Massively parallel display of genomic DNA fragments by rolling-circle amplification and strand displacement amplification on chip

Massively parallel genomic DNA fragments display on chip plays a key role in the new generation DNA sequencing. Here, we developed a new technology to display the parallel genomic DNA fragment massively based on two-step reaction with Ф29 DNA polymerase. The genomic DNA fragments were firstly amplified by rolling-circle amplification (RCA) reaction in liquid phase, and then amplified further on the chip by the strand displacement of Ф29 DNA polymerase. In our experiments, through DNA colonies produced by two-step amplification reaction T7 genomic DNA fragments are displayed massively and parallely on the chip, which has been verified through hybridizing the probe labeled with fluorescence or extension reaction with fluorescent-dNTP. The significant difference of fluourescence signals between background and displayed DNA fragments could be obtained. Our results show that the method has good reproducibility in experiments, which may be hopeful to serve the high-throughput sequencing.     

Stereoselective synthesis of the cytotoxic macrolide FD-891

[reaction: see text] A total synthesis of the naturally occurring, cytotoxic macrolide FD-891 is described. Three fragments were first stereoselectively constructed using mainly asymmetric aldol and allylation reactions. The complete framework was then assembled using two Julia-Kocienski olefinations to connect the three fragments and a Yamaguchi reaction to close the macrolactone ring.     

Total synthesis of (-)-mucocin

[reaction: see text] An enantioselective total synthesis of (-)-mucocin has been completed. A combination of asymmetric glycolate aldol additions and ring closing metathesis reactions were exploited to construct the C18-C34 and C7-C17 fragments. A selective cross-metathesis reaction was employed as the key step to couple two complex fragments.     

Cu-mediated Stille reactions of sterically congested fragments: towards the total synthesis of zoanthamine

A study on the Stille reaction of alkenyl iodides and stannanes with structural resemblance to retrosynthetic fragments of a projected total synthesis of the marine alkaloid zoanthamine was carried out. A range of reaction conditions was examined, and a protocol developed by Corey utilizing excess copper(I) chloride and lithium chloride was found to be most efficient. The methodology was successfully applied to join two major fragments of the zoanthamine skeleton.     

Multi C−H Functionalization Reactions of Carbazole Heterocycles via Gold-Catalyzed Carbene Transfer Reactions

Herein we describe a multiple C−H functionalization reaction of carbazole heterocycles with diazoalkanes. We show that gold catalysts play a distinct role in enabling a multiple C−H functionalization reaction to introduce up to six carbene fragments onto molecules containing multiple carbazole units or to link multiple carbazole units into a single molecule. A one-pot stepwise approach enables the introduction of two different carbene fragments to allow orthogonal deprotection and straightforward derivatization.     

Protein‐Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background‐Free Amidation Reaction

Protein‐templated reactions enable the target‐guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non‐catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background‐free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein–inhibitor complex was elucidated.     

Self-assembly of a group I intron from inactive oligonucleotide fragments

The Azoarcus group I ribozyme was broken into four fragments, 39-63 nucleotides long, that can self-assemble into covalently contiguous ribozymes via RNA-directed recombination events. The fragments have no activity individually yet can cooperate through base pairing and tertiary interactions to produce stable trans complexes at 48 degrees C. These complexes can then catalyze a sequence of energy-neutral recombination reactions utilizing other oligomers as substrates, assembling covalent versions of the ribozyme. Up to 17% of the original fragments are converted into approximately 200 nucleotide products in 8 hr. Assembly occurs primarily by only one of many possible pathways, and the reaction is driven in the correct and forward direction by the burial of key base-pairing regions in stems after recombination. Autocatalysis, and hence self-replication, is inferred by a reaction rate increase upon doping the reaction with full-length RNA.     

Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.     

Quantitation of PCR Products by Capillary Electrophoresis in a Single Run

IntroductionItisnecessarytoevaluatetheamountofDNAfragmentinsuchcasesasgeneexpressioninvestigati0n.HybridizationwithradioactivitytaggedoligonucleotidesfollowedbyphotograPhyanddensitometricscanisoftenemployedtomeettheneedsofsuchrequirementS,whichislaborintensive,timec0nsumingandunautomated-CaPillaryelectrophoresis(CE)tendst0beastrongtoolintheanalysis0fDNAfragmentSwiththeadvantagesofhighsensitivity,highres0luti0nandautomation.QuantitativeanalysisofDNAfragmentshasbeendonebysomeauthorsusingC…     

Femtosecond multichannel photodissociation dynamics of CH3I from the A band by velocity map imaging

The reaction times of several well-defined channels of the C-I bond rupture of methyl iodide from the A band, which involves nonadiabatic dynamics yielding ground state I(2P3/2) and spin-orbit excited I*(2P1/2) and ground and vibrationally excited CH3 fragments, have been measured by a combination of a femtosecond laser pump-probe scheme and velocity map imaging techniques using resonant detection of ground state CH3 fragments. The reaction times found for the different channels studied are directly related with the nonadiabatic nature of this multidimensional photodissociation reaction.     

Stereoselective synthesis of (+)-SCH 351448: a unique ligand system for sodium, calcium, and other cations

(+)-SCH 351448 (Na+ salt A) was synthesized employing ring-closing olefin metathesis reaction of an open diene diester intermediate for construction of the 28-membered macrodiolide structure. The open diene diester was prepared from the monomeric hydroxy carboxylic acid and two different olefin fragments. The monomeric hydroxy acid was synthesized via Julia-Julia coupling reaction of intermediates derived from the same olefinic fragments. Oxane units in these fragments were prepared by radical cyclization reactions of beta-alkoxyacrylates. Analogous SCH 351448 salts incorporating other mono- and divalent cations may be prepared. Under acidic conditions, SCH 351448 (Na+ salt A) was the most stable complex, but SCH 351448 (Ca2+ salt) and (Na+ salt B) appear to be physiologically important species.     

Curing of diglycidylamine-based epoxides with amines: Kinetic model and simulation of structure development

A complex mechanism of the reaction of diglycidylamine (DGA)-based epoxides with primary amines is described and the kinetic model is developed. The reaction mechanism involves the addition of amine, etherification, and also formation of small cycles and anionic homopolymerization of the epoxide compound including transfer and termination. The polymer structure was theoretically described by distribution of structural fragments determined using the kinetic model. Simulation of the structure evolution during the DGA-aniline reaction at varying molar ratios of reagents and dilution of the system was performed. The effect of the reaction conditions on the distribution of structural fragments, such as branching units and cyclic structures, was determined. © 1995 John Wiley & Sons, Inc.     

Recent Progress in the Construction of Natural De-O-Sulfonated Sulfonium Sugars with Antidiabetic Activities

A group of sulfonium salts equipped with a polyhydroxylated side-chain structure have been isolated and identified as potent α-glycosidase inhibitors. Consequently, they have become an attractive target in diverse research disciplines, including organic synthesis, drug discovery, and chemical biology. To this end, the development of practical and effective synthetic strategies, especially for more bioactive de-O-sulfonated sulfonium salts, is a significant research area in organic synthesis. An ideal synthetic methodology should provide easily accessible intermediates with high chemical stability for the key coupling reaction to diastereoselectively construct the sulfonium cation center. This minireview summarizes recently developed strategies applied in the construction of natural de-O-sulfonated sulfonium sugars: 1) acid-catalyzed de-O-sulfonation of sulfonium sulfate inner salts, 2) a coupling reaction between side-chain fragments containing leaving groups and a thiosugar, 3) a coupling reaction between side-chain fragments containing epoxide structures and a thiosugar, and 4) a two-step sequential S_N2 nucleophilic substitution between side-chain fragments containing thiol groups and a diiodide derivative.     

The oxidation of cinnamaldehyde with alkaline hydrogen peroxide

The oxidation of cinnamaldehyde (3-phenyl-2-propenal) by alkaline peroxide results in epoxidation of the double bond to form cinnamaldehyde epoxide (3-phenyl-2,3-epoxy-propanal) which undergoes further reaction by ring opening and side chain cleavage to yield benzaldehyde and acidic fragments. The reactions are first-order in the organic substrates and perhydroxyl anion and second-order overall. In the presence of alkali alone, two further reactions take place in which cinnamaldehyde and cinnamaldehyde epoxide side chains are cleaved by reaction with hydroxide ion to form benzaldehyde and side chain fragments. These reactions are first-order in the organic substrates and hydroxide ion and second-order overall. Increasing solvent polarity accelerates the rates of reaction and reaction mechanisms have been proposed to describe the observed kinetic behavior. The stereoselectivity of the epoxidation reaction has been examined in terms of an existing model for epoxidation of α, β-unsaturated ketones by alkaline peroxide. © 1993 John Wiley & Sons, Inc.     

Dual kinetically controlled native chemical ligation using a combination of sulfanylproline and sulfanylethylanilide peptide

Dual kinetically controlled native chemical ligation using a newly developed sulfanylproline-mediated reaction in combination with an N-sulfanylethylanilide peptide was successfully applied to a previously unreported sequential coupling of peptide fragments added simultaneously to the reaction.     

Electron impact mass spectra of bifunctional steroids. The interaction between ionic and neutral fragments derived from the same parent ion

Apparent ‘long distance’ H transfers in the fragmentation of bifunctional steroids under electron impact result from an ‘intramolecular’ reaction between ionic and neutral fragments. These transfers are made possible by the independent rotation of the fragments and result from a compromise between their cross-section and the velocity of separation of the fragments. They are compared with other processes previously described as involving the formation of a ‘complex’ between ionic and neutral fragments derived from the same parent ion.     

Synthesis and Properties of Copolymers Having Polyconjugated Blocks in the Macromolecules. III Thermal Behavior of Polyisoprene-Containing Polyazophenylene Fragments in the Main Chains

The thermal and thermooxidative stability of polyisoprene containing polyazophenylene fragments of different composition was investigated. Elevated inhibiting activity of polyazophenylene -methane fragments was found to be caused by the intramolecular synergism due to the combination of the reaction of peroxy radicals with mobile hydrogen atoms of methylene bridge groups and decomposition of hydroperoxides under the influence of polyconjugated blocks.     

Solid products from thermal decomposition of polyethylene terephthalate: Investigation by CP/MAS 13C-NMR and fourier transform-IR spectroscopy

The solid reaction products from pyrolysis of polyethylene terephthalate in the presence and absence of red phosphorus were characterized by CP/MAS ¹³C-NMR, FR-IR, and MAS ³¹P-NMR spectroscopy. Over the temperature range of 300–400°C, polyethylene terephthalate was converted in a sealed vial to a highly crosslinked polymer of terephthalic acid. Pyrolysis in the presence of red phosphorus, which functions as a flame retardant by increasing the amount of char, yielded an intractible polyaromatic phosphate ester. After thermal cleavage of polyethylene terephthalate with formation of free carboxyl and vinyl ester groups, there are two competing reaction pathways. The smaller molecular weight fragments may enter the vapor phase where they undergo further degradation primarily to CO₂, CO, and acetaldehyde, as described by others. However, if volatilization of the oligomeric fragments is inhibited, an alternate reaction pathway gives rise to the formation of highly crosslinked char. Red phosphorus decreases the volatility of the oligomeric fragments by converting them to phosphates and thereby enhances char formation.     

3 + 2] Annulation of allylic silanes in acyclic stereocontrol: total synthesis of (9S)-dihydroerythronolide A

The [3 + 2] annulation reactions of allylic silanes can be utilized to achieve acyclic stereocontrol. This method was employed as a key step in an enantioselective total synthesis of (9S)-dihydroerythronolide A. The key annulation reaction served to establish most of the stereochemistry of the target, including the two tetrasubstituted carbon stereocenters. The symmetry of the target molecule allowed it to be disconnected into two equally sized fragments, both of which were generated from the same annulation reaction. The two fragments were coupled using a tin(II)-mediated chelation-controlled aldol reaction of an alpha-benzyloxy ethyl ketone. This convergent total synthesis of (9S)-dihydroerythronolide A was accomplished with the longest linear sequence of 29 steps and in 5.4% overall yield.     

DNA-templated dimerization of hairpin polyamides

Double-helical DNA accelerates the rate of ligation of two six-ring hairpin polyamides which bind adjacent sites in the minor groove via a 1,3-dipolar cycloaddition to form a tandem dimer. The rate of the templated reaction is dependent on DNA sequence as well as on the distance between the hairpin-binding sites. The tandem dimer product of the DNA-templated reaction has improved binding properties with respect to the smaller hairpin fragments. Since cell and nuclear uptake of DNA-binding polyamides will likely be dependent on size, this is a minimum first step toward the design of self-assembling small gene-regulating fragments to produce molecules of increasing complexity with more specific genomic targeting capabilities.