Methods for synthesizing nucleosides, nucleoside derivatives...

Details Methods for synthesizing nucleosides, nucleoside derivatives... Methods for synthesizing nucleosides, nucleoside derivatives...

2001-08-31

2004-02-03

Barts, Samuel (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S027300, C536S124000, C536S026100

Reexamination Certificate

active

06686463

ABSTRACT:

TECHNICAL FIELD OF INVENTION
This invention relates to the chemical synthesis of nucleosides, non-nucleosides and derivatives thereof, including nucleoside and non-nucleoside phosphoramidites and succinates.
BACKGROUND OF THE INVENTION
The following is a brief description of the synthesis of nucleosides. This summary is not meant to be complete but is provided only for understanding the invention that follows. This summary is not an admission that the work described below is prior art to the claimed invention.
Structural modifications of oligonucleotides are becoming increasingly important as their possible clinical applications emerge (Usman et al, 1996, Ed., Springer-Verlag, Vol. 10, 243-264; Agrawal, 1996,
Trends Biotech.,
14, 376-387; Christoffersen and Marr, 1995,
J. Med. Chem.,
38, 2023-2037). The efficient synthesis of nucleic acids that are chemically modified to increase nuclease resistance while maintaining potency is of importance to the potential development of new therapeutic agents.
Research into the study of structure-function relationships in ribonucleic acids has in the past, been hindered by limited means of producing such biologically relevant molecules (Cech, 1992,
Nucleic Acids Research,
17, 7381-7393; Francklyn and Schimmel, 1989,
Nature,
337, 478-481; Cook et al., 1991,
Nucleic Acids Research,
19, 1577-1583; Gold, 1988,
Annu. Rev. Biochemistry,
57, 199-233). Although enzymatic methods existed, protocols that allowed one to probe structure function relationships were limited. Only uniform post-synthetic chemical modification (Karaoglu and Thurlow, 1991,
Nucleic Acids Research,
19, 5293-5300) or site-directed mutagenesis (Johnson and Benkovic, 1990,
The Enzymes,
Vol. 19, Sigman and Boyer, eds., 159-211) were available In the latter case, researchers were limited to using natural bases. Fortunately, adaptation of the phosphoramidite protocol for RNA synthesis has greatly accelerated our understanding of RNA. Site-specific introduction of modified nucleotides at any position in a given RNA has now become routine. Furthermore, one is not confined to a single modification but can include many variations in each molecule.
While it is seemingly out of proportion that one small structural modification can have such an impact, the presence of a single hydroxyl at the 2′-position of the ribofuranose ring has been the major reason that research in the RNA field has lagged so far behind comparable DNA studies. Progress has been made in improving methods for DNA synthesis that have enabled the production of large amounts of antisense deoxyoligonucleotides for structural and therapeutic applications. Only recently have similar gains been achieved for RNA (Wincott et al., 1995,
Nucleic Acids Research,
23, 2677-2684; Sproat et al., 1995,
Nucleosides and Nucleotides,
14, 255-273; Vargeese et al., 1998,
Nucleic Acids Research,
26, 1046-1050).
The chasm between DNA and RNA synthesis is due to the difficulty of identifying orthogonal protecting groups for the 5′- and 2′-hydroxyls. Historically, two standard approaches have been taken by scientists attempting to solve the RNA synthesis problem, The first approach involves developing a method that seeks to adapt to state-of the-art DNA synthesis, while the second approach involves designing a method specifically suited for RNA. Although adaptation of the DNA process provides a more universal procedure in which non-RNA phosphoramidites can easily be incorporated into RNA oligomers, the advantage to the latter approach is that one can develop a process that is optimal for RNA synthesis and as a result, better yields can be realized. However, in both cases similar issues exist, including, for example, the identification of protecting groups that are both compatible with synthesis conditions and capable of being removed at the appropriate juncture. This problem does not refer only to the 2′- and 5′-OH groups, but includes the base and phosphate protecting groups as well. Consequently, the accompanying deprotection steps, in addition to the choice of ancillary agents, are critical. Another shared obstacle is the need for efficient synthesis of the monomer building blocks.
The most common paradigm has been to apply DNA synthesis methods to RNA. Consequently, it is critical to identify a 2′-hydroxyl protecting group that is compatible with DNA protecting groups yet can easily be removed once the oligomer is synthesized. Due to constraints placed by the existing amide protecting groups on the bases and the 5′-O-dimethoxytrityl (DMT) group (or in some cases the 9-(phenyl)xanthen-9-yl (Px) group), the 2′-blocking group must be stable to both acid and base. In addition, the 2′blocking group must also be inert to the oxidizing and capping reagents. Although the most widely used 2′-hydroxyl protecting group is tert-butyldimethylsilyl (TBDMS) ether, many others have been explored. These alternative 2′-protecting groups include acetal groups, such as the tetrahydropyranyl (THP), methoxytetrahydropyranyl (mthp), 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), 1-(2-chloroethoxy)ethyl, 2-hydroxyisophthalate formaldehyde acetal, and 1-{4-[2-(4-nitrophenyl)ethoxycarbonyloxy]-3-fluorobenzyloxy}ethyl groups. In addition, photolabile groups, such as the o-nitrobenzyl, o-nitrobenzyloxymethyl, and p-nitrobenzyloxymethyl groups have been used. Other groups include the 1,1-dianisyl-2,2,2-trichloroethyl group, the p-nitrophenylethyl sulfonyl group, and the 2′-O-triisopropylsilyl-oxy-methyl group. Additional 2′-protecting groups that have been studied are reviewed in Gait et al., 1991;
Oligonucleotide Synthesis,
In
Oligonucleotides and Analogues, A Practical Approach
(F. Eckstein, ed.), 25-48, and Beaucage and Iyer, 1992,
Tetrahedron,
48, 2223-2311.
By far the most popular 2′-protecting group is the tert-butyldimethylsilyl group, developed principally by Ogilvie and co-workers (Usman et al., 1987,
J.A.C.S.,
109, 7845-7854). Recent advances in silyl chemistry in both the synthesis (Wincott et al., 1995,
Nucleic Acids Research,
23, 2677-2684, Sproat et al., 1995,
Nucleosides and Nucleotides,
14, 255-273, Vargeese et al., 1998,
Nucleic Acids Research,
26, 1046-1050) and deprotection (Wincott et al., supra; Sproat et al., supra) arenas have made it's use an even more viable approach to the production of oligoribonucleotides.
The introduction of the tert-butyldimethylsilyl group at the 2′-position of a ribonucleotide is usually effected by the reaction of a 5′-O-dimethoxytrityl-nucleoside with tert-butyldimethylsilyl chloride in the presence of either silver nitrate or imidazole. The resulting mixture of 2′-O-tert-butyldimethylsilyl, 3′-O-tert-butyldimethylsilyl and bis-substituted (3′,2′-di-O-tert-butyldimethylsilyl) products must be purified to isolate the desired 2′-O-tert-butyldimethylsilyl derivative, usually by column chromatography. Treatment of the isolated 3′-O-tert-butyldimethylsilyl derivative by equilibration in triethylamine/methanol or pyridine/water can effect migration of the silyl ether, resulting in the capability of isolating additional 2′-O-tert-butyldimethylsilyl product. Multiple re-equilibrations can be utilized to obtain smaller and smaller quantities of the desired 2′-O-tert-butyldimethylsilyl product, however, this process is time-consuming and requires a separate purification step after each equilibration. Therefore, even though formation of the 2′-O-tert-butyldimethylsilyl isomer is sometimes kinetically favored, the resulting isolated yield of the desired 2′-O-tert-butyldimethylsilyl isomer is generally diminished due to formation of the competing 3′-O-tert-butyldimethylsilyl and bis-substituted isomers. Accordingly, there exists a need for a general method for nucleoside phosphoramidite synthesis useful in the selective introduction of silyl protection at the 2′-hydroxyl of a nucleoside.
The utilizatio

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