Process for the preparation of 2′-O-alkyl-guanosine,...

Details Process for the preparation of 2′-O-alkyl-guanosine,... Process for the preparation of 2′-O-alkyl-guanosine,...

1999-12-09

2001-06-05

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025300, C536S025310, C536S025320, C536S025330

Reexamination Certificate

active

06242592

ABSTRACT:

FIELD OF INVENTION
This application is directed to processes for the preparation of 2′-O-alkyl guanosine, uridine and cytidine phosphoramidites.
BACKGROUND OF THE INVENTION
A number of oligonucleotide analogs have been made. One class of oligonucleotides that have been synthesized are the 2′-O-substituted oligonucleotides. Such oligonucleotides have certain unique and useful properties. In U.S. patent application Ser. No. 07/814,961, filed Dec. 24, 1991, abandoned entitled Gapped 2′ Modified Phospharothioate Oligonucleotides, assigned to the same assignee as this application, the entire contents of which are herein incorporated by reference, 2′ substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand.
In a recent article, Sproat, B. S., Beijer, B. and Iribarren, A.,
Nucleic Acids Research,
1990, 18:41, the authors noted further use of 2′-O-methyl substituted oligonucleotides as “valuable antisense probes for studying pre-mRNA splicing and the structure of spliceosomes”.
2′-O-Methyl and ethyl nucleotides have been reported by a number of authors. Robins, et al.,
J. Org. Chem.,
1974, 39, 1891; Cotten, et al.,
Nucleic Acids Research,
1991, 19, 2629; Singer, et al.,
Biochemistry
1976, 15, 5052; Robins,
Can. J. Chem.
1981, 59, 3360; Inoue, et al.,
Nucleic Acids Research,
1987, 15, 6131; and Wagner, et al.,
Nucleic Acids Research,
1991, 19, 5965;112.
Sproat, B. S. and Lamond, A. I., in “2′-O-Methyloligoribonucleotides: synthesis and applications,
Oligonucleotides and Analogs A Practical Approach;
Eckstein, F. Ed.; IRL Prest, Oxford, 1991, describe syntheses of 2′-O-methylribonucleoside-3′-O-phosphoramidites. The uridine phosphoramidite synthesis described therein requires both base and sugar protection of the starting nucleoside prior to alkylation. Only after the base and sugar protecting groups are in place on the uridine is it then alkylated. Post alkylation, the base protecting group is removed followed by 5′-O-dimethoxytritylation and phosphitylation. The cytidine phosphoramidite synthesis described by Sproat and Lamond utilizes (and thus requires) the base and sugar blocked 2′-O-methyl uridine analog. This analog is then converted to a blocked cytidine analog, the blocking group is removed from the sugar, the analog is dimethoxytritylated and finally phosphitylated. The guanosine phosphoramidite synthesis taught by Sproat and Lamond starts from a 2-amino-6-chloronucleoside having 3′ and 5′ sugar hydroxy groups blocked. This nucleoside is converted to a 2,6-dichloro derivative. The dichloro compound is then 2′-O alkylated. Following O-alkylation, the dichloro compound is converted to a diazido intermediate. The diazido intermediate is in turn converted to a diamino intermediate. The diamino intermediate is then deaminated to the guanosine analogue. The 2-amino group of the guanosine analogue is blocked followed by dimethoxytritylation and finally phosphitylation. This guanosine procedure is also published in Sproat, et. al.,
Nucleic Acids Research,
1991 19:733.
The above synthetic procedures involve multiple steps and numerous reagent treatments—9 different reagent treatments for uridine, 10 for cytidine and 12 for guanosine. For the cytidine and guanosine compounds at least one of the reagents that is required is not readily available and thus is a very expensive reagent.
Certain oligonucleotides containing 2′-O-alkyl substituted nucleotides are promising candidates for use as human pharmaceuticals. For use in large scale therapeutic testing and eventually for human pharmaceutical use, large amounts of these oligonucleotides must be synthesized. The large amounts of oligonucleotides in turn requires large amounts of the 2′-O-alkyl nucleoside phosphoramidites used in synthesizing the oligonucleotides. Consideration must therefore be given to both cost and purity of the starting phosphoramidites used in the synthesis of such oligonucleotides. As a general premise, as the number of synthetic steps increases, the cost of manufacture increases. Further as the number of steps increases, quality control problems escalate. In view of this, it is evident that there is a great need for new and improved procedures for preparing nucleoside phosphoramidites.
OBJECTS OF THE INVENTION
It is an object of this invention to provide new and improved synthetic methods for the preparation of 2′-substituted nucleoside phosphoramidites.
It is a further object of this invention to provide new and improved synthetic methods for the preparation of 2′-O-alkyl nucleoside phosphoramidites.
A further object of this invention is to provide new and improved syntheses of 2′-O-alkyl guanosine phosphoramidites.
A further object of this invention is to provide new and improved syntheses of 2′-O-alkyl cytidine phosphoramidites.
A further object of this invention is to provide new and improved syntheses of 2′-O-alkyl uridine phosphoramidites.
A further object of this invention is to provide new and improved syntheses of 2,6-diamino-9-(2′-O-alkyl-&bgr;-D-ribofuranosyl)purine phosphoramidites.
A further object of this invention is to provide new and improved oligonucleotide syntheses that utilize the improved phosphoramidite syntheses of the invention.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and appended claims.
SUMMARY OF THE INVENTION
Previous methods for the preparation of 2′-O-alkylated nucleoside phosphoramidites involved numerous steps and reagents, resulting in decreased efficiency and increased cost.
In accordance with this invention there are provided improved processes for the preparation of 2′-O-alkylated nucleoside phosphoramidites including 2′-O-alkylated guanosine, cytidine and uridine phosphoramidites. Further in accordance with this invention there are provided processes for the preparation of oligonucleotides that include at least one 2′-O-alkylated nucleotide incorporated within the oligonucleotide.
In accordance with the invention there are provided processes for preparing a 2′-O-alkylated guanosine 3′-O-phosphoramidite comprising the steps of alkylating a 2,6-diamino-9-(ribofuranosyl)purine to form a 2,6-diamino-9-(2′-O-alkylated ribofuranosyl)purine; deaminating said 2,6-diamino-9-(2′-O-alkylated ribofuranosyl)purine to form a 2′-O-alkylated guanosine; blocking the 5′-hydroxyl moiety of said 2′-O-alkylated guanosine; and phosphitylating the 3′-position of said 5′-blocked 2′-O-alkylated guanosine.
Further in accordance with the invention there are provided processes for preparing a 2′-O-alkylated cytidine 3′-phosphoramidite that include the steps of alkylating an unblocked cytidine to form a 2′-O-alkylated cytidine; blocking the 5′-hydroxyl moiety of said 2′-O-alkylated cytidine; and phosphitylating the 3′-position of said 5′-blocked 2′-O-alkylated cytidine.
Further in accordance with the invention there are provided processes for preparing a 2′-O-alkylated uridine 3′-O-phosphoramidite that include the steps of treating a uridine with a dialkyltin oxide to form a 2′,3′-O-dialkylstannylene derivative of uridine; alkylating said stannylene derivative of uridine to form a 2′-O-alkylated uridine; blocking the 5′-hydroxyl moiety of said 2′-O-alkylated uridine; and phosphitylating the 3′-position of said 5′-blocked 2′-O-alkylated uridine.
Further in accordance with the invention there are provided processes for preparing a 2′-O-alkylated 2,6-diamino-9-(&bgr;-D-ribofuranosyl)purine 3′-O-phosphoramidite that include the steps of alkylating a 2,6-diamino-9-(&bgr;-D-ribofuranosyl)-2′-purine to provid

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