Flash Silica Research Articles

Expanded Fluorescent Nucleoside Analog as Hybridization Probe

The design and synthesis of fluorescent nucleosides has been the subject of intensive research because these nucleoside derivatives can be used as tools in molecular biology and diagnostics. Fluorescent nucleoside analogs that are sensitive to the local environment in DNA duplexes are attractive candidate probes for DNA hybridization and for investigating nucleic acid structure. They display a strong signal change upon hybridization with a target DNA, and structural changes in DNA such as the formation of Gquadruplexes and i-motifs. Although a broad range of substituted fluorescent dyes are suitable for labelling nucleic acids, rapid growth in this area requires new and efficient fluorescent nucleoside analogs. Our strategy to synthesize an efficient fluorescent analog is based on the use of expanded nucleobase analogs, which have good fluorescent properties such as high quantum efficiency and high sensitivity to the microenvironment. This system does not change the conformation of stable BDNA and also yields higher duplex stability owing to the bulky hydrophobic planar structure. In this context, we synthesized a new fluorescent nucleoside analog, 1-(2Deoxy-β-D-erythro-pentofuranosyl) benzothieno[3,2,-d]pyrimidine 2,4(3H)-dione( U). Scheme 1 shows our synthetic strategy to prepare the expanded fluorescent nucleoside U. Compound 2 was prepared from 1 in three steps, as shown in Scheme 1. The initial step is nucleophilic displacement of o-halobenzonitrile (2-fluorobenzonitrile) 1 with thioglycolate anion, followed by spontaneous base-induced aldol cyclization with ethyl carboxyisocyanate and base treatment to produce 2 as a white solid in an overall yield of 40% starting from compound 1. Compound 2 was coupled to a sugar moiety to afford an unresolved anomeric mixture of compound 3 in 72%. After detoluoylation, attempts to protect the 5'position of the α/β anomeric mixture with DMT-Cl failed. Thus, instead of DMT-Cl, 4,4'-dimethoxytrityl tetrafluoroborate (DMTBF4) in pyridine was employed for selective protection of the 5'-hydroxyl group of the nucleoside to obtain the protected product. The 5'-ODMT anomeric mixture was separated on a flash silica gel column. A small amount of each anomer was deprotected using aqueous acetic acid to afford the free nucleosides, which were subjected to extensive spectroscopic analysis to determine the anomeric configuration unambiguously and to measure the quantum yield. ROESY showed a correlation spot for H-1' and H-4', as expected for the β configuration of 6. The β anomer of 4 was treated with standard phosphoramidite reagent to yield the nucleoside 3'-O-phosphoramidite derivative, 5. To evaluate the sensitivity of U to its microenvironment, fluorescence spectra of U were measured in different solvents (Figure 1A). The emission intensity of U was markedly affected by solvent polarity. U showed higher quantum efficiency in polar than in non-polar solvents (Table 1). We also measured the emission change for U at different pH values to evaluate its sensitivity. U exhibited different signals, depending on the pH (acidic, neutral, basic) (Figure 1B). This difference may arise from O-4 protonation in

A multigram chemical synthesis of the γ-secretase inhibitor LY411575 and its diastereoisomers

An improved chemical synthesis of N-2((2 S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)- N1-((7 S)-5-methyl-6-oxo-6,7-dihydro-5 H-dibenzo[ b, d]azepin-7-yl)- l-alaninamide (LY411,575, 9a), a known γ-secretase inhibitor, is described. The key synthetic steps, which used no chiral chromatography in the entire sequence, involved (1) improved microwave-assisted synthesis of a seven-membered lactam (±)-(5,7-dihydro-6 H-dibenz-[ b, d]azepin-6-one 2, and (2) convenient isolation of pure LY411575 from a mixture of four diastereomers by simple flash silica gel chromatography. Starting from the resolved aminolactams 5a and 5b, all four diastereomers were produced in enantiomerically pure form.

Two new monogalactosyl diacylglycerols from brown alga Sargassum thunbergii.

Two new monogalactosyl diacylglycerols (MGDGs) along with two known glycolipids were isolated from the moderate polar fraction of the methanolic extract of the brown alga Sargassum thunbergii by using reversed silica flash chromatography. Two new MGDGs were identified as (2S)-1-O-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-2-O-(9Z,12Z,15Z-octadecatrienoyl)-3-O-beta-D-galactopyranosyl-sn-glycerol (1) and (2S)-1-O-(9Z,12Z,15Z-octadecatrienoyl)-2-O-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-beta-D-galactopyranosyl-sn-glycerol (2) by FAB tandem mass spectrometry, NMR techniques, and specific enzyme-catalyzed hydrolysis of the sn-1 fatty acyl linkage. The regiochemical attachment of the acyl chains in the glycerol moiety was established by 2D NMR correlations and confirmed by enzymatic hydrolysis.

Evidence for Four-Component Close-Range Sex Pheromone in the Parasitic Wasp Glyptapanteles flavicoxis

Females of the parasitic wasp Glyptapanteles flavicoxis (Hymenoptera: Braconidae) deposit a close-range sex pheromone from their abdominal tip that attracts conspecific males and elicits wing-fanning behavior. In this study, we isolated the pheromone components and determined their role in the males' behavior. In coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of the females' body extract, four components (below GC detection) elicited strong responses from male antennae. Monitored by GC-EAD, the components were separated by flash silica gel and high-performance liquid chromatography. Y-tube olfactometer experiments with one or more components revealed that all are necessary to elicit short-range attraction and wing-fanning responses by males. These components remained below detection threshold of the mass spectrometer (approximately 10 pg) even when 4500 female equivalents were analyzed in a single injection, which attests to the potency of the pheromone and the insects' sensitivity to it.

Pharmacokinetics of Dihydroergocristine and Its Major Metabolite 8- Hydroxy-Dihydroergocristine in Human Plasma

Dihydroergocristine (DHEC) is a semi-synthetic drug mainly used for age-related cognitive impairment. In this study, its major metabolite 8'-hydroxy-dihydroergocristine (8'-OH-DHEC) was produced in incubates of a bovine liver preparation using dihydroergocristine mesylate (DHECM) as substrate. Purification was achieved by flash silica gel column and reverse phase liquid chromatographies, and identification was based on accurate molecular mass measurements, mass fragmentation spectra and NMR ((1)H/(13)C) chemical shifts. By using the substance produced in vitro, a fast, sensitive, specific and robust LC/MS/MS method for the simultaneous determination of DHEC and its major metabolite in human plasma was developed and validated. Bromocriptine was used as internal standard and limits of quantification for DHEC and 8'-OH-DHEC were 10 pg/ml and 20 pg/ml, respectively. Pharmacokinetic parameters were investigated on 12 male healthy volunteers to whom a single dose of 18 mg DHECM was administered in tablets (Iskevert). The peak of DHEC was 0.28 +/- 0.22 microg/l, the t(max) 0.46 +/- 0.26 h, the AUC(last) 0.39 +/- 0.41 microg/l.h and the terminal elimination half-life 3.50 +/- 2.27 h. The peak of 8'-OH-DHEC was 5.63 +/- 3.34 microg/l, the t(max) 1.04 +/- 0.66 h, the AUC(last) 13.36 +/- 5.82 microg/l.h and the terminal elimination half-life 3.90 +/- 1.07 h. Dosing of 18 mg DHECM was well tolerated, causing no adverse events.

Readily Available Carbohydrate‐Derived Imines and Amides as Chiral Ligands for Asymmetric Catalysis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Readily available carbohydrate-derived imines and amides as chiral ligands for asymmetric catalysis

An easy and diastereoseletive strategy for the synthesis of a new tetrahydrofuranic chiral aldehyde and a new tetrahydrofuranic acid derived from d-ribose is reported. These compounds have been shown to be useful starting materials for obtaining a never described before class of iminic- and amidic-carbohydrate-based chiral ligands, endowed with stability to purification on silica flash column and to storage, and characterised by the presence of a stereogenic centre α to the iminic or amidic bond. The first results obtained from the application of these chiral ligands in a model reaction, conjugate addition of Et 2Zn to cyclohexenone, are discussed.

ChemInform Abstract: Synthesis of 2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl Cyanide by Phase Transfer Reaction.

The potential antiviral, antibacterial, and antitumorous activities shown by C-nucleosides which have the carbon-carbon glycosidic bond have incited many attempts at the organic synthesis. 2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl cyanide (2) has been used as a potentially versatile intermediate for the synthesis of C-nucleoside derivatives. Ribofuranosyl cyanide 2 was prepared primarily from the reaction of 1-halo2,3,5-tri-O-benzoyl-β-D-ribofuranose which was prepared from the reaction of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-Dribofuranose (1) with mercuric cyanide. An efficient procedure for the synthesis of 2 was accomplished by the reaction of 1-O-acetate ribose 1 with cyanotrimethylsilane by Utimoto and Horiie. Our synthetic approach was to prepare 2,3,5-tri-O-benzoyl-β-D-ribofuranosyl cyanide (3) from the reaction of 1-O-acetyl-2,3,5-tri-O-benzoylβ-D-ribofuranose (1) with potassium cyanide instead of TMSCN (Scheme 1) because potassium cyanide is cheaper and more easily commercially available than TMS-CN. Potassium cyanide could provide the cyanide ion in the presence of 18-crown-6 in nonpolar solvent, which is called phase transfer reaction. After 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (1) was dissolved in dry methylene chloride, stannic chloride, 18-crown-6, and potassium cyanide were added to the solution and then the reaction mixture was heated under reflux for 24 h. Ribofuranose 1 was converted to 2,3,5-tri-O-benzoyl-β-D-ribofuranosyl cyanide (3) via 1,2-acyloxonium intermediate 2 which allowed a naked cyanide ion to attact on the β-face. From the H NMR spectrum of the obtained product 3, the anomeric proton at C1 was identified as a doublet at δ = 5.49 ppm (J12 = 4.0 Hz). For an adequate choice of reagent ratio, several reactions were carried out. The optimized condition was obtained from the reaction with a molar ratio of compound 1/KCN/stannic chloride/18-crown-6 = 1.0 : 2.0 : 1.5 : 0.15. 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose (4) was also tested under the phase transfer reaction condition to synthesize 2,3,5tri-O-acetyl-β-D-ribofuranosyl cyanide. The reaction of 4 in the similar manner as compound 3 by using stannic chloride as a Lewis acid gave no desired product. On the contrary, the reaction of compound 4 in the presence of BF3·OEt2 provided two different products, 5 and 6, separated by flash silica gel column chromatography (Scheme 2). H NMR spectrum of the isolated product 5 showed two anomeric protons at 5.92 and 5.95 (J12 = 3.9 and 4.2 Hz). The separated product 5, therefore, was expected to be a mixture of 3,5-di-O-acetyl-1,2-O-(1-endo-cyanoethylidene)α-D-ribofuranose and its exo-isomer which could be also obtained from the reaction of 1-O-acetate 4 with TMS-CN in the presence of BF3·OEt2. H NMR spectrum of product 6, identified as a 3,5-di-Oacetyl-1,2-O-(1-endo or exo-acetoxy-ethylidene)-α-D-ribofuranose, presented four methyl groups at the range of δ = 2.02-2.07 ppm and a C1 proton with a doublet at δ = 6.36 (J12 = 4.2 Hz), whereas the starting material 4 showed a C1 proton at δ = 6.09 ppm.

Separation of acid diterpenes of Copaifera cearensis huber ex ducke by flash chromatography using potassium hydroxide impregnated silica gel

Various acid diterpenes of Copaifera cearensis Huber ex Ducke were isolated from the crude extract by flash Silica Gel/Potassium Hydroxide Chromatography. The main components were identified by GC-MS analyses. Further fractionation by reversed phase (RP) semi-preparative HPLC allowed isolation and identification of minor components and provided additional spectral data of those compounds. It was possible to detect the presence of eleven acid diterpenes in addition to a sesquiterpene acid. In addition to potassium hydroxide deposition on the silica surface, the presence of Si-OK residues, resulting from a cation exchange process, was established.

Optimization of β- d-glucuronide synthesis using UDP-glucuronyl transferase

The optimization of alkyl and aryl β- d-glucuronide synthesis using UDP-glucuronyl transferase and UDP-glucuronic acid was undertaken to develop a synthetic method suitable for preparation of multimilligram quantities of glucuronides as analytical standards. The two most important factors in yield optimization appeared to be having the right amounts of enzyme and co-factor present in the reaction. The enzyme concentration showed a clear optimum. Too much enzyme could seriously reduce yields (by as much as 70% for 4-methyl phenol, for example). The optimal UDPGA concentration appeared to be approximately twice that of the substrate, whatever the latter may have been. Higher substrate concentrations (up to 8 m m) appeared to be beneficial, provided that excessive quantities of co-solvent were not required to solubilize the substrate. The choice of co-solvent was also important, acetonitrile and DMSO were considerably better than ethanol. A simple, effective means of isolating and purifying both the glucuronide and any unreacted starting material using C-18-derivatized flash silica gel has been demonstrated. Since this synthetic approach generally resulted in little loss of unreacted starting material, it should be well suited to substrates that are scarce and expensive, especially isotopically labeled compounds .

Two novel beta-carboline compounds from the Maillard reaction between xylose and tryptophan.

Two nonvolatile beta-carboline alkaloids were isolated from the Maillard reaction between xylose and tryptophan by solvent extraction and flash silica gel column chromatography. Their structures were elucidated by spectral methods (UV, MS, and NMR) as 1-(1,4-dihydroxybutyl)-beta-carboline and 1-(1,3, 4-trihydroxybutyl)-beta-carboline.

Soluble Synthetic Multiporphyrin Arrays. 1. Modular Design and Synthesis

A set of porphyrin building blocks has been developed for the construction of light-harvesting model compounds and related molecular photonic devices. The porphyrins are facially encumbered to enhance solubility in organic solvents, are employed in a defined metalation state (free base (Fb) or zinc chelate), and bear peripheral functional groups such as iodo or ethyne for joining the porphyrins via covalent bonds. The coupling of an iodophenylporphyrin and an ethynylphenylporphyrin via mild Pd-mediated reactions (2−4 mM of each porphyrin in toluene/triethylamine (5:1) with Pd2(dba)3 and AsPh3 at 35 °C for 2 h) yields the corresponding diphenylethyne-linked multiporphyrin array in 70−80% yield. The arrays are easily purified by a sequence of flash silica chromatography, preparative size exclusion chromatography, and gravity elution silica chromatography. The diphenylethyne linkers give a center-to-center separation of the porphyrins of ∼20 A. Model light-harvesting compounds are easily prepared using Zn an...

Flash chiral chromatography with cellulose tris(3,5-dimethylphenylcarbamate)-coated phases improved resolution of basic analytes

The preparative resolution of some chiral basic analytes on a flash chiral chromatography column packed with a cellulose carbamate-coated phase is described. Compared with separations obtained, on a column packed with 20% (w/w) cellulose tris(3,5-dimethylphenylcarbamate) coated onto underivatised flash silica (40–63 μm Davisil, 150 Å), a substantial improvement was observed when the support medium was changed to octadecylated silica (37–55 μm Bondapak ODS, 300 Å). The ODS phase is extremely effective for the preparative separation of basic analytes such as β-blockers and the optimum conditions can be readily established by the use of a related high-pressure liquid chromatography column containing the chiral carbamate coated onto Nucleosil-ODS (5 μm, 300 Å).

Diastereoselective synthesis of all four isomers of 3-(4-chlorophenyl) glutamic acid: identification of the isomers responsible for the potentiation of L-homocysteic acid-evoked depolarizations in neonatal rat motoneurons.

All four isomers of 3-(4-chlorophenyl)glutamic acid (5-8) were prepared by diastereoselective synthesis. Addition of (6S)-(+)-bis-lactim ether 15 to cis-4-chlorocinnamate 12 gave a mixture comprising mainly the (2R,3S)- and (2R,3R)-isomers 5 and 6, respectively (in a ratio of 56:40), while addition of (6R)-(-)-bis-lactim ether 16 to 4-chlorocinnamate 12 gave a mixture comprising mainly the (2S,3R)- and (2S,3S)-isomers 8 and 7, respectively (in a ratio of 56:42). The four stereoisomers (5-8) were therefore conveniently prepared by addition of either 3-lithio-(6S)- or -(6R)-bis-lactim ether (15 or 16, respectively) to 4-chlorocinnamate 12 and separation of the resultant mixtures of diastereoisomers (23-26) by flash silica gel chromatography. The absolute configurations of 6 and 7 were confirmed by X-ray crystallography. Both the (2S,3S)- and (2S,3R)-isomers (7 and 8, respectively) at a concentration of 100 microM significantly potentiated depolarizations induced by 10 microM L-homocysteic acid (L-HCA) (% control +/- sem: 130.4 +/- 3.6, n = 20 and 114.5 +/- 2.4, n = 11, respectively) while the (2R,3S)-isomer 5 significantly reduced L-HCA response amplitude (94.2 +/- 1.4, n = 9) and the (2R,3R)-isomer 6 was inactive. Experiments designed to compare the agonist-potentiating actions of 7 and 8 in the neonatal rat spinal cord with L-trans-pyrrolidine-2,4-dicarboxylic acid, the well-known L-Glu uptake inhibitor, provided additional evidence for the selective enhancement of depolarizations due to L-HCA and not those due to L-Glu. This selective action supports the existence of multiple excitatory amino acid uptake sites.

Ortho- and meta-(Diethynylcyclopentadienyl)tricarbonylmanganese: Building Blocks toward the Construction of Metal Fragment Supported Fullerenynes?

Starting from [(trimethylsilyl)ethynyl]cymantrene (1), the two isomeric 1,2- and 1,3-[bis-(trimethylsilyl)ethynyl] cymantrenes (3a, 4a ) were prepared by a deprotonation/iodination/coupling sequence. The coupling reaction was performed on the mixed ortho-/meta-iodo[(trimethylsilyl)ethynyl]cymantrenes (2) by using Stille methodology (tin alkynes and PdCl{sub 2}(CH{sub 3}CN){sub 2} as catalyst in ethyl ether/DMF). Separation of the isomers (3a, 4a) is achieved by chromatography over flash silica gel. The ortho-diethynylcymantrene 3a is isolated in 42%, while the corresponding meta compound 4a is formed in 26%. If the deprotonation reaction of 1 in pentane/TMEDA is performed under carefully controlled conditions (-78{degree}C), the formation of only ortho 2 is observed in 67% yield. Coupling with trimethylstannyl(trimethylsilyl)acetylene furnished 3a in 57%. Crystal structures have been carried out for complexes 3a. Removal of the alkyne-bound trimethylsilyl groups is achieved in yields of >80% by treatment of 3a and 4a with potassium carbonate in methanol. The parent diethynylcymantrenes (3b, 4b) are stable compounds. Treatment of 1,2-diethynylcymantrene 3b under Hay conditions gives coupled material, a polymer poly[(ortho-cymantrene)butadiynylene] (6) in 68% yield (M{sub n} = 9300). 15 refs., 4 figs., 1 tab.

Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae.

A radiolabeled component of a membrane extract of Rhizobium meliloti 2011 cells grown in the presence of 35S-labeled sulfate was isolated by silica flash chromatography and purified by high performance liquid chromatography (HPLC). Based on 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analyses, the structure of the compound was determined to be sulfoquinovosyl diacylglycerol (SQDG). NMR analyses indicated substantial heterogeneity in the fatty acid composition and that an important group was the cyclopropyl fatty acids. This first report of the occurrence of SQDG outside of the plant kingdom, photosynthetic bacteria or diatoms deserves special attention as, in this case, the bacterium is one that can fix nitrogen in symbiosis with plants. The origins of the bacterium's ability to synthesize this class of membrane lipids is an important question. Membrane extracts of other strains of the family Rhizobiaceae were screened for the presence of SQDG. The occurrence of SQDG in the symbiotic organisms was confirmed, while no SQDG was detected in either the Agrobacterium tumefaciens or the Escherichia coli strains tested. The current function of these lipids in symbiosis and the commonality of the ability of bacteria that function as plant symbionts to synthesize such molecules are all germane to studies of the Rhizobium/legume symbiosis.

Synthesis of Casein-Related Peptides and Phosphopeptides. VI. The Efficient Global 'Phosphite-Triester' Phosphorylation of Multiple-Serine-Containing Peptides by Using Dibenzyl N,N-Diethylphosphoramidite

The peptide Ac-Ser(P)- NHMe and the multiple-Ser(P)-containing peptides Ac-Ser(P)-Ser(P)- NHMe and Ac-Ser(P)-Ser(P)-Ser(P)- NHMe were prepared in good yield by a global phosphorylation approach which employed benzyl phosphate protection. The three serine-containing peptides were phosphorylated by the use of dibenzyl N,N-diethylphosphoramidite/1H-tetrazole followed by in situ oxidation of the resultant dibenzyl phosphite-triesters with m- chloroperoxybenzoic acid. The protected Ser(PO3Bzl2)-containing peptides were purified by C18 reverse-phase silica flash column chromatography and the benzyl phosphate groups were cleaved from the protected Ser(PO3Bzl2) peptides by palladium- catalysed hydrogenolysis.