A sensitive method for the quantitation of lysophosphatidylcholine in canine heart

We have developed a procedure for the determination of small amounts of lysophosphatidylcholine in cardiac tissue. Lysophosphatidylcholine from canine heart was separated from the major phospholipids by column chromatography, and then acetylated with labeled acetic anhydride. The acetylated lysophosphatidylcholine was isolated by thin-layer chromatography and the lysophosphatidylcholine content was calculated from the radioactivity associated with the acetylated product. Although the sensitivity of the assay depends on the specific radioactivity of the acetic anhydride used, as low as 0.5 nmol of lysophospholipid in tissue samples can be readily quantitated. The results obtained from the control and ischemic canine cardiac tissues by this assay compares favorably with those obtained by lipid-phosphorus assay. The sensitivity and specificity of the present procedure allows us and other investigators to assay for lysophosphatidylcholine content in very small (10 mg wet weight) tissue samples.     

Transesterification/acetylation of long chain alcohols with alkyl acetate

Gas chromatographic characterizations of fatty alcohols are generally carried out as the free alcohols, trimethyl silyl or acetyl derivatives. In this study, transesterification/acetylation of long chain fatty alcohols is simply carried out by dissolving the alcohol in ethyl/methyl acetate and passing through a micro-column packed with solid NaOH. Reaction times are slightly different for alcohols of different chain length. Rice bran alcohols of 24–34 carbon atom are successfully acetylated. Also, castor oil methyl ester can be interesterified but with longer reaction time.     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法。并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响。对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定。研究证明, 在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果。适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析。     

Applications of chromic acid-Celite columns to micro- and semimicro preparations of fatty aldehydes.

In a convenient two-phase procedure, columns of Celite 545 charged with aqueous chromic acid are used to oxidize microgram and milligram amounts of fatty alcohols to the corresponding aldehydes with approximately a 70% yield. The original configuration and position of the double bond is fully maintained during the oxidation of unsaturated alcohols.     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法.并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响.对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定.研究证明,在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果.适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析.     

Mode of action of a family 75 chitosanase from Streptomyces avermitilis

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).     

Zeolite-catalyzed Helferich-type glycosylation of long-chain alcohols. Synthesis of acetylated alkyl 1,2-trans glycopyranosides and alkyl 1,2-cis C2-hydroxy-glycopyranosides

Zeolite-catalyzed glycosylation of long-chain alcohols, using the inexpensive and readily available peracetylated beta-D-gluco- and galactopyranoses as glycosyl donors under solvent free conditions, has been explored for the first time. Among the various forms (H-, Na-, Fe- and Zn) of beta zeolite examined as catalysts in the reaction of 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose with cetyl alcohol, Fe-beta zeolite gave the maximum yield of 63% of cetyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and cetyl 3,4,6-tri-O-acetyl-alpha-D-galactopyranoside. Fe-beta Zeolite-catalyzed glycosylation was found to be general affording the title compounds in each case in a moderate yield, but with a good stereoselectivity. The yield of synthetically valuable acetylated long-chain alkyl 1,2-cis C2-hydroxy-glycopyranosides obtained in the present single-step procedure is considerably higher than that of the previously reported multi-step method employing the Stork silicon tether approach.     

Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing

Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.     

A simple and rapid preparation of alditol acetates for monosaccharide analysis

A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.     

High-yield method for isolation and culture of endothelial cells from rat coronary blood vessels suitable for analysis of intracellular calcium and nitric oxide biosynthetic pathways

We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This method makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed.     

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α-N-Acetyl Derivative

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH₂ terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ?15 fmol of β-endorphin per incubation tube, i.e., ? 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH₂ terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.     

Polyamines in Entamoeba invadens

The polyamine content of Entamoeba was measured by a procedure that involved benzoylation followed by high performance liquid chromatography (h.p.l.c.). A high concentration of putrescine and significant amounts of spermidine and spermine were found in actively growing trophozoites and in the cyst forms of the organism. In contrast, trophozoites in stationary phase had greatly reduced amounts of putrescine and exhibited peaks in h.p.l.c., possibly indicative of acetylated polyamines. alpha-D,L-difluoromethylornithine (DFMO) lowered the concentration of polyamines in growing trophozoites, but did not inhibit the degree of proliferation. There is evidence for pathways of polyamine biosynthesis in Entamoeba other than through ornithine decarboxylase (ODC).     

A new procedure for the acetylation of lipids

Long-chain alcohols and other lipids containing hydroxy groups are acetylated at room temperature by reaction with acetic anhydride in acetonitrile in the presence of an ion exchanger.     

TRANSPROTECTION OF SILYL ETHERS OF NUCLEOSIDES IN FeCl3 BASED IONIC LIQUIDS

Ionic liquid mediated deprotection of tert-butyldimethyl silyl (TBDMS) ethers derived from various primary and secondary alcohols have been studied and the reaction conditions optimized. Deprotection of the silyl ethers in FeCl3 based ionic liquids in presence of acetic anhydride yielded the acetate esters of the corresponding alcohols in good yields. The transprotection methodology was extended to the silyl ethers of nucleosides to yield the corresponding acetylated products.     

Transprotection of silyl ethers of nucleosides in FeCl3 based ionic liquids

Ionic liquid mediated deprotection of tert-butyldimethyl silyl (TBDMS) ethers derived from various primary and secondary alcohols have been studied and the reaction conditions optimized. Deprotection of the silyl ethers in FeCl3 based ionic liquids in presence of acetic anhydride yielded the acetate esters of the corresponding alcohols in good yields. The transprotection methodology was extended to the silyl ethers of nucleosides to yield the corresponding acetylated products.     

Determination of the configuration of 3,6-anhydrogalactose and cyclizable alpha-galactose 6-sulfate units in red seaweed galactans

A combination of two reported procedures was used in order to determine the configuration of the 3,6-anhydrogalactose present in red seaweed polysaccharides. A mild hydrolysis (to cleave only 3,6-anhydrogalactosyl linkages) was followed by a reductive amination with a chiral amine. Then, the total hydrolysis proceeded, followed by a new step of reductive amination. In this way, using (S)-alpha-methylbenzylamine as the chiral amine, it was possible to separate and quantitate both enantiomers of 3,6-AnGal and its 2-O-methyl ether as their diastereomeric acetylated aminoalditols. On the other hand, using (S)-1-amino-2-propanol, even though the derivatives of both enantiomers of 3,6-AnGal are not separated, the mixture can be safely quantitated with respect to galactose. Furthermore, a one-pot technique was developed to carry out an alkaline treatment of the polysaccharides, followed by the double hydrolysis-reductive amination procedure, which is useful to determine the proportions of both enantiomers of 6-sulfated 4-linked galactose units in the native polysaccharides. The unexpected presence of small amounts of units of this type belonging to the D-series in a porphyran sample is revealed by this novel procedure.     

High-valent tin(IV) porphyrin: An efficient and reusable catalyst for tetrahydropyranylation of alcohols and phenols under mild conditions

In this paper, rapid and highly efficient tetrahydropyranylation of alcohols and phenols with 3,4-dihydro-2H-pyran (DHP) in the presence of catalytic amounts of high-valent tin (IV) tetraphenylporphyrinato trifluoromethanesufonate, [Sn^IV(TPP)(OTf)₂] is reported. In this catalytic system, primary, secondary and tertiary alcohols as well as phenols were converted to their corresponding tetrahydropyranyl ethers (THP-ethers) in excellent yields and short reaction times at room temperature. It is noteworthy that this method can be used for chemoselective tetrahydropyranylation of primary alcohols in the presence of secondary and tertiary alcohols and phenols. The catalyst was reused several times in the protection reactions without loss of its catalytic activity.     

C15 acetogenins and terpenes of the dictyoceratid sponge Spongia zimocca of Il Rogiolo: A case of seaweed-metabolite transfer to,and elaboration within,a sponge?

• 1.1. In the narrow Mediterranean area of Il Rogiolo, the acetates of the secondary alcohols of β-chamigrene (−)-1b, branched lauroxepane (−)-2b, and isopimarane (+)-3b are found in the dictyoceratid sponge Spongia zimocca, whereas the corresponding free alcohols (+)-1a, (−)-2a, and probably also (+)-3a, are found in the red seaweed Laurencia microcladia.
• 2.2. No one of these acetylated metabolites could be detected either in this seaweed or in the global algal mass of Il Rogiolo.
• 3.3. Terpenes bearing tertiary alcoholic functionalities of either L. microcladia (−)-4E, (−)-4Z or another red seaweed of the same area, Sphaerococcus coronopifolius [(+)-5, (−)-6], are found unaltered also in S. zimocca.
• 4.4. These findings imply transfer of the hydroxyl-bearing metabolites from the seaweeds to the sponge and it is tempting to speculate that the secondary alcoholic function is acetylated in the sponge while tertiary alcohols and the secondary alcohol (+)-7 escape acetylation, the first ones as sterically-hindered alcohols and the latter one as a sponge metabolite residing in a special cell compartment.

     

Esterification of beta-chitin via intercalation by carboxylic anhydrides

beta-chitin is known to form intercalation complexes with aliphatic alcohols and amines. We found that it also forms complexes with carboxylic anhydrides. When the beta-chitin-acetic anhydride complex was heated to 105 degrees C, the hydroxyl groups of chitin were acetylated by a host-guest reaction, maintaining the host's crystal structure. Structures of complex and acetylated products were analyzed by X-ray diffraction, (13)C CP/MAS NMR, and infrared spectroscopy. The maximum degree of substitution (DS) was close to 1.0, suggesting regioselective esterification at the C6 position of chitin. Partially acetylated beta-chitin with a DS of 0.4 could incorporate various guest species that are difficult to be incorporated by original beta-chitin. In contrast, beta-chitin acetate with a DS of 1 lost the ability to form a complex. Intercalation complexes of beta-chitin with cyclic anhydrides (succinic and maleic) also underwent esterification by heating, and the products with a DS of approximately 1 dissolved in aqueous alkali, apparently as the result of the dissociation of introduced carboxyl groups. These phenomena are potentially useful in controlling the complexation ability of beta-chitin and the preparation of regioselectively esterified chitin derivatives.     

Fluorescent high-performance liquid chromatography assay for lipophilic alcohols

A new ultrasensitive fluorescent derivatization procedure for chromatographic analysis of primary, secondary, and nonpolar tertiary alcohols is described. The procedure uses Bodipy FL in basic dichloromethane solution with Mukaiyama’s reagent (2-chloro-1-methylpyridinium iodide) to form highly fluorescent ester derivatives that can be separated by silica normal-phase high-performance liquid chromatography (HPLC). Rhodamine WT and Oregon green 488 were also useful derivatization reagents. The detection limit for detection of cholesterol and bryostatin by Bodipy FL was less than 1 fmol. The reaction conditions are gentle enough that low concentrations of unstable alcohols such as bryostatin 1 can be measured.