Building blocks for the solution phase synthesis of...

Details Building blocks for the solution phase synthesis of... Building blocks for the solution phase synthesis of...

2001-03-30

2004-01-13

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06677120

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for the preparation of 3′-O and 5′-O-levulinyl nucleosides from common precursors using an enzymatic approach. These methods are useful for the large-scale synthesis of oligonucleotides.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to affect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as “antisense” agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities.
Three principal methods have been used for the synthesis of oligonucleotides. The phosphotriester method, as described by Reese,
Tetrahedron
1978, 34, 3143; the phosphoramidite method, as described by Beauage, in
Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs
; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as described by Froehler in
Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs
Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63-80.
The phosphotriester approach has been widely used for solution phase synthesis, whereas the phosphoramidite and H-phophonate strategies have found application mainly in solid phase syntheses. Recently, Reese reported a new approach to the solution phase synthesis of oligonucleotides on H-phosphonate coupling. See, Reese et al.
Nucleic Acids Research,
1999, 27, 963-971, and Reese et al.
Biorg. Med. Chem. Lett.
1997, 7, 2787-2792, which is incorporated herein by reference. Solution phase synthesis is the method of choice in producing large-scale quantities of oligonucleotides.
These solution phase methods require the use of nucleoside monomer building blocks bearing protecting groups on the 3′-O and/or the 5′-O positions. The protecting groups should be stable to coupling conditions and selectively cleaved without affecting other protecting groups in the molecule. One such protecting group is the levulinyl group, —C(O)—(CH
2
)
2
—C(O)—CH
3
. However, the preparation of nucleosides bearing these protecting groups involves several tedious chemical protection/deprotection and/or purification steps.
For example, the 3′,5′-di-O-levulinyl protection of nucleosides can be accomplished using a well-established method wherein nucleosides are selectively acylated at their hydroxyl sites by reacting the nucleosides with levulinic acid in the presence of DCC (dicyclohexylcarbodiimide). Despite the utility of this method, it suffers from at least one significant problem. The method requires a large excess of DCC to achieve optimal yields. The excess DCC is converted to DCU (dicyclohexylcarbodiimide) upon completion of the reaction, which must be separated from the reaction mixture. Unfortunately, for large-scale syntheses, the separation step requires considerable time and expense.
Prior to the present invention, synthesis of 5′-O-levulinyl nucleosides was accomplished by reacting parent nucleosides with levulinic acid and 2-chloro-1-methylpyridinium iodide. Iwai et al.,
Nucleic Acids Res.
1988, 16, 9443-9456; Iwai et al.
Tetrahedron
1990, 46, 6673-6688. However, because this method does not afford selective acyaltion of the 5′-hydroxyl function, additional purification and deprotection steps are necessary because both 3′-acyl and 3′,5′-diacyl derivatives are formed in the reaction. After the 3′,5′-diacyl derivatives are separated by chromatography, the residue must be treated with DMTrCl to remove the 3′-acyl compound. Finally, an additional purification by chromatography isolates the 5′-O-levulinyl derivatives in very low yields.
Before now, the synthesis of 3′-O-levulinyl nucleosides (2′-deoxy or 2′-protected) was accomplished by the treatment of parent nucleosides with levulinic acid or levulinic anhydride and DCC. One of the major drawbacks of this method is that it requires that the 5′-hydroxyl function be protected as a 5′-O-DMTr group prior to acylation with levulinic acid. The 5′-O-DMTr group must then be removed in an acid medium to afford the 3′-O- protected nucleosides. See, Reese et al.,
Nucleic Acids Res.
1999, 27, 963-971, and Reese et al.,
J. Chem. Soc., Perkin Trans.
1 1999, 1477-1486.
Commercially viable methods for the large-scale synthesis of oligonucleotides are constantly being explored. It has been found that the application of biocatalysts in organic synthesis has become an attractive alternative to conventional chemical methods. See, Carrea, et al.
Angew. Chem. Int. Ed.
2000, 39, 2226-2254; Bornscheuer, et al.
Hydrolases in Organic Synthesis. Regio- and Stereoselective Biotransformations
; Wiley-VCH: Weinheim, 1999. Enzymes catalyze reactions with high chemo-, regio-, and stereoselectivity. See, Ferrero et al.
Chem. Rev.
2000, 100, 4319-4347; Ferrero et al.,
Monatsh. Chem.
2000, 131, 585-616. It has previously been reported that
Candida antarctica
lipase B (CAL-B) catalyzes acylation at the 5′-hydroxyl group of nucleosides with high selectivity.
Pseudomonas cepacia
lipase (PSL) shows unusual regioselectivity towards the secondary alcohol at the 3′-position of 2′-deoxynucleosides. Moris et al.,
J. Org. Chem.
1993, 58, 653-660; Gotor et al.
Synthesis
1992, 626-628.
In the last few years the use of antisense oligonucleotides has emerged as an exciting new therapeutic paradigm. As a result, very large quantities of therapeutically useful oligonucleotides are required in the near future. In view of the considerable expense and time required for synthesis of oligonucleotide building blocks, there has been a longstanding effort to develop successful methodologies for the preparation of oligonucleotides with increased efficiency and product purity.
SUMMARY OF THE INVENTION
Applicants have discovered methods that are useful in, for example, the large-scale synthesis of oligonucleotides. The methods of the present invention help to minimize the number of steps required to yield desired results using an enzymatic approach. Applicants have found that both 3′-O-levulinyl nucleosides and 5′-O-levulinyl nucleosides can be prepared from a common precursor the regioselecti

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