Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1993-07-28
2003-08-12
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024500, C536S025300, C514S04400A
Reexamination Certificate
active
06605708
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to synthetic oligonucleotides. More particularly, the invention relates to synthetic oligonucleotides having modified internucleoside linkages that confer resistance to nucleolytic degradation.
2. Summary of the Related Art
In recent years there has been considerable interest in the use of synthetic oligonucleotides as modulators of gene expression. (See, for example, Agrawal, Trends in Biotechnology 10:152-158 (1992)). At least part of this interest has been directed toward the development of modified oligonucleotides having greater resistance to nucleolytic degradation than is possessed by conventional oligonucleotide phosphodiesters. Included among such oligonucleotides are chimeric oligonucleotides having different types of modified internucleotide linkages. For example, Pederson et al., U.S. Pat. No. 5,149,797, discloses chimeric oligonucleotides having regions that activate RNase H and regions that do not activate RNase H.
Among the modified internucleoside linkages that have been incorporated into oligonucleotides are phosphate-based and non-phosphate-based linkages. The former group includes the well known phosphorothioate, phosphorodithioate, methylphosphonate and phosphoramidate linkages, among others. The latter group includes sulfone, sulfate and carbamate linkages. Such linkages modify oligonucleotides in a manner that can affect the oligonucleotide in its abilities to resist nucleolytic degradation, undergo normal hydrogen bonding and base stacking, bind to a complementary target nucleic acid, and be taken up by cells.
Certain types of carbamate internucleoside linkages are known in the art. Gait et al., J. Chem. Soc., Perkin I:1684-1686 (1974) first disclosed a dinucleotide containing a carbamate internucleotide linkage of the structure 5′-nucleoside-3′-OCONH-5′-nucleoside-3′. Mungall and Kaiser, J. Org. Chem. 42:703-706 (1977), discloses synthesis of modified dinucleotides and trinucleotides having carbamate linkages of the same structure. Coull et al., Tetrahedron Lett. 28:745-748 (1987), teaches that a hexameric oligonucleotide carbamate having the same internucleoside linkage does not undergo base stacking, even though its size and shape should not produce steric hindrance. Stirchak et al., Nucleic Acids Res. 17:6129-6141 (1989), discloses that a hexameric poly C morpholine oligonucleotide carbamate having the same internucleoside linkage forms a multistrand complex, rather than a Watson-Crick duplex, with complementary poly (dG). Wang and Weller, Tetrahedron Lett. 32:7385-7388 (1991) disclose solid phase synthesis of oligonucleotide or morpholine oligonucleotide carbamates having this same internucleotide linkage.
Although oligonucleotide carbamates are of interest for use as modulators of gene expression due to their stability over a wide pH range and their resistance to enzymatic degradation, the absence of base stacking and the formation of multi-strand complexes by unknown mechanisms may hinder such a use as a practical matter. Consequently, there is a need for new linkages having similar resistance to degradation, but without hindering the ability of oligonucleotides to undergo base stacking and, at least in some configurations, without significantly destabilizing the double helix formed between the oligonucleotide and its complementary target nucleic acid.
BRIEF SUMMARY OF THE INVENTION
The invention provides convenient building blocks for assembling oligonucleotides containing a novel carbamate linkage. The carbamate linkage in building blocks and oligonucleotides according to the invention has the structure 5′-nucleoside-3′-NHCOO-5′-nucleoside-3′, which is the reverse of the structure of known carbamate internucleoside linkages. Surprisingly, oligonucleotides containing this novel carbamate linkage do undergo base stacking, unlike known carbamate-linked oligonucleotides. In addition, the stability of double helices involving oligonucleotides according to the invention is not significantly impaired when the novel carbamate linkages are present in certain preferred positions within the oligonucleotides.
In a first aspect, the invention provides dimeric, trimeric and tetrameric building blocks in which at least two nucleosides are linked by the novel carbamate linkage 5′-nucleoside-3′-NHCOO-5′-nucleoside-3′. Such building blocks preferably have appropriate protecting or leaving groups for oligonucleotide synthesis at their 5′ and 3′ ends. These building blocks can be easily made using solution phase chemistry, and can be used for oligonucleotide assembly just like the protected monomers currently used.for solid phase oligonucleotide synthesis. Such building blocks according to the invention are useful for assembling oligonucleotides having novel carbamate internucleoside linkages at various positions within the oligonucleotide.
In a second aspect, the invention provides a method of using building blocks according to the invention to assemble oligonucleotides containing novel carbamate linkages. In this aspect, conventional oligonucleotide synthesis chemistries are utilized with the building blocks according to the invention.
In a third aspect, the invention provides novel oligonucleotides containing the carbamate internucleoside linkage 5′-nucleoside-3′-NHCOO-5′-nucleoside-3′ at one or more positions within the oligonucleotide. Such oligonucleotides are readily assembled using conventional solid phase chemistry, but using dimeric, trimeric, or tetrameric building blocks according to the invention during one or more cycles of assembly. Such oligonucleotides according to certain embodiments of the invention are useful for studying the position-dependence of the effect of carbamate internucleoside linkages on destabilizing a double helix. In certain preferred embodiments of oligonucleotides according to the invention, the novel carbamate linkages are in positions that do not result in significant destabilization of a double helix formed between the oligonucleotide and a complementary target nucleic acid. Most preferably, such novel carbamate linkages are in positions in which they confer upon the oligonucleotide increased resistance to nucleolytic degradation. Oligonucleotides according to such an embodiment are useful for modulating gene expression, both in in vitro experimental systems and in therapeutic uses for plant, animal or human diseases.
REFERENCES:
patent: 5149797 (1992-09-01), Pederson et al.
patent: 0464638 (1992-01-01), None
patent: 9106629 (1991-05-01), None
patent: WO 9220822 (1992-11-01), None
patent: WO 9220823 (1992-11-01), None
Agrawal, “Antisense Oligonucleotides as Antiviral Agents,”Trends in Biotechnology, 10, 152-158 (May, 1992).*
R. S. Root-Bernstein (I), “AIDS IS More Than HIV: Part I,”Genetic Engineering News, Sep. 1, 1992, pp. 4-6.*
R. S. Root-Bernstein (II), “AIDS IS More Than HIV: Part II,”Genetic Engineering News, Sep. 15, 1992, pp. 4-5.*
T. Beardsley, “Trends in Cancer Epidemiology—A War Not Won,”Scientific American, 270(1), 130-138 (Jan., 1994).*
G. Zon, “Oligonucleotide Analogues as Potential Chemotherapeutic Agents,”Pharmaceutical Research, 5(9), 539-549 (1988).*
Gait et al.,J. Chem. Soc., Perkin I, 1684-1686 (1974).
Mungall and Kaiser,J. Org. Chem.42, 703-706 (1977).
Coull et al.,Tetrahedron Lett. 28, 745-748 (1987).
Stirchak et al.,Nucleic Acids Res. 17, 6129-6141 (1989).
Wang and Weller,Tetrahedron Lett. 32, 7385-7388 (1991).
Damha et al.,Nucleic Acids Res. 18, 3813 (1990).
Caruthers et al.,Methods in Enzymology154, 287 (1987).
Stirchak et al.,J. Org. Chem. 52, 4202 (1987).
Weller et al.,J. Org. Chem. 56, 6000 (1991).
Nyilas et al.,Tetrahedron46, 2149 (1990).
Agrawal Sudhir
Habus Ivan
Crane L. Eric
Hale and Dorr LLP
Hybridon, Inc.
Wilson James O.
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