ester was hydrolyzed about 4.6 times more slowly than the beta-alanine ester and had a half-life of about 31 min. These data indicate that the fluorescein with two appended carboxylates may be a useful membrane-impermeant fluorophore for fluorogenic probe applications inside living cells.     

Alkyl ester prodrugs for improved topical delivery of ibuprofen

Topical delivery of ibuprofen directly to the site of inflammation can overcome gastrointestinal side effects associated with its long term oral administration. The set of physicochemical properties necessary for optimum topical delivery of ibuprofen can be imparted by formation of its ester prodrugs. Various alkyl ester prodrugs (methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-buty, n-pentyl, hexyl, heptyl, octyl, lauryl, cetyl and octadecyl esters) were synthesised and studied for their physicochemical properties and activity in the carrageenan induced rat paw oedema by topical route. Favourable shift in lipophilicity and self penetration enhancing effect of prodrugs responded in improved topical activity over the parent drug ibuprofen.     

Regioselective synthesis of cellulose ester homopolymers

Regioselective synthesis of cellulose esters is extremely difficult due to the small reactivity differences between cellulose hydroxyl groups, small differences in steric demand between acyl moieties of interest, and the difficulty of attaching and detaching many protecting groups in the presence of cellulose ester moieties without removing the ester groups. Yet the synthesis of homopolymers of particular regioselectively substituted anhydroglucose esters is of critical importance to allow us to determine the analytical characteristics of such homopolymers, their structure-property relationships, and to obtain guidance that may ultimately enable identification and synthesis of cellulose derivatives with superior properties for various applications. We report here a new, general synthesis of both cellulose-2,6-O-diesters and cellulose-2,6-A-O-3-B-O-triesters with a high degree of regioselectivity, employing 3-O-allylcellulose as a key protected precursor. 3-O-Allylcellulose was identified as a protected intermediate with high potential for the synthesis of these derivatives with the aid of molecular modeling of corresponding glucose analogs. We report also the first analytical and structure property studies of these regioselectively substituted cellulose esters.     

Predominant role of basicity of leaving group in alpha-effect for nucleophilic ester cleavage

It has been found that alpha-effects in nucleophilic reactions, unexpectedly large nucleophilicity due to adjacent unpaired electrons, are strongly dependent on the structure of substrate. The nucleophilic cleavages of 4-nitrobenzoate esters and 4-methylbenzoate esters by HOO- have been systematically investigated in detail. When the leaving groups of substrates are sufficiently good (aryl, 2,2,2-trifluoroethyl, and 2,2-dichloroethyl esters), alpha-effect is evident. However, this effect drastically decreases as the leaving group gets poorer, and is only marginal for the cleavages of 2-fluoroethyl and methyl esters. In the nucleophilic cleavages by salicylaldoxime and acetohydroxamic acid, alpha-effect is also notable only for the esters having good leaving groups. These enormous dependences of alpha-effects on the substrate-structure have been interpreted in terms of the difference in the position of transition-state in the reaction coordinate.     

Formate ester formation in amide solutions

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100°C and more rapidly at 130°C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.     

Activity correlations in the phorbol ester series

Activity correlations in the phorbol ester series. The production of inflammation by phorbol esters on mammalian skin correlates on a structural basis with in vitro measurements of lymphocyte mitogenesis and mobilization of prostaglandins. All of the pro-inflammatory phorbol esters tested in our laboratory have been shown to activate the enzyme protein kinase C, and such an interaction could in large part explain the induction of an inflammatory response in vivo. Certain of these compounds additionally induce aggregation of human and rabbit blood platelets. This activity does not structurally correlate with the induction of inflammation but may correlate with the known tumour-promoting actions of phorbol derivatives. Compounds which induce platelet aggregation stimulate the secretion of a biologically active substance which we have termed 'Factor W'. The production of Factor W occurs into human plasma following platelet stimulation by phorbol tumour-promoting agents. It is an unstable substance, distinct in its aggregating properties from phorbol esters, ADP, 5-hydroxytryptamine, thrombin, platelet aggregating factor and the products of arachidonate oxidative metabolism.     

Interaction of cholesterol ester and triglyceride in human plasma very low density lipoprotein.

The properties of human plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and their extracted lipids were compared using calorimetric, X-ray scattering, and polarizing microscopy techniques. Intact LDL, and cholesterol esters isolated from LDL and VLDL each undergo reversible changes in their physical state around body temperature. These transitions are associated with ordered liquid crystalline to liquid phase changes of the cholesterol esters. In contrast to LDL, VLDL has no reversible transitions and shows no evidence of ordered liquid crystalline structures between 10 and 45 degrees C. Therefore, unlike LDL, VLDL does not contain a separate cholesterol ester region capable of undergoing cooperative melting. Solubility studies at 37 degrees C of cholesterol esters and triglyceride isolated from VLDL show that even at a weight ratio of 1:1, which greatly exceeds the relative amount of cholesterol esters in VLDL, cholesterol ester is completely soluble in triglyceride. Thus, the cholesterol ester in VLDL is not sequestered in a separate domain within VLDL, but is dissolved in the liquid core of the particle.     

Purification and properties of an esterase B4 from human liver.

A butyryl esterase, designated B4, has been purified from human liver and some of its properties described. The activity of this enzyme comprises 0.48% of the total butyryl esterase activity found in human liver. Esterase B4 has been distinguished from other butyryl esterases by its preference for the esters of the fluorogenic compounds 4-methyl umbilliferone and fluorescein over naphthyl esters as substrates. Other distinguishing features of this esterase include a relatively high pI (pH 8.7) A monomeric structure of low molecular weight (20 000) and high solubility in solutions of ammonium sulphate.     

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.     

Biologically active ester derivatives as potent inhibitors of the soluble epoxide hydrolase

Substituted ureas with a carboxylic acid ester as a secondary pharmacophore are potent soluble epoxide hydrolase (sEH) inhibitors. Although the ester substituent imparts better physical properties, such compounds are quickly metabolized to the corresponding less potent acids. Toward producing biologically active ester compounds, a series of esters were prepared and evaluated for potency on the human enzyme, stability in human liver microsomes, and physical properties. Modifications around the ester function enhanced in vitro metabolic stability of the ester inhibitors up to 32-fold without a decrease in inhibition potency. Further, several compounds had improved physical properties.     

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts

Summary Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acidN-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.     

Synthesis, photophysical, photochemical, DNA cleavage/binding and cytotoxic properties of pyrene oxime ester conjugates

A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting from N-O bond homolysis. Photoproduct formation and their distribution were found to be solvent dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause cytotoxicity upon irradiation of light.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Glycerol-derived ester oligomers from cork suberin

The cork suberin polyester was partially depolymerized by a methanolysis reaction catalyzed by calcium hydroxide. The methanolisate was analysed by ESI-MS/MS in the form of [M+Li](+) adduct-ions. This reaction solubilized a mixture of monomers and oligomers, including a set of glycerol-derived dimeric and trimeric esters. Four types of glycerol esters were identified: monoacylglycerols of alpha,omega-diacids, of omega-hydroxyacids and of monoacids; diglycerol diesters of alpha,omega-diacids; diacylglycerols of alpha,omega-diacids; monoacylglycerols of linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the glycerol esters are the main ones found as cork suberin monomers. It is concluded that suberin is a glycerol-derived lipid of polymeric dimensions. Due to the protective and insulating role that it plays in plants, suberin should be considered together with the other known glycerolipids that build up biological membranes.     

Convenient synthesis of L-proline benzyl ester

Mesylates or tosylates of delta-hydroxy-L-norvaline esters spontaneously afford L-proline esters upon exposure to aqueous buffer in near quantitative yield. This novel reaction has led to the development of a simple route to optically active proline esters.     

Intravascular metabolism of the cholesteryl ester moiety of rat plasma lipoproteins

The intravascular metabolism of the cholesteryl ester moiety of rat plasma LDL, HDL1, and HDL2 was determined in intact male rats. Biosynthetically labeled lipoproteins were prepared by zonal ultracentrifugation from the plasma of rats injected with [3H]cholesterol. The lipoproteins were concentrated by vacuum ultrafiltration as other procedures were found to alter the biological properties of the lipoproteins. After injection of labeled LDL, [3H]cholesteryl esters remained with the injected lipoprotein and decayed from plasma with a t1/2 of 7-8 hours. [3H]Cholesteryl esters in HDL1 behaved similarly and decayed with a t1/2 of 10.5 hours. With HDL2, however, a different metabolic pattern was observed with intraplasma conversion of some [3H]cholesteryl ester HDL2 particles to HDL1. Since such conversion of HDL2 to HDL1 was not observed after in vitro incubations of rat plasma, this process seems to depend on metabolic events that occur in vivo. [3H]Cholesteryl esters disappeared from HDL2 with a t1/2 of 6-7 hours, while the esters that were transferred to HDL1 decayed with a t1/2 of 10-11 hours, similar to labeled cholesteryl esters injected with HDL1. The study demonstrated that the high apoE content of rat plasma HDL1 is not associated with rapid catabolism of the lipoprotein and that a major source of HDL1 in the rat is the intraplasma conversion of HDL2 particles to HDL1.