Substituted purines and oligonucleotide cross-linking

Details Substituted purines and oligonucleotide cross-linking Substituted purines and oligonucleotide cross-linking

1998-08-04

2001-05-15

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025310, C536S025320, C536S025330, C536S025340, C544S264000

Reexamination Certificate

active

06232463

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to novel substituted purine compounds that may be incorporated into oligonucleotides and may serve as cross-linkers for complementary oligonucleotides and oligonucleotide analogs. Oligonucleotides and their analogs are used for a variety of therapeutic and diagnostic purposes, such as treating diseases, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in site specific fashions. The compounds of the invention include novel heterocyclic bases, nucleosides, and nucleotides. When incorporated into oligonucleotides, the compounds of the invention are useful, inter alia, for modulating RNA activity. The compounds are also useful as research reagents.
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 focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, attempts have been made to moderate the 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 achieve therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate expression of genes which are responsible for the formation of undesired protein.
One method for inhibiting specific gene expression is by the use of oligonucleotides or modified oligonucleotides as “antisense” agents. As so used, oligonucleotides or modified oligonucleotides are selected to be complimentary to a specific, target, messenger RNA (mRNA) sequence. 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. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides or modified oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted.
The use of oligonucleotides, modified oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic and diagnostic use is actively being pursued by many commercial and academic groups. While the initial suggested mode of activity of antisense agents was via hybridization arrest, several additional mechanisms or terminating antisense events have also been studied in relation to antisense use in therapeutics. In addition to hybridization arrest these include cleavage of hybridized RNA by the cellular enzyme ribonuclease H (RNase H), RNA catalytic or chemical cleaving and cross-linking. Various reviews in the scientific literature summarize these studies. See, for example,
Oligonucleotides: Antisense Inhibitors of Gene Expression
, CRC Press, Inc., Boca Raton, Fla. (Cohen ed., 1989); 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; Walder, et al.,
Proc. Natl. Acad. Sci., USA
, 1988, 85, 5011; and Dagle, et al.,
Antisense Research
&
Development
, 1991, 1, 11.
Hybridization arrest denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides, such as those of Miller, et al.,
Anti
-
Cancer Drug Design
, 1987, 2, 117-128, and &agr;-anomer oligonucleotides are two extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
In the RNase H terminating event, activation of RNase H by a heteroduplex formed between a DNA type oligonucleotide or oligonucleotide analog and the targeted RNA results in cleavage of target RNA by the enzyme, thus destroying the normal function of the RNA. To date, the RNAse H enzyme has been found to be activated only by either natural phosphodiester DNA oligonucleotides or phosphorothioate DNA oligonucleotides. Walder, supra and Stein, et al.,
Nucleic Acids Research
, 1988, 16, 3209-3221 describe the role that RNase H plays in the antisense approach.
oligonucleotides or modified oligonucleotides acting as chemical or catalytic RNA cleavers require either the attachment of pendent groups with acid/base properties to oligonucleotides or the use of ribozymes, i.e. RNAs having inherent catalytic properties. In the pendent group approach, the pendent group is not involved with the specific Watson-Crick hybridization of the oligonucleotide or oligonucleotide analog with the mRNA but is carried along by the oligonucleotide or oligonucleotide analog to serve as a reactive or non-reactive functionality. The pendent group is intended to interact with the mRNA in some manner to more effectively inhibit translation of the mRNA into protein. Such pendent groups have also been attached to molecules targeted to either single or double stranded DNA. Such pendent groups include intercalating agents, cross-linkers, alkylating agents, or coordination complexes containing a metal ion with associated ligands.
Cross-linking of a nucleic acid with a complimentary oligonucleotide or modified oligonucleotide is used to modulate RNA activity by disrupting the function of nucleic acids. To date this has primarily been achieved by cleaving the target. The known approaches using cross-linking agents, as well as alkylating agents and radical generating species, as pendent groups on oligonucleotides for antisense diagnostics and therapeutics have had several significant shortcomings. It is known to cross-link nucleic acids by exposure to UV light; however, such cross-linking is positionally uncontrollable. To overcome this lack of specificity, some workers have covalently cross-linked complementary strands of oligonucleotides at a specific site utilizing controlled chemistry. These workers attached a nitrogen mustard to either the 3′ terminal ribose unit of an oligonucleotide or oligonucleotide analog via an acetal linkage or to the 5′ end of an oligonucleotide or oligonucleotide analog via a phosphoramide linkage. On hybridization, the reactive mustards covalently cross-linked to the complementary strand via alkylation of the ternary heteroaromatic nitrogen atom at the 7-position of guanine or adenine: see Grineva et al.,
FEBS
., 1973, 32, 351-355. Other workers have attached an &agr;-bromomethylketone to the 4-position of a cytidine nucleotide which spans the major groove and alkylates the 7-position of a complementary guanine residue in a targeted strand: see Summerton et al.,
J. Mol. Bio
., 1978, 122, 145-162
; J. Theor. Biology
, 1979, 78, 61-75; and U.S. Pat. No. 4,123,610. The alkylated bases formed under these conditions are quaternary charged species that are subject to rapid chemical degradation via imidazole ring opening followed by cleavage of the targeted strand. Meyer et. al.,
J. Am. Chem. Soc
., 1989, 111, 8517, described attaching an iodoacetamidopropyl moiety to the 5-position of a thymidine nucleotide of DNA that alkylated the 7-position of a guanine nucleotide at a position two base pairs down the complementary strand.
Cross-linking may also be achieved by hybridization. For example, an N6,N6-ethano-adenine or N4,N4-ethanocytosine alkylates an appropriately positioned nucleophile in a complementary strand. This process has been

Affiliated with

Also associated with

Rating

Substituted purines and oligonucleotide cross-linking does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Substituted purines and oligonucleotide cross-linking, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Substituted purines and oligonucleotide cross-linking will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2562250