Process for selective N-acylation of purine nucleosides

Details Process for selective N-acylation of purine nucleosides Process for selective N-acylation of purine nucleosides

2001-04-17

2003-04-01

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S027100, C536S027200, C536S027210

Reexamination Certificate

active

06541626

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for selectively protecting the exocyclic amino function of purine nucleosides.
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 Beaucage, 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.
The synthesis of oligonucleotides requires the rapid and quantitative coupling of building blocks, such as nucleosides. Protecting groups are routinely used during these coupling reactions to allow selective reaction between two functional groups while protecting all other functionalities present in the reacting molecules. One such functional group that requires protection is the exocyclic amino function of a nucleobase. Amino protection helps to achieve quantitative coupling by inhibiting the risk of depurination of a growing oligonucleotide, which is facilitated under acidic conditions. In a partially depurinated oligomer chain, cleavage takes place at the site of depurination through double &bgr;-elimination, thereby generating truncated sequences.
Amino protecting groups, or N-acyl groups, are typically derived from carboxylic acids, which are used to acylate the amino functions, thereby forming amide bonds. Prior to the present invention, the methods used to acylate or protect the amino functions resulted in acylation of the hydroxyl functions present on the nucleoside as well. These methods involve various protection/deprotection steps resulting in decreased yields and increased product impurities. For example, the classical procedure used to protect amino groups on nucleosides involves per-acylatation of the nucleosides. The per-acylated nucleoside is then selectively hydrolyzed at the esters leaving the N-acylated nucleosides. See, Schaller, et al.
J. Am. Chem. Soc.
1963, 85, 3821. According to another general procedure, the hydroxyl functions are first protected by silylating the groups prior to acylating the amino function. See, Ti et al.
J. Am. Chem. Soc.
1982, 104, 1316. These groups must then be removed from the hydroxyl functions.
Another problem with conventional methods for protecting amino functions is that all the reagents used for protection are either in chloride or anhydride form. The preparation of acid chlorides from their respective acids is only about 50% and harmful chlorinating agents are required, such as POCl
3
and SO
2
Cl
2
. Additionally, these methods require a heating step which further increases costs for large-scale production.
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
According to one embodiment of the present invention, methods are provided for selectively protecting the exocyclic amino function of a nucleoside without compromising the hydroxyl groups of the sugar moieties. The methods comprise contacting an amino protecting reagent with an activating agent in the presence of a coupling agent to form an activated ester and reacting the activated ester with the purine nucleoside for a time and under conditions effective to covalently attach a protecting group onto the exocyclic amino function of the nucleoside.
In some embodiments of the present invention, the protecting group is formyl, isobutyryl, methoxyacetyl, allyloxycarbonyl, isopropoxyacetyl, levulinyl, 4-pentenoyl, 4-nitrophenylethyloxycarbonyl, phenyl acetyl, (4-t-butylphenyl)acetyl, 9-fluorenylmethoxycarbonyl, &agr;-phenylcinnamoyl, phenoxyacetyl, 2-chlorophenoxyacetyl, 2-chloro-4-(tert-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, benzoyl, 4-methoxybenzoyl, 4-chlorobenzoyl, 4-nitrobenzoyl, 4-dimethylaminobenzoyl, 4-t-butylbenzoyl, 2-(methyl)sulfonylethoxycarbonyl, 2-(4-chloro)sulfonylethoxycarbonyl, 2-(4-nitro)sulfonylethoxycarbonyl, diphenylacetyl, 3,4-dichlorobenzoyl, 3-methoxy-4-phenoxybenzoyl, 2-(acetoxymethyl)benzoyl, benzoyloxymethylbenzoyl, 1,8-naphthaloyl, or 2-(t-butyldiphenylsilyloxymethyl)benzoyl. Other amino protecting groups are amenable to the present invention and within the purview of the skilled artisan.
Activating agents include, among others, O-nitrophenol, 2,4-dinitrophenol, 2,4,5-trichlorophenol, pentachlorophenol, N-hydroxyphthalimide, N-hydroxysuccinimide, N-hydroxyphthalimide, hydroxypiperidine, 5-chloro-8-hydroxy-quinoline, and compounds having one of the following formulae:
According to one embodiment of the present invention, methods are provided for recycling a polymer bound coupling agent from the reaction mixture containing the N-protected nucleoside prepared according to the methods described herein. Upon N-acylation, the reacted polymer bound coupling agent is filtered from the reaction mixture and recycled. The recycling is effected by dehydrating the polymer support with a dehydrating agent such as tosyl chloride or POCl
3
in the

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