Beckman Instruments, Inc.

Beckman Coulter, Inc.

Naeem B Hanna

Firdous Farooqui

M Parameswara Reddy

Parameswara Meda Reddy

Meda Parameswara Reddy

Maged A Michael

Activating groups based on N-hydroxynaphthalimide, are disclosed herein. The activating groups can mediate the coupling of labeling moieties, such as biotin or cyanine dyes, to a variety of components, including chain terminators, nucleoside triphosphates, and oligonucleotides, which are used in nucleotide sequencing. From these activating groups, activated esters of the labeling moieties can be prepared. The activated esters react with a component, for example a derivatized nucleotide chain terminator, to give a labeled component. In additions, methods of the present invention provide for labeling a nucleoside triphosphate in organic media. The activating groups and methods of the present invention allow the activation and coupling reactions to occur at a much higher yield, compared with the prior art.

Efficient activated cyanine dyes

A compound of the general formula ##STR1## wherein R is alkyl of 1 to about 10 carbons and R' is selected from the group consisting of trityl and pixyl, for use in the synthesis of 2'-OMe RNA sequences. Fast cleavage and deprotection of oligonucleotides is facilitated by the use of a reagent comprising methylamine as active component in place of the traditional reagent ammonium hydroxide.

2'-OMe C.sup.AC phosphoramidite and methods for preparation and use

A compound of general formula ##STR1## wherein R is methyl R' is selected from the group consisting of trityl and pixyl, and R' is H or OMe. These compounds may advantageously be employed in the synthesis of oligonucleotides by conventional methods, such as automated solid phase synthesis. Use of ethylene diamine in the cleavage and deprotection procedure substantially eliminates the formation of undesirable side products.

C.sup.ac -methylphosphonamidites and methods for preparing

Disclosed herein are N.sup.4 protected deoxycytidines for use in the synthesis of oligonucleotides, the protecting groups being represented by the formula: --CO--(CH.sub.2).sub.0-9 --CH.sub.3. Preferred embodiments are N.sup.4 acetyl deoxycytidines and include N.sup.4 acetyl deoxycytidine phosphoramidites and N.sup.4 acetyl deoxycytidine H-phosphonates. When used to prepare oligonucleotides the protected deoxycytidine compounds provide high quality oligonucleotide products with little side product from cleavage and deprotection reactions carried out with alkyl amine compounds.

CAC H-phosphonate and its use in the synthesis of oligonucleotides

Deoxyribonucleotide and ribonucleotide derivatives of the general formula I ##STR1## wherein R.sup.1 represents --CR'R'--Ar, in which Ar is substituted aryl (as hereinafter defined) and R' and R' are independently selected from the group consisting of hydrogen and lower alkyl; one of --R.sup.2 and R.sup.3 is a hydroxyl-protecting group and the other is a group suitable for synthesis of polynucleotides or for attachment of the nucleotide to a solid support; R.sup.4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base. When synthesis is carried out using these derivatives, the deprotection procedure is reduced to an essentially instantaneous process. The derivatives have acceptable shelf life and are very stable to conventional DNA synthesis conditions. Particularly preferred are those compounds wherein Ar is mono- and dihalo-substituted phenyl.

Synthesis of DNA using substituted phenylacetyl-protected nucleotides

Disclosed are reagents of methods for cleaving and deprotecting insolubilized and protected synthetic oligonucleotides. In a particularly preferred embodiment, the reagent comprises methylamine and t-butylamine.

Methods and reagents for cleaving and deprotecting oligonucleotides

Disclosed herein are protecting groups for exocyclic amino groups of the bases adenine, guanine and cytosine for use in the synthesis of oligonucleotides, the protecting groups being represented by the formula: --CO-- (CH.sub.2).sub.0-9 --CH.sub.3. In a particularly preferred embodiment, the base cytosine is protected with acetyl (--CO--CH.sub.3), and the oligonucleotide incorporating said protected cytosine is subjected to a cleavage/deprotection reagent comprising at least one straight chain alkylamine having from 1 to about 10 carbon atoms.

Growing community of inventors

Fullerton, CA, United States of America

Naeem B Hanna