Allied Corporation

Allied Chemical Corporation

Genetics Institute, Inc.

Steven E Diamond

Frank Mares

Benjamin S Tovrog

Calvin P Vary

Neil M Wolfman

Mary Collins

Jon I Williams

Edward F Fritsch

Marian S Ellwood

Andrew Szalkiewicz

Deborah A Muccigrosso

Joseph G Brewen

Jeffrey P Solar

Francis J Regina

Reginald T Tang

James E Galle

Astrid P Koudelka

Jeffrey Corsi

A probe polynucleotide binds to a target nucleotide sequence in the nucleic acid of a biological sample, and then is enzymatically extended in the 3'-direction with a mixture of nucleoside triphosphates including at least one nucleoside triphosphate that has been detectably labeled. After separating extended hybrid from unreacted nucleoside triphosphates, detectably-modified nucleotides which have been incorporated are determined. In some forms, the 3'-terminal nucleotide of the probe polynucleotide is selected to form a matched pair with some sample strands, but a mismatched pair with other sample strands. In such cases, if the primer dependent enzyme used for extension is one lacking 3'-exonuclease activity, then only those hybrids forming such a matched pair will be extended and subsequently determined.

Method and kit for polynucleotide assay including primer-dependant DNA

A reagent complex is disclosed containing (1) a probe polynucleotide having a target binding region complementary to a target nucleotide sequence and (2) a signal strand polynucleotide bound by base pairing to a portion of the target binding region. The signal strand is displaced from the reagent complex by the target nucleotide sequence and separated from intact reagent complexes. A digestable ribonucleotide segment of the displaced signal strand, such as a 3' , terminal segment, is digested to ribonucleotide phosphates, which are phosphorylated to ATP. The ribonucleotide phosphates or ATP are detected.

Diagnostic reagent, kit and method employing polynucleotide

A diagnostic reagent is disclosed containing a complex of a probe polynucleotide (P) bound via purine/pyrimidine hydrogen bonding to a labeled polynucleotide (L). The probe (P) contains a target binding region (TBR) capable of binding to a target nucleotide sequence (G) of a biological sample. A method is disclosed in which contact with a sample containing the target nucleotide sequence (G) causes binding, initially between G and a single-stranded portion (IBR) of the target binding region (TBR). Thereafter the labeled polynucleotide (L) is displaced from the complex by branch migration of (G) into the (P)/(L) binding region. A volume excluding polymeric agent such as poly(ethylene oxide) (PEO or PEG) or other polyethers enhances the rate of appearance of displaced labeled polynucleotide. Determination of displaced labeled polynucleotide (L) gives a value which is a function of the presence and concentration of target nucleotide sequence (G) in the sample.

Displacement polynucleotide assay employing polyether and diagnostic kit

A diagnostic reagent is disclosed containing a complex of a probe polynucleotide (P) bound via purine/pyrimidine hydrogen bonding to a labeled polynucleotide (L). The probe (P) contains a target binding region (TBR) capable of binding to a target nucleotide sequence (G) of a biological sample. A method is disclosed in which contact with a sample containing the target nucleotide sequence (G) causes binding, initially between G and a single-stranded portion (IBR) of the target binding region (TBR). Thereafter the labeled polynucleotide (L) is displaced from the complex by branch migration of (G) into the (P)/(L) binding region. Determination of displaced labeled polynucleotide (L) gives a value which is a function of the presence and concentration of target nucleotide sequence (G) in the sample.

Displacement polynucleotide assay method and polynucleotide complex

RNA such as messenger RNA is digested to nucleotide phosphates including AMP or ADP. The ATP or a byproduct of the phosphorylation, e.g., pyruvate, is detected. Exemplary enzymes used (with appropriate co-reactants and co-factors) are: (1) polynucleotide phosphorylase, pyruvate kinase and luciferase, or (2) phosphodiesterase (or RNase), myokinase, pyruvate kinase and luciferase. The phosphorylation to ATP (e.g., with pyruvate kinase) is preferably coupled with the previous (reversible) enzymatic step.

Method and kit for detecting polyriboadenosine segments and messenger RNA

At the conclusion of a selective binding assay (e.g., immunological or nucleic acid), an enzyme is present in modulated concentration and/or activity to moderate a chemical reaction such as the cleavage of o-nitrophenyl-.beta.-D-galactopyranoside. After a controlled period, the enzymatic product is transferred to the gas phase and concentrated relative to other components in the enzymatic reaction mixture, such as by extraction into ethyl ether, injection into a gas chromatography column and detection by flame ionization or electron capture. Kits for such assays are also disclosed.

Binding assay with amplified read-out and gas-phase detection

A process for the oxidation of olefinically unsaturated substrates of the formula R--CH.dbd.CH-R' wherein R and R' are independently selected from members of the group consisting of H, alkyl, alkenyl, cycloalkyl, aryl and halo-, cyano- and ether-substituted alkyl, alkenyl, cycloalkyl and aryl which comprises reacting in the liquid phase said substrate with a rhodium nitro complex of the formula L.sub.n RhNO.sub.2, wherein n is 4 or 5 and wherein L.sub.n represents any combination of monodentate, bidentate, tridentate and tetradentate ligands such as to provide four or five bonding sites, for a time sufficient to produce an oxidized product of the formula RCH.sub.2 C(O)R' and a reduced product of the formula L.sub.n RhNO is disclosed. A preferred embodiment of the process wherein the rhodium nitro complex contains at least one ligand replaceable by olefin to be oxidized and operates to produce the oxidized product RCH.sub.2 C(O)R' in the absence of other Group VIII metals as co-catalyst is also disclosed.

Oxidation of olefins using rhodium nitro complex

A dinitrile is hydrogenated to an omega-aminonitrile with hydrogen with a supported highly dispersed rhodium catalyst and only ammonia present. The catalyst is prepared by hydrolyzing a rhodium(III) halide or nitrate with strong aqueous base at elevated temperatures, drying the supported rhodium hydroxide at elevated temperatures and intimately contacting with hydrogen the dried product with hydrogen at 260.degree.-360.degree. C. High conversions, selectivity to aminonitrile and long catalyst service times and catalyst recyclability are achieved in the absence of an aprotic solvent.

Selective hydrogenation of aliphatic dinitriles to omega-aminonitriles

A dinitrile is hydrogenated to an omega-aminonitrile with hydrogen in an aprotic solvent with a supported rhodium catalyst and ammonia present. The catalyst is prepared by hydrolyzing a rhodium (III) halide with strong aqueous base at elevated temperatures, drying the supported rhodium hydroxide at elevated temperatures and hydrogenating the dried product at elevated temperatures. High conversions, selectivity to aminonitrile and catalyst recyclability are achieved.

Selective hydrogenation of dinitrile to omega-aminonitrile and supported

A dinitrile is reacted with hydrogen in the presence of a solvent and a catalyst to form an omega-aminonitrile such as epsilon-aminocapronitrile at high selectivity. The catalyst is a rhodium complex of the formula (RR'Rh)A reacted with a strong base such as sodium hydroxide. R is derived from a diene such as 1,5-hexadiene. R' is a heterocyclic containing an aromatic ring nitrogen. A is a complex monovalent anion such as hexafluorophosphate.

Selective hydrogenation of dinitrile to omega-aminonitrile catalyzed by

An olefinically unsaturated substrate is reacted with a transition metal nitro complex, having the transition metal bonded to the nitrogen of the nitro radical, in the presence of a thallium(III) compound as cocatalyst to cause the production of oxirane products, rather than aldehydes or ketones. The process is applicable to the production of ethylene oxide, propylene oxide, epichlorohydrin, epoxidized fatty acids and oxiranes from various other internal and terminal olefins.

Producing epoxide from olefin using metal nitro complexes

Hydrogen peroxide production by oxidation of a 2-alkyl-9,10-dihydroxyanthracene, especially the 2-(t-butyl) compound, and catalytic hydrogenation of the resulting 2-alkylanthraquinone back to the dihydroxy compound, using a water-insoluble (bipyridyl) rhodium complex as hydrogenation catalyst. Especially such process carried continuously in a cell in which an organic phase is separated by a porous membrane from an aqueous phase, and the anthraquinone is hydrogenated on the organic side of the cell and hydrogen peroxide is obtained from the aqueous phase. Preferably the rhodium complex is formed with a bipyridyl ligand attached to polystyrene beads, whereby to assure that rhodium is not carried into the hydrogen peroxide production side of the cell.

Process for hydrogen peroxide production from hydrogen and oxygen

Direct reaction of anilines with olefins occurs at elevated temperature in solution containing dissolved complex of the aniline and an eighth group metal, especially rhodium. The products are N-alkylanilines and alkylquinolines.

Direct reaction of anilines with olefins

A process of reacting in the liquid phase a hexacoordinated ruthenium(II) complex with oxygen in the presence of a source of hydrogen ions under conditions forming hydrogen peroxide and the corresponding hexacoordinated ruthenium(III) complex and reacting the hexacoordinated ruthenium(III) complex with hydrogen sulfide to form elemental sulfur and regenerate the hexacoordinated ruthenium(II) complex, the hexacoordinated ruthenium(III) complex having a reduction potential of not more than +0.68. Five or six of the ligands of the ruthenium complexes are non-labile in both valence states.

Hexacoordinated ruthenium moderated production of hydrogen peroxide and

A new liquid phase oxidation process in which a metal nitro complex transfers an oxygen atom from the nitro ligand to a substrate; especially such process conducted cyclically or catalytically using molecular oxygen as the oxygen source. Metal nitrosyl complex is formed as a coproduct together with an oxidation product of the substrate. In a catalytic process, molecular oxygen maintains a concentration of nitro ligand in the reaction mixture. In a cyclic process, nitrosyl ligand of the metal nitrosyl coproduct is reoxidized by molecular oxygen in presence of a monodentate base such as pyridine to nitro ligand; and the nitro complex, thus regenerated, can be used again to oxidize the substrate, In particular the metal is a Group VIII metal, especially cobalt and the nitro complex is pyridine cobalt nitro saloph. To accelerate oxidation of the substrate at given temperature, a cocatalyst is included in the reaction mixture, e.g. an organic complex of a transition metal such as Mo(VI), Pd(II), or a compound of a trivalent metallic element with an element of Group VII, such as the compound BF.sub.3.

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Springfield, NJ, United States of America

Steven E Diamond