Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-04-30
2003-01-21
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025310, C536S023100, C435S006120, C435S091100, C435S375000, C435S372000, C435S442000
Reexamination Certificate
active
06509459
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities and processes useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g.,
Methods in Molecular Biology, Vol
20:
Protocols for Oligonucleotides and Analogs
pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993);
Oligonucleotides and Analogues: A Practical Approach,
pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer,
Curr. Op. in Biotech.
6: 12 (1995); and
Antisense Research and Applications
(Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al.,
J. Molec. Biol.
72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese,
Tetrahedron Lett.
34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers,
Tetrahedron Lett.
22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild,
Tetrahedron Lett.
28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al.,
Biochemistry
23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al.,
Biochemistry
27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al.,
Proc. Antl. Acad. Sci. USA
85: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3′-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3′ functional group of the incoming nucleoside and the 5′ hydroxyl group of the 5′-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 &mgr;mol to 1 mmol and higher). See Padmapriya et al.,
Antisense Res. Dev.
4: 185 (1994). Several modifications of the standard phosphoramidite processes have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al.,
Tetrahedron
50: 9255 (1994); Theisen et al.,
Nucleosides
&
Nucleotides
12: 43 (1994); and Iyer et al.,
Nucleosides
&
Nucleotides
14: 1349 (1995)) and isolation (Kuijpers et al.
Nucl. Acids Res.
18: 5197 (1990); and Reddy et al.,
Tetrahedron Lett.
35: 4311 (1994)) of oligonucleotides.
The routine synthesis of oligonucleotides is presently carried out using various N-acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine), and benzoyl for adenine and cytosine. After the synthesis of the oligonucleotides is carried out using either phosphoramidite chemistry or H-phosphonate chemistry, the protecting groups are removed by treatment with ammonia at 55-60° C. for 5-10 hours. Using these protecting groups, PO oligonucleotides and other modified oligonucleotides can be synthesized. However, it would be greatly advantageous to be able to carry out such synthesis more rapidly, which would be possible if the time required for removal of the protecting groups could be reduced.
In addition, when currently available deprotection conditions are used, in certain instances where modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out. Examples of such base-sensitive modified oligonucleotides include, ribonucleoside-containing oligonucleotides, methylphosphotriester oligonucleotides, phosphoramidates, etc.
One such example is the large-scale synthesis of RNA which is required for the ribozyme-based therapeutic strategies. Such synthesis presents special challenges due to two factors. These are, first, 3′-5′ to 2′-5′ internucleotide chain migration during preparation of nucleoside monomer precursors, during synthesis, and during removal of protecting groups from the RNA, and second, degradation of RNA. Use of classical protecting groups compounds these factors. For successful RNA synthesis, it is essential that the 2′ hydroxyl protecting group remains intact until the final deprotection step and that following its removal, the 2′ hydroxyl group does not attack the vicinal phosphodiester groups and thereby promote cleavage or migration of the internucleotidic linkages.
Another example is that current synthesis procedures allow the synthesis of some, but not all possible oligonucleotide phosphoramidates, because some of these compounds are labile under the highly alkaline conditions required for deprotection of the nucleoside base. Oligonucleotides containing primary phosphoramidate internucleoside linkages, for example, have not previously been possible to synthesize for this reason. In the case of the oligonucleotide phosphoramidates, this inability to synthesize oligonucleotides containing primary phosphoramidate internucleoside linkages has probably slowed their development as optimally useful compounds for molecular biology applications and the antisense therapeutic approach. This is likely because the oligonucleotide phosphoramidates that have been developed all have relatively large chemical substituents in place of one of the nonbridging oxygen atoms on the phosphate backbone, which may lead to steric hindrance in the ability of the oligonucleotide to bind to its target. It would be valuable to have internucleotidic primary phosphoramidate linkages, since incorporation of such non-ionic linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides. For example, Galbraith et al., Antisense Research and Development 4: 201-206 (1994) disclose complement activation by oligonucleotides. Henry et al., Pharm. Res. 11: PPDM8082 (1994) discloses that oligonucleotides may potentially interfere with blood clotting.
Yet another example is the synthesis of oligonucleotides containing methylphosphonate internucleoside linkages. Various methodologies have been used to synthesize such oligonucleotides. Miller et al., Biochemistry 25: 5092-5095 (1986), discloses an early methodology using a polymer support. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches a more generally applicable phosphoramidite approach using a controlled pore glass (CPG) support. All of the existing approaches, however, are inherently limited by the susceptibility of the methylphosphonate linkage to hydrolysis by base, which precludes the use of the usual deprotection step, which employs prolonged treatment with 28% ammonium hydroxide. Some attempts to deal with this problem have included the use
Agrawal Sudhir
Habus Ivan
Iyer Radhakrishnan P.
Yu Dong
Hale and Dorr LLP
Hybridon, Inc.
Wilson James O.
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