Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-01-06
2004-01-06
Low, Christopher S. F. (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S312700, C548S313100, C548S314700, C548S334500, C548S557000, C536S022100, C536S023100, C536S025300, C536S025330, C536S025600, C536S026100
Reexamination Certificate
active
06673940
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to cyclic compounds that undergo nucleotide base pair-specific interactions with double stranded nucleic acids. The invention also concerns methods of using such cyclic compounds, as well as methods relating to their solid state synthesis.
BACKGROUND OF THE INVENTION
None of the following discussion of the background of the invention, which is provided solely to aid the reader in understanding the invention, is admitted to be or to describe prior art to the invention.
The design of synthetic ligands capable of reading information stored in the DNA double helix has been a long-standing goal of chemistry and molecular biology. Cell-permeable small molecules, which target predetermined nucleotide sequences in double-stranded nucleic acids, particularly double-stranded DNA (dsDNA), are useful in regulating, or modulating, gene-expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. See Moser, et al.,
Science,
vol. 238:645-650 (1987), Duvalvalentin, et al.,
Proc. Nat'l Acad. Sci. USA,
vol. 89:504-508 (1992), Maher, et al.,
Biochemistry,
vol. 31:70-81 (1992). Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach so far has been limited to purine tracks and suffers from poor cellular uptake.
Another recent approach to targeting specific nucleotide sequences in dsDNA has involved molecules known as “polyamides.” See U.S. Ser. No. 08/607,078, PCT/US97/03332, U.S. Ser. Nos. 08/837,524, 08/853,525, PCT/US97/12733, U.S. Ser. No. 08/853,522, PCT/US97/12722, PCT/US98/06997, PCT/US98/02444, PCT/US98/02684, PCT/US98/01006, PCT/US98/03829, and PCT/US98/0714. As described in the foregoing references, polyamides comprise polymers of amino acids covalently linked by amide bonds. Preferably, the amino acids used to form these polymers include N-methylpyrrole (Py) and N-methylimidazole (Im).
Wade, et al. (
J. Am. Chem. Soc.,
vol. 114:8783-8794 (1992)) reported the design of polyamides that bind in the minor groove of dsDNA at 5′-(A,T)G(A,T)C(A,T)-3′ sequences by a dimeric, side-by-side motif; Mrksich, et al. (
Proc. Natl. Acad. Sci. USA,
vol. 89:7586-7590 (1992)), reported an antiparallel, side-by-side polyamide motif for sequence-specific recognition in the minor groove of dsDNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin; and Trauger, et al. (
Nature,
vol. 382:559-561 (1996)) reported the recognition of a targeted dsDNA by a polyamide at subnanomolar concentrations. The particular order of amino acids in such polyamides, and their pairing in dimeric, antiparallel complexes formed by association of two polyamide polymers, determines the sequence of nucleotides in dsDNA with which the polymers preferably associate.
The development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provided a useful code to control target nucleotide base pair sequence specificity. Specifically, an Im/Py pair in adjacent polymers was fond to distinguish G•C from C•G and both of these from A•T or T•A base pairs. A Py/Py pair was found to specify A•T from G•C but could not distinguish A•T from T•A. White, et al. (
Biochemistry,
vol. 35:12532-12537 (1996)) reported the effects of the A•T/T•A degeneracy of Py/Im polyamide recognition in the minor groove of dsDNA. White, et al. (
Chem.
&
Biol.
vol. 4:569-578 (1997)) reported the pairing rules for recognition in the minor groove of dsDNA by Py/Im polyamides and the 5′→3′, N→C orientation preference for polyamide binding in the minor groove of dsDNA.
More recently, it has been discovered that inclusion of a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp)(made by replacing a single hydrogen atom in Py with a hydroxy group), in a polyamide and paired opposite Py enables A•T to be discriminated from T•A by an order of magnitude. Utilizing Hp together with Py and Im in polyamides provides a code to distinguish all four Watson-Crick base pairs (i.e., A•T, T•A, G•C, and C•G) in the minor groove of dsDNA, as described in Table 1.
TABLE 1
Pairing Code for Minor Groove
Recognition
Pair
G · C
C · G
T · A
A · T
Im/Py
+
−
−
−
Py/Im
−
+
−
−
Hp/Py
−
−
+
−
Py/Hp
−
−
−
+
Favored (+), disfavored (−)
As discussed above, a number of different polyamide motifs have been reported in the literature, including “hairpins,” “H-pins,” “overlapped,” “slipped,” and “extended” polyamide motifs. Specifically, hairpin polyamides are those wherein the carboxy terminus of one amino acid polymer is linked via a linker molecule, typically aminobutyric acid or a derivative thereof to the amino terminus of the second polymer portion of the polyamide. Indeed, the linker amino acid &ggr;-aminobutyric acid (&ggr;), when used to connect first and second polyamide polymer portions, or polyamide subunits, C→N in a “hairpin motif,” enables construction of polyamides that bind to predetermined target sites in dsDNA with more than 100-fold enhanced affinity relative to unlinked polyamide subunits. See Trauger, et al.,
Nature,
vol. 382:559-561 (1996), Swalley, et al.,
J. Am. Chem. Soc.,
vol. 119:6953-6961 (1997), Turner, et al.,
J. Am. Chem. Soc.,
vol. 119:7636-7644 (1997), Trauger, et al.,
Angew. Chemie. Int. Ed. Eng.,
vol. 37:1421-1423 (1997), Turner, et al.,
J. Am. Chem. Soc.,
vol. 120:6219-6226 (1998), Kelly, et al.,
Proc. Nat'l Acad. Sci. USA,
vol. 93:6981-6985 (1996), Trauger, et al.,
J. Am. Chem. Soc.,
vol. 118:6160-6166 (1996), Geierstanger, et al.,
Nature Struct. Biol.,
vol. 3:321-324 (1996), Swalley, et al.,
Chem. Eur. J.,
vol. 3:1600-1607 (1997), and Trauger, et al.,
J. Am. Chem. Soc.,
vol. 120:3534-3535 (1998). Moreover, eight-ring hairpin polyamides (comprised of two four amino acid polymer portions linked C→N) have been found to regulate transcription and permeate a variety of cell types in culture. See Gottesfield, J. M.; et al.,
Nature,
vol. 387:202-205 (1997).
An H-pin polyamide motif, i.e., wherein two paired, antiparallel polyamide subunits are linked by a linker covalently attached to an internal polyamide pair, have also been reported. Another polyamide motif that can be formed between linked or unlinked polyamide subunits is an “extended” motif, wherein one of the polyamide subunits comprises more amino acids than the other, and thus has a single-stranded region. See U.S. Ser. No. 08/607,078. In contrast, an “overlapped” polyamide is one wherein the antiparallel polyamide subunits completely overlap, whereas in a “slipped” binding motif, the two subunits overlap only partially, with the C-terminal portions not associating with the N-terminal regions of the other subunit. See U.S. Ser. No. 08/607,078.
The literature has also reported the synthesis of a six-ring Py/Im-containing cyclic polyamide (cyclo-(Im-Py-Py-&ggr;-Py-Py-Py-&ggr;)) that bound its designated five base pair (bp) dsDNA target sequence (5′-TGTTA-3′) at subnanomolar concentrations, and with 40-fold higher affinity relative to a hairpin analog (Im-Py-Py-&ggr;-Py-Py-Py-Dp) containing the same amino acid pairings. See Cho, et al.,
Proc. Nat'l Acad. Sci. USA,
vol. 92:10389-10392 (1995). It was postulated that closing the ends of the hairpin to form a cyclic compound would restrict conformational space for the DNA-binding molecule. Significantly, however, the hairpin analog more strongly bound to its match sequence versus single base pair mismatch sites by a factor of 20, whereas the cyclic polyamide bound match versus mis-matched sites with only 4-17-fold specificity, suggesting that an energetic price was paid by forming a cyclic molecule which had more restricted conformational flexibility as compared to non-
Baird Eldon E.
Dervan Peter B.
California Institute of Technology
Foley & Lardner
Kam Chih-Min
Low Christopher S. F.
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