Versatile methods for the synthesis of mixed-acid 1,2-diacylglycerols

Two methods for synthesizing mixed-acid 1,2-diacylglycerols starting from 2,3-epoxy-1-propanol (glycidol) or 3-chloro-1,2-propanediol have been described. This first method involves fatty acid addition to a protected glycidol derivative and solid-state isomerization. The second approach exploits the specificity of the trityl group for primary alcohols and the nucleophilic replacement of chlorine by a carboxylate ion in an aprotic solvent. The second method proves to be more general: with 3-chloro-1-O-trityl-1,2-propanediol as an intermediate compound apparently all types of mixed-acid, saturated and unsaturated, chiral and racemic 1,2-diacylglycerols can be prepared in good yields. In the first method tritylglycidol is a good starting compound. The use of this method, however, is restricted because only the 2-position of glycerol can be occupied with an unsaturated fatty acid. For de-blocking protected 1,2-diacylglycerols, the trityl group and other protecting groups were exchanged for the trifluoroacetyl group, which group could then be removed without any detectable acyl migration (< 1%). To this end, the 1,2-diacyl-3-trifluoroacetyl-glycerols were dissolved at room temperature in methanol containing pyridine, whereby the trifluoroacetyl group was split off, giving the 1,2-diacylglycerol.     

Structure and polymorphism of saturated monoacid 1,2-diacyl-sn-glycerols

The 1,2-diacyl-sn-glycerols (1,2-DGs) are the predominant naturally occurring isomer found in cell membranes, lipid droplets, and lipoproteins. They are involved in the metabolism of monoacylglycerols, triacylglycerols, and phospholipids. The 1,2-DGs participate in the activation of protein kinase C, in phosphorylation of target proteins, and in transduction of extracellular signals into the cell. We have undertaken a study of the physical properties of a homologous series of synthetic optically active diacylglycerols. Stereospecific 1,2-diacyl-sn-glycerols were synthesized with saturated fatty acyl chains of 12, 16, 18, 22, and 24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The solvent-crystallized form for all the 1,2-DGs packs in the orthorhombic perpendicular subcell (beta') and melts with a single sharp endotherm to an isotropic liquid. On quenching, the C12, C16 and C18 compounds pack in a hexagonal subcell (alpha), whereas the C22 and C24 pack in a pseudohexagonal subcell (sub-alpha). The sub-alpha phase reversibly converts to the alpha phase. The long spacings of these compounds in both the alpha and beta' phases increase with chain length. In the alpha and beta' phases, the acyl chain tilts were found to be 90 degrees and 62 degrees from the basal methyl plane. The polymorphic behavior of 1,2-diacyl-sn-glycerol is quite different from that of the corresponding monoacid saturated 1,3-diacylglycerols which form two beta phases with triclinic parallel subcells.     

Acid binding and detritylation during oligonucleotide synthesis.

Under the conditions normally used for detritylation in oligonucleotide synthesis, the haloacetic acid binds strongly to the oligonucleotide. Acetonitrile also forms a complex with the deblocking acid, in competition with the oligonucleotide, and drastically slows detritylation. Incomplete removal of acetonitrile during the deblock step may slow the kinetics enough to result in incomplete detritylation of the oligonucleotide. Acid binding to the growing oligonucleotide causes striking chromatographic effects in the presence of high oligonucleotide mass densities. In packed-bed column reactors, at low linear velocities, the acid binding almost completely depletes free acid from the deblocking solution. This results in an advancing zone within which the oligonucleotide is saturated with acid. Detritylation occurs mostly in a narrow band at the front of the advancing saturated zone. Increasing the DCA concentration in order to achieve quick saturation can give faster and more complete detritylation while minimizing the exposure time of the oligonucleotide to acid.     

Synthesis of 2,3- or 1,2-unsaturated derivatives of 2-deoxy-2-trifluoromethylhexopyranoses

The attempted conversion, by treatment with CsF/TBFA in MeCN, of acetylated derivatives of 2-chlorodifluoromethyl-2-deoxyhexopyranoses into their corresponding 2-trifluoromethyl derivatives was always accompanied by an elimination reaction. Thus, representative educts with the D-gluco- and D-manno-configuration gave derivatives of 2,3-dideoxy-2-trifluoromethyl-D-erythro-hex-2-enopyranose and 1,5-anhydro-2-deoxy-2-trifluoromethyl-d-arabino-hex-1-enitol, respectively. X-ray analyses are given for 1,3,4,6-tetra-O-acetyl-2-chlorodifluoromethyl-2-deoxy-alpha-D-mannopyranose and 4,6-di-O-acetyl-2,3-dideoxy-2-trifluoromethyl-alpha-D-erythro-hex-2-enopyranose.     

O-ethylidene-D-allopyranoses: 1,2-O-,1,2:4,6-, and 2,3:4,6-di-O-ethylidene derivatives

Solutions of 1,2-O-acetoxonium chlorides derived from O-acetylated D-allopyranose derivatives were treated with sodium borohydride to give three pairs of previously unknown 1,2-O-ethylidene-α-D-allopyranose diastereoisomers: 3,4,6-tri-O-acetyl-1,2-O-ethylidene-α-D-allopyranoses; 4,6-di-O-acetyl-3-O-benzyl-1,2-O-ethylidene-α-D-allopyranoses; and 3-O-benzyl-1,2:4,6-di-O-ethylidene-α-D-allopyranoses. Examples of a second class of novel O-ethylidene-D-allopyranoses, the diastereoisomeric methyl 2,3:4,6-di-O-ethylidene-α-D-allopyranosides, were prepared by treating methyl 4,6-O-benzylidene-α-D-alloside with acetaldehyde-sulfuric acid. Assignments of dioxolane ring configurations and pyranose conformations were made by n.m.r. analyses.     

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.     

Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis.

Fully protected CPG-immobilized monomer, dimer and trimer oligonucleotides were used to study depurination during the chemical synthesis of oligonucleotides. Disappearance of the oligonucleotide during acid exposure time relative to an internal thymidine standard not subject to depurination was monitored by reverse phase HPLC analysis. Depurination half-times obtained for dichloroacetic acid (DCA) and trichloroacetic acid (TCA) in methylene chloride were found to be 3% DCA >> 15% DCA > 3% TCA. In order to understand the implications of depurination during DNA synthesis, the detritylation kinetics of model compounds DMT-dG-pT dimer and DMT-[17mer] mixed-base sequence were also measured. These results improve our ability to properly balance the contradictory goals of obtaining maximum detritylation with minimum depurination in oligonucleotide synthesis.     

Enzymatic epoxidation: synthesis of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-Epoxyoctane by Pseudomonas oleovorans.

The kinetics of the enzymatic formation of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-epoxyoctane by growing and resting cell suspensions of Pseudomonas oleovorans are described. Formation of 1,2-epoxyoctane occurs concurrently with exponential growth on 1-octene, providing that 1-octene is in excess. Conversion of 1,7-octadiene to 7,8-epoxy-1-octene by cells growing on octane lags behind exponential growth and continues into the stationary phase, terminating upon cell death. Formation of 1,2-7,8-diepoxyoctane does not begin until the cells are well into the stationary phase and also continues until cell death. Results with growing and resting cell suspensions suggest that the various substrates compete for the same enzyme system; that viable cells are essential for substrate transport and epoxidation by whole cells; and that whole cells may concentrate and sequester the epoxides, rendering them unrecoverable by our current methods.     

New selective nonsteroidal aromatase inhibitors: synthesis and inhibitory activity of 2,3 or 5-(alpha-azolylbenzyl)-1H-indoles

Six azolyl substituted indoles were synthesized and tested for their activity to inhibit two P450 enzymes: P450 arom and P450 17a. It was observed that the introduction of alpha-imidazolylbenzyl chain at carbon 3 or 5 on indole nucleus led to very active molecules. Compounds 22, 23 and especially 33 demonstrate very high potential against P450 arom. Under our assay conditions of high substrate concentration the IC50 are 0.057, 0.0785 and 0.041 microM, respectively. These compounds are moderate inhibitors against P450 17alpha.     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.     

Synthesis of 2,3-O-(propane-1,2-diyl)- and 2,3-O-(3-hydroxypropane-1,2-diyl)-cyclomaltoheptaose

2¹,3¹-O-(Propane-1,2-diyl)cyclomaltoheptaose has been prepared from 2-O-allylcyclomaltoheptaose by mercuration in trifluoroacetic acid, followed by reduction with sodium borohydride. 2-O-(2,3-Epoxypropyl)cyclomaltoheptaose, prepared from 2-O-allylcyclomaltoheptaose by oxidation with dimethyldioxirane, was converted into 2¹,3¹-O-(3-hydroxypropane-1,2-diyl)cyclomaltoheptaose by treatment with trifluoroacetic acid. Both derivatives containing fused 1,4-dioxane rings are mixtures of stereoisomers, in which the methyl and hydroxymethyl group, respectively, that is linked to this ring, occupies an axial or an equatorial position.     

Facile Synthesis of 1,2: 5,6-Di-O-cyclohexylidene-d-mannitol and 2,3-O-Cyclohexylidene-d-glyceraldehyde

The reaction of cyclohexanone diethyl acetal with d-mannitol yielded quantitatively 1,2: 5,6-di-O-cyclohexylidene-d-mannitol (1) and its isomer (2). From 1, 2,3-O-cyclohexylidene-d-glyceraldehyde (3) was obtained in a quantitative yield without racemization.     

Stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane by alkene-utilizing bacteria

Resting cells of ethene grown Mycobacterium 2W produced 1,2-epoxypropane stereospecifically from propene as revealed by optical rotation, ¹H n.m.r. using a chiral shift reagent, and also by complexation gas chromatography involving a glass capillary column coated with an optically active metal chelate. The gas-liquid chromatography method allowed the rapid screening of 11 strains with regard to stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane. Bacteria grown on either ethene, propene or butadiene all predominantly produced the R form of 1,2-epoxypropane from propene and 1,2-epoxybutane from 1-butene while the strains tested for 1-chloro-2,3-epoxypropane production from 3-chloro-1-propene predominantly accumulated the S enantiomer.     

Stereoselective synthesis of 2-S-ethyl(phenyl)-2-thio-beta-glucopyranosides via 1,2-migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-mannopyranosides.

1,2-Migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-D- and L-mannopyranosides under the action of TMSOTf readily afforded the corresponding 2-S-ethyl(phenyl)-2-thio-beta-glucopyranosides, ready precursors to 2-deoxy-arabino-hexopyranosides (2-deoxy-beta-glucopyranosides).     

Regulation of protein synthesis in rabbit reticulocyte lysates by 2,3-bisphosphoglycerate.

2,3-Bisphosphoglycerate was the most potent effector of glycolytic intermediates tested for their effects on protein synthesis in gel-filtered lysates from rabbit reticulocytes. 2,3-Bisphosphoglycerate at low levels was stimulatory but became inhibitory at high levels. Both effects were dependent on Mg²⁺ concentrations. The higher the concentration of Mg²⁺, the higher the concentration of 2,3-bisphosphoglycerate required for maximal activation. 2,3-Bisphosphoglycerate concentrations required to exhibit an inhibitory effect increased as Mg²⁺ concentration increased. Both effects of 2,3-bisphosphoglycerate are discussed in terms of regulation of hemoglobin synthesis during maturation of erythroid cells.     

Differentiating the 2,3-diols of glucopyranosides by 4,6-O-benzylidene-protected-1,2-D-glucopyranosylorthoesters strategy

A facile and efficient method to differentiate the 2,3-diols of glucopyranosides based on 1,2-orthoesters strategy was developed. Stable thioglucosides were employed as the starting materials to prepare the corresponding 1,2-orthoesters. When treated with HCl aqueous solution and followed with Et(3)N, differentiation of the 2,3-diols was efficiently achieved along with the generation of a convertible anomeric hydroxyl group. In addition, an easy and practical method based on NOE was proposed to determine whether the 1,2-orthoesters were endo-type or exo-type.