Benzazolides and their employment in phosphate ester oligonucleotide synthesis processes

Claim 2

A benzazolide of the formula ##STR15## wherein X is N or CH, A, B, C, and D are independently selected from aong hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower alkyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato amino and cyano, R is a hydrogen or a base labile phosphate protecting group, P g is a selectively removable protecting group selected from levulinyl and acid labile protecting groups and B* is a base selected from 1-thyminyl, 1-(N-protected)cytosinyl, 9-(N-protected)adeninyl and 9-(N-protected)quaninyl.

Claim 3

The benzazolide of claim 1 characterized as a benzotriazolide wherein X is N.

Claim 4

The benzotriazolide of claim 2 wherein B, C and D are hydrogens.

Claim 5

The benzotriazolide of claim 3 wherein R is a base-labile phosphate protecting group.

Claim 6

The benzotriazolide of claim 4 wherein R is selected from among 1 to 4 carbon alkyls, 6 to 12 carbon aryls, alkaryls and aralkyls and halo-substituted 6 to 12 carbon aryls, alkaryls and aralkyls.

Claim 7

The benzotriazolide of claim 5 wherein the N-protected base is protected with a group selected from among benzoyl, isobutryl and anisoyl.

Claim 8

A benzazolide of the formula ##STR16## wherein X is N or CH, A, B, C, and D are independently selected from among hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower akyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato amino and cyano, R is a base labile phosphate protecting group, P g is a selectively removable protecting group selected from levulinyl and acid labile protecting groups and B* and B*' are independently selected from 1-thyminyl, 1-(N-protected)cytosinyl, 9-(N-protected)adeninyl and 9-(N-protected)guaninyl and n is an integer 1 or greater.

Claim 9

A benzazolide of the formula ##STR16## wherein X is N or CH, A, B, C, and D are independently selected from among hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower akyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato amino and cyano, R is a base labile phosphate protecting group, P g is a selectively removable protecting group selected from levulinyl and acid labile protecting groups and B* and B*' are independently selected from 1-thyminyl, 1-(N-protected)cytosinyl, 9-(N-protected)adeninyl and 9-(N-protected)guaninyl and n is an integer 1 or greater.

Claim 10

In a phosphate triester process for coupling deoxynucleotides wherein a first nucleotide having a protected 3' phosphate diester group is reacted under coupling conditions with a second nucleoside or nucleotide having an available free 5' hydroxyl group in the presence of a coupling agent, the improvement comprising employing as said coupling agent an arylsulfonylbenzazolide of the formula ##STR17## wherein X is CH or N and A, B, C, D, and A', B', C', D' and E' are independently selected from among hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower alkyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato, amino and cyano.

Claim 11

In a phosphate triester process for coupling deoxynucleotides wherein a first nucleotide having a protected 3' phosphate diester group is reacted under coupling conditions with a second nucleoside or nucleotide having an available free 5' hydroxyl group in the presence of a coupling agent, the improvement comprising employing as said coupling agent an arylsulfonylbenzazolide of the formula ##STR17## wherein X is CH or N and A, B, C, D, and A', B', C', D' and E' are independently selected from among hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower alkyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato, amino and cyano.

Claim 12

The process of claim 8 wherein B, C and D are hydrogens, B' and D' are hydrogens and A', C' and E' are hydrogens or the same alkyl of 1 to 4 carbon atoms.

Claim 13

The process of claim 9 wherein X is N.

Claim 14

The process of claim 10 being further characterized as a flow process in which controlled amounts of solutions of said first nucleotide, said second nucleotide or nucleoside and said coupling agent are serially fed for controlled periods into a reaction zone for a reaction period.

Claim 15

A process for synthesizing an oligonucleotide comprising a. reacting a nucleotide or nucleoside with a phosphorodibenzazolide of the formula ##STR18## wherein X is N or CH; A, B, C and D are independently selected from hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower alkyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato amino and cyano, and R is a base-labile phosphate protecting group to yield a phosphorylated nucleotide, and b. coupling said phosphorylated nucleotide with a second nucleotide or nucleoside.

Claim 16

A process for synthesizing an oligonucleotide comprising a. reacting a nucleotide or nucleoside with a phosphorodibenzazolide of the formula ##STR18## wherein X is N or CH; A, B, C and D are independently selected from hydrogen, chloro, bromo, iodo, fluoro, lower alkyls of 1 to 4 carbon atoms, substituted lower alkyls, benzyl, phenyl, substituted benzyl, substituted phenyl, lower alkoxies of 1 to 4 carbon atoms, nitro, nitroso, sulfonato amino and cyano, and R is a base-labile phosphate protecting group to yield a phosphorylated nucleotide, and b. coupling said phosphorylated nucleotide with a second nucleotide or nucleoside.

Claim 17

A process of claim 12 wherein X is CH.

Claim 18

A process of claim 12 wherein X is N.

Claim 19

A process for preparing a blocked nucleotide triester which comprises a. reacting a nucleotide or nucleoside with a benzotriazolide of claim 6 to yield a phosphorylated nucleotide, and b. reacting said phosphorylated nucleotide with a blocking alcohol to give a blocked nucleotide triester.