Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Patent
1997-09-30
2000-01-25
Gerstl, Robert
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
435 6, 514 44, 514410, 514453, 536 231, 536 245, 536 253, C07D31922, C07H 2100
Patent
active
060180587
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to biochemical reagents used in the manipulation of genetic material, and, particularly, to compounds which can cleave DNA or RNA molecules at specifically defined locations.
BACKGROUND OF THE INVENTION
It is well known that deoxyribonucleic acid (DNA) is a helically-shaped double stranded molecule having complementary base pairs connecting the two phosphate-sugar backbone strands. The ability to cleave DNA or RNA, both single strand and double strand, is important. It is particularly important that it be possible to direct this cleavage such that it occurs at specifically-defined sites in the DNA or RNA molecule. Such DNA and RNA cleavage agents could be useful in a laboratory setting for biological or pharmaceutical research or as part of a diagnostic tool. It is also possible that such agents could be used in vivo as part of a gene therapy regimen, for example in the treatment of cancer.
At the present time, there are several types of reagents that can be used to cleave DNA either thermally or photochemically. These include (1) dienyne natural products and their synthetic derivatives; (2) other reagents that generate biradicals, e.g., azoalkenes; (3) organoplatinum reagents; (4) anthraquinones; and (5) rhodium and ruthenium complexes. The dienyne products and their derivatives are very complex molecules that will cleave DNA either thermally or photochemically. This DNA cleaving activity is thought to be the source of their activity against certain types of cancer. These compounds, however, are not readily available. The need to isolate these materials from their natural sources makes it difficult to obtain dienynes in large quantities at reasonable prices. Even if these compounds were synthesized chemically, the synthesis would involve multiple step reactions just to prepare the active molecules, not to mention the additional synthetic effort that would be necessary to attach them to DNA sequence specific groups. Thus, the dienyne materials do not offer a cost-effective way of cleaving DNA molecules.
Azoalkenes that form trimethylenemethane biradicals (see Bregant, T. M.; Groppe, J.; Little, R. D.; J. Am. Chem. Soc. 1994, 116: 3635-3636) are thermally unstable and decompose over several days at room temperature.
Anthraquinones are para-quinones that are highly reactive and sensitive to visible light. They will destroy their own sequence-recognition chains unless stored in the cold and rigorously protected from visible light.
Organoplatinum reagents have also been shown to be effective for the photochemical cleavage of DNA. Here again, these reagents are relatively expensive to produce in bulk quantities and are not particularly amenable to the synthetic modification which is necessary if DNA sequence recognition properties are to be incorporated into them. Furthermore, the toxicological properties of these materials can become a problem when used in vivo, for example in the treatment of cancer.
Rhodium and ruthenium complexes have also been shown to be effective as photochemical agents for the cleavage of DNA. However, like the organoplantinum complexes, they are expensive to manufacture and could pose significant toxicological risks if they are to be used in vivo.
There is, therefore, a need for DNA cleavage reagents which are easy to synthesize in bulk quantities, which are relatively stable on storage and will survive to reach their target site in the cell, which do not involve the cost, availability or toxicological issues present with the precious metal compounds, and which may easily be modified to bind to specific DNA sequences and, therefore, cleave DNA at specifically defined sites. It is this problem which the present invention seeks to address.
Dervan and Becker, J. Am. Chem. Soc. (1978) 100: 1968-1970, describes the synthesis and use of bis(methidium) spermine as a polyintercalating molecule (i.e., a molecule which simultaneously binds to adjacent sites of DNA). The approach taken in this work was to form a dimer of ethidium bromide, a known interca
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Harsch Andreas
Keller Stephen J.
Schlemm Donna J.
Schnapp Karlyn A.
Wilson Robert M.
Gerstl Robert
University of Cincinnati
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