Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1997-01-10
1999-10-12
Marschel, Ardin H.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 6, 436501, 514 44, 536 241, 536 243, 536 2431, 536 2432, 536 2433, 536 253, 935 77, 935 78, C12Q 168
Patent
active
059657200
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to the compounds and compositions of N3'.fwdarw.P5' phosphoramidates, synthesis methods and hybridization and nuclease resistance methods employing oligonucleotide N3'.fwdarw.P5' phosphoramidates.
BACKGROUND OF THE INVENTION
Oligonucleotides have been proposed as potent diagnostic compounds and as new rationally designed therapeutic agents (Uhlman, 1990; Helene, et al., 1990; Helene, 1991). The mechanism of action of these compounds is based on their specific interaction with RNA or DNA regions of interest.
Several modifications of the natural phosphodiester internucleoside bond {phosphomono- (Eckstein, et al., 1985; Cohen, 1993) or dithioate (Marshall, et al., 1993), methylphosphonate (Miller, 1991), phosphodiester amidate (Letsinger, et al., 1988; Froehler, et al., 1988)} have been introduced to improve (i) the stability of the oligomers in biological media, and (ii) the hybridization properties of the oligomers.
Unfortunately, the vast majority of these analogs exhibit reduced binding with target RNA or DNA strands via duplex or triplex formation (Kibler-Herzog, et al., 1991). Moreover, the presence of the stereoisomers at phosphorous in some of these analogs may complicate the binding patterns with complimentary nucleic acids (LaPlauche, et al., 1986; Bower, et al., 1987; Tidd, et al., 1988).
SUMMARY OF THE INVENTION
Methods, compounds and compositions of the present invention relate to oligodeoxyribonucleotides having contiguous nucleoside subunits joined by intersubunit linkages. In the oligonucleotides, at least 2 contiguous subunits are joined by N3'.fwdarw.P5' phosphoramidate intersubunit, or greater than 3 of the total intersubunit linkages are N3'.fwdarw.P5' phosphoramidate intersubunit linkages.
An exemplary N3'.fwdarw.P5' phosphoramidate intersubunit linkage is: ##STR1## Where X is --O.sup.-, --OR or --R, and R is selected from the group consisting of alkyl, alkenyl, aryl, and aralkyl.
An exemplary N3'.fwdarw.P5' phosphoramidate oligonucleotide is: ##STR2## Where X is --O.sup.-, --OR or --R, and R is selected from the group consisting of alkyl, alkenyl, aryl, and aralkyl, n is from 4 to 100 and B is a base. Normally the 5' oxygen is either bonded to a hydrogen or another nucleotide in the oligonucleotide and the 3' oxygen is bonded to another nucleotide in the oligonucleotide. For definitions of these exemplary substituent groups see the Definitions section below.
The nucleoside subunits making up the oligodeoxyribonucleotides of the present invention can be selected to be in a defined sequence: such as, a sequence of bases complementary to a single-strand nucleic acid target sequence or a sequence that will allow formation of a triplex between the oligodeoxyribonucleotide and a target duplex.
In one embodiment the oligodeoxyribonucleotide has at least 3 contiguous subunits joined by N3'.fwdarw.P5' phosphoramidate linkages. This grouping of linkages can, for example, be located at the 3' end of the oligodeoxyribonucleotide. At this location the N3'.fwdarw.P5' phosphoramidate linkages confer nuclease resistance to the oligodeoxyribonucleotide.
In another embodiment of the present invention, all of the intersubunit linkages are N3'.fwdarw.P5' phosphoramidate linkages.
The present invention includes intermediates used to make the oligodeoxyribonucleotides and methods for synthesizing such intermediates. The intermediates are of the following formulas: ##STR3##
The present invention includes a method for generating a duplex nucleic acid molecule by forming an oligodeoxyribonucleotide having contiguous nucleoside subunits joined by intersubunit linkages, where the oligodeoxyribonucleotide contains intersubunit linkages of Formula 1 or such linkages in combination with other linkages discussed herein. The oligodeoxyribonucleotide has a sequence of nucleoside subunits to form a duplex with a target nucleic acid molecule when the oligodeoxyribonucleotide is contacted with the target nucleic acid molecule under conditions to allow effective fo
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Zielinski, W.S., and Orgel, L.
Chen Jer-Kang
Gryaznov Sergei M.
Schultz Ronald G.
Lynx Therapeutics, Inc.
Macevicz Stephen C.
Marschel Ardin H.
Mendlein John D.
Powers Vincent M.
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