Standard Reaction Conditions Research Articles

Direct and Potent Regulation of γ-Secretase by Its Lipid Microenvironment

gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.

Carbonyl Propargylation from the Alcohol or Aldehyde Oxidation Level Employing 1,3‐Enynes as Surrogates to Preformed Allenylmetal Reagents: A Ruthenium‐Catalyzed CC Bond‐Forming Transfer Hydrogenation

Carbonyl Propargylation from the Alcohol or Aldehyde Oxidation Level Employing 1,3‐Enynes as Surrogates to Preformed Allenylmetal Reagents: A Ruthenium‐Catalyzed CC Bond‐Forming Transfer Hydrogenation

SPYING ON CATALYSTS

FOR THE FIRST TIME, researchers have applied infrared microspectroscopy to monitor catalytic reactions as they occur within the pores of zeolite crystals ( Angew. Chem. Int. Ed. , DOI: 10.1002/anie.200705562). The technique provides scientists with a new procedure for probing the detailed relationship between a catalyst’s structure and its function. The method also offers a means for elucidating reaction pathways mediated by industrially relevant catalysts such as zeolites. Like law enforcement agents who search for ways to spy on criminals so they can catch the perpetrators “in the act,” chemists try to develop methods to monitor catalysts under typical conditions and catch the catalysts promoting chemical reactions. Developing analytical methods that are compatible with elevated temperatures and pressures and other standard catalysis reaction conditions is challenging. Yet a few in situ microscopy and spectroscopy methods that can scrutinize the internal surfaces of porous catalyst materials during the ...

First heterogeneously palladium-catalysed fully selective C3-arylation of free NH-indoles

A simple heterogeneously palladium-catalysed procedure for the selective C3-arylation of indoles is reported. Under relatively standard reaction conditions (Pd-catalyst, K 2CO 3, dioxane, reflux), using only 1 mol % [Pd(NH 3) 4]/NaY as the catalyst, indoles substituted or not at position 2 gave up to 92% conversion (i.e., 85% isolated yield) towards the expected C3-arylated indole.

Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials

The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study.

Iron‐mediated degradation kinetics of substituted dispiro‐1,2,4‐trioxolane antimalarials

The iron-mediated reactivity of various dispiro-1,2,4-trioxolanes was determined by automated kinetic analysis under standard reaction conditions. The active antimalarial compounds underwent peroxide bond cleavage by Fe(II) resulting in products indicative of carbon-centered radical formation. The rate of reaction was heavily influenced by the presence of spiro-substituted adamantane and cyclohexane rings, and was also significantly affected by cyclohexane ring substitution. Steric hindrance around the peroxide oxygen atoms appeared to be the major determinant of reaction rate, however polar substituents also affected reactivity by an independent mechanism. A wide range of reaction rates was observed within this class of peroxide antimalarials, however iron-mediated reactivity did not directly correlate with in vitro antimalarial activity.

Synthesis of a 5-Spirocyclopropyl Deoxyrhamnojirimycin as a Constrained Naringinase Inhibitor

: A spirocyclopropyl analogue of deoxyrhamnojirimycin has been synthesized in 11 steps and 20% overall yield from L-arabinose. The key step of the reaction path involves titanium-mediated aminocyclopropanation of an L-arabinononitrile derivative. Improvements of the standard reaction conditions were investigated. The use of methyltitanium triisopropoxide as the titanium source allowed the reaction to take place at room temperature and afforded the corresponding cyclopropylamine in 80% yield.

Hydroformylation of naphthas with a rhodium complex in biphasic medium

The chlorocarbonyl bis-[butylphenyl (meta-sulfonate-phenyl)phosphine] rhodium (I) complex shows catalytic hydroformylation activity in toluene/water biphasic medium for 1-hexene, cyclohexene, 2,3-dimethyl-2-butene and 2-methyl-2-pentene, their binary mixtures and a real Venezuelan naphtha, under standardized reaction conditions (1000 psi of syngas (1:1 H2/CO), 100°C, substrate/catalyst molar ratio (600:1) and 4 h reaction time), obtaining high percent conversion to oxygenated products.

Cy2NH·HX-Promoted Cyclizations of o-(Alk-1-ynyl)benzoates and (Z)-Alk-2-en-4-ynoate with Copper Halides to Synthesize Isocoumarins and α-Pyrone

Cy 2 NH·HX was found to improve the cyclization reactions of o-(alk-1 -ynyl)benzoates and (Z)-alk-2-en-4-ynoate with CuX 2 (X = Cl, Br) to synthesize the corresponding 4-haloisocoumarins and 5-bromo-2-pyrone, respectively. In the presence of two equivalents of CuCl 2 , cyclization of methyl 2-(2-phenylethynyl)benzoate was conducted smoothly to afford the corresponding desired product in 83% yield after 24 hours, whereas the yield was enhanced to 91% in eight hours when 0.1 equivalent of Cy 2 NH·HCl was added. Under the standard reaction conditions, a variety of o-(alk-1 -ynyl)benzoates and (Z)-alk-2-en-4-ynoate underwent the cyclization reaction with CUX 2 to provide the corresponding 4-haloisocoumarins and 5-bromo-2-pyrone, respectively, in moderate to excellent yields.

Regioselective Trapping of Terminal Di-, Tri-, and Tetraynes with Benzyl Azide

[Structure: see text] The reaction of benzyl azide with terminal di-, tri-, and tetraynes appended with a range of functional groups has been explored. Standard reaction conditions for BnN3 catalyzed by CuSO4.5H2O gave alkynyl, butadiynyl, and hexatriynyl triazoles in moderate to good yields. The reaction proceeds regioselectively as determined by the X-ray crystallographic analysis of three derivatives (1c, 1d, and 3c), and no evidence of multiple azide addition to the polyyne framework is observed.

Revisiting the Reaction Between Diaminomaleonitrile and Aromatic Aldehydes: a Green Chemistry Approach

The reaction between diaminomaleonitrile (DAMN) and aldehydes and the resulting monoimines are well known. Since the standard reaction conditions involve the use of toxic solvents (typically methanol), we have sought to apply green chemistry principles to this reaction by either using water as the solvent without any catalysts or employing "solvent-free" conditions. The monoimines derived from DAMN are of interest as precursors for obtaining different heterocyclic systems and linear polymers. The methodologies used have significant advantages with regards to cost and environmental considerations.

Rapid Expression of Functional Genomic Libraries

Genomic-scale analysis of protein function is currently limited by the ability to rapidly express the enormous diversity of protein targets in their active form. We describe a method to construct transcriptionally active expression templates (ETs) in parallel using a single PCR step wherein the overlap-extension reaction for addition of transcription regulatory elements is separated from the amplification of the full-length product by using a GC-rich single primer. Over 90% of 55 diverse genomic targets were extended with T7 regulatory elements to form ETs in high yield and purity. The unpurified ETs directed protein expression using a cell-free protein synthesis (CFPS) system supplemented with cofactors and metal ions to activate a variety of enzymes. Higher activities were obtained in the modified CFPS reactions compared to standard reaction conditions. Protein purification was avoided because the expressed enzyme activity was significantly greater than the background activity associated with the cell extract. These improvements in the parallel synthesis of linear ETs combined with enhanced in vitro enzyme activation help to make CFPS systems more attractive platforms for high-throughput evaluation of protein function.

Identification of Separate Structural Features That Affect Rate and Cation Concentration Dependence of Self-Cleavage by the Neurospora VS Ribozyme

The cleavage site of the Neurospora VS ribozyme is located in an internal loop in a hairpin called stem-loop I. Stem-loop I undergoes a cation-dependent structural change to adopt a conformation, termed shifted, that is required for activity. Using site-directed mutagenesis and kinetic analyses, we show here that the insertion of a single-stranded linker between stem-loop I and the rest of the ribozyme increases the observed self-cleavage rate constant by 2 orders of magnitude without affecting the Mg(2+) requirement of the reaction. A distinct set of mutations that favors the formation of the shifted conformation of stem-loop I decreases the Mg(2+) requirement by an order of magnitude with little or no effect on the observed cleavage rate under standard reaction conditions. Similar trends were seen in reactions that contained Li(+) instead of Mg(2+). Mutants with lower ionic requirements also exhibited increased thermostability, providing evidence that the shifted conformation of stem-loop I favors the formation of the active conformation of the RNA. In natural, multimeric VS RNA, where a given ribozyme core is flanked by one copy of stem-loop I immediately upstream and another copy 0.7 kb downstream, cleavage at the downstream site is strongly preferred, providing evidence that separation of stem-loop I from the ribozyme core reflects the naturally evolved organization of the RNA.

Investigation of acetyl migrations in furanosides

Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside) derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

In/Co-ferrierite: A highly active catalyst for the CH4-SCR NO process under presence of steam

A series of monometallic (In, Co) and bimetallic (In/Co and Co/In) catalysts supported on ferrierite type zeolite were tested in the selective catalytic reduction of nitric oxide, in the presence of methane and excess of oxygen. All the catalysts were prepared by contact-induced ion exchange. A strong synergistic effect was observed for the catalysts containing both indium and cobalt, in comparison with the monometallic samples. For these bimetallic catalysts, a very high selectivity to nitrogen and rather efficient fuel economy were observed under the standard reaction conditions (NO=1000ppm, CH4=2000ppm, O2=4%, H2O=2500ppm). Moreover, the In/Co-ferrierite catalyst displays significant stability under a prolonged test (∼200h) in the presence of 2.5% of steam: the activity dropped rather moderately but was completely restored if steam supply was cut off, while the selectivity of the reaction was not affected in the whole temperature range scanned (300–500°C). The study suggests that a redox-type promotional effect of Co species on NO oxidation may be responsible of the strong synergistic effect detected in bimetallic In–Co formulations.

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12 h at 50 °C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.

Rolling circle amplification-RACE: a method for simultaneous isolation of 5′ and 3′ cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize

Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification

In this review I will describe the development of a technique that enables genotyping of individual DNA molecules in the context of morphologically preserved fixed cells, from the fundamental concept published in 1994 to the present status. The review describes enzyme-assisted histochemistry approaches to achieve highly specific molecular identification reactions coupled to efficient signal amplification. The primary molecular identification is accomplished through circularization of oligonucleotide probes, called padlock probes. The circularization reaction is catalyzed by a DNA ligase, which provides robust distinction between single-nucleotide variants under standard reaction conditions. To generate a detectable signal from individual circularized probe molecules, a DNA polymerase is added that replicates probe circles, generating a long tandem-repeated DNA product, easily visualized using a standard epi-fluorescence microscope. Individual signals are recorded as bright dots, providing digital information about the abundance of specific sequences and opportunities for simultaneous detection of several targets using spectral multiplexing. The importance of strictly target-dependent signal amplification will be discussed.

Thiazolium-Catalyzed Additions of Acylsilanes: A General Strategy for Acyl Anion Addition Reactions

A strategy utilizing N-heterocyclic carbenes (NHCs) derived from thiazolium salts has been developed for the generation of carbonyl anions from acylsilanes. Synthetically useful 1,4-diketones and N-phosphinoyl-alpha-aminoketones have been prepared in good to excellent yields via NHC-catalyzed additions of acylsilanes to the corresponding alpha,beta-unsaturated systems and N-phosphinoylimines. These organocatalytic reactions are air- and water-tolerant methods to execute robust carbonyl anion addition reactions. Additionally, polysubstituted aromatic furans and pyrroles have been efficiently synthesized in a one-pot process using this carbonyl anion methodology. The addition of alcohols to the reaction renders the process catalytic in thiazolium salt. In an effort to synthesize a potential intermediate along the proposed reaction pathway, silylated thiazolium carbinols have been identified to provide good yields of carbonyl anion addition products when subjected to the standard reaction conditions in the presence of suitable electrophiles.

Lipase-mediated Transformation of Vegetable Oils into Biodiesel using Propan-2-ol as Acyl Acceptor

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 degrees C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7(th) cycle when methanol was used as an acyl acceptor, under standard reaction conditions.