Diribonucleoside Phosphoramidites

Details Diribonucleoside Phosphoramidites Diribonucleoside Phosphoramidites

2003-01-17

2004-08-24

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

Reexamination Certificate

active

06780989

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to methods for synthesis of phosphorothioate oligonucleotides and to novel synthetic intermediates useful in the methods. The methods comprise the in-situ generation of amidites on a solid support. The methods are useful, inter alia, for the preparation of phosphorothioate oligonucleotides which, in turn, are useful as diagnostic reagents, research reagents and therapeutics agents.
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.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs that are designed to bind in a specific fashion to a specific mRNA by hybridization (i.e., oligonucleotides that are specifically hybridizable with a target mRNA). Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA by any of a number of mechanisms, i.e., to interfere with translation reactions by which proteins coded by the mRNA are produced. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits; however there are still problems to be solved. See generally, Cook, P. D.
Anti
-
Cancer Drug Design
1991, 6, 585; Cook, P. D.
Medicinal Chemistry Strategies for Antisense Research, in Antisense Research
&
Applications
, Crooke, et al., CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al.,
A. Chem. Rev
. 1990, 90, 543.
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, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides and oligonucleotide analogs toward nuclease degradation.
A serious deficiency of unmodified oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes.
A number of chemical modifications have been introduced into antisense agents (i.e., oligonucleotides and oligonucleotide analogs) to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense agents, to stabilize the antisense agents from nucleases and other enzymes that degrade or interfere with their structure or activity in the body, to enhance the antisense agents' binding to targeted RNA, to provide a mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, and to improve the antisense agents' pharmacokinetic and pharmacodynamic properties. It is unlikely that unmodified, “wild type,” oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases.
Oligonucleotides which have been modified to contain phosphorothioate linkages are capable of terminating RNA by activation of RNase H upon hybridization to RNA. These oligonucleotide analogs have been demonstrated to be sequence specific regulators of gene expression in eukaryotic and procaryotic systems, and are the most promising candidates to date for practical application as “antisense” therapeutic agents. See Eckstein,
Oligonucleotide and Analogs, A Practical Approach
, 1991, IRL Press, pp. 87-103.
Potential applications of phosphorothioate oligonucleotides as drugs have created a new challenges in the large-scale synthesis of these compounds. Thus, there remains a need for improved methods of synthesizing phosphorothioate oligonucleotides. The present invention addresses these, as well as other needs.
SUMMARY OF THE INVENTION
The present invention is directed to novel methods for the preparation of oligomeric compounds having phosphorothioate linkages. The present invention discloses solid support oligonucleotide synthetic methods which involve the generation of support-bound phosphoramidites. In preferred embodiments, the methods comprise the steps of:
reacting a phosphordiamidite of formula:
wherein:
R
1
is a protecting group;
R
2
and R
3
are dialkylamino or morpholino;
B is a nucleosidic base; and
R
6
is halogen, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, or a polyether of the formula (O-alkyl)
m
, where m is 1 to about 10;
with a support-bound synthon of formula:
wherein:
R
4
is a linker connected to a solid support;
R
5
is a phosphoryl protecting group; and
n is 0 to 100;
to form a support-bound phosphoramidite. The support-bound phosphoramidite has the formula:
The support-bound phosphoramidite is protected, preferably by reaction with a reagent of formula R
5
—OH, to form a support-bound phosphite of formula:
The support-bound phosphite is then sulfurized to form a protected phosphorothioate, and then the protected phosphorothioate is deprotected to form a further support-bound synthon wherein n is increased by 1.
Throughout, it is understood that variable substituents R
1-6
may be the same or different in differing oligomeric subunits.
In some preferred embodiments, R
2
and R
3
are diisopropylamino. In other preferred embodiments R
5
is 2-cyanoethyl, 4-cyano-2-butenyl, or diphenylmethylsilylethyl. In further preferred embodiments the reaction of the phosphite compound with the support-bound synthon is preformed in the presence of an organic base, preferably tetrazole. In other preferred embodiments the support-bound phosphite is oxidized with a sulfurization reagent such as Beaucage reagent, tetraethylthiuram disulfide, dibenzoyl tetrasulfide, phenacetyl disulfide, 1,2,4-dithiuazoline-5-one, 3-ethoxy-1,2,4-dithiuazoline-5-one, a disulfide of a sulfonic acid, sulfur, or sulfur in combination with a ligand such as triaryl, trialkyl, triaralkyl, or trialkaryl phosphines.
The present methods provide for the synthesis of oligonucleotides consisting of a wide variety of nucleosidic bases, including naturally occurring nucleosidic bases such as adenine, guanine, thymine, cytosine, and uracil, as well a

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