Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2002-01-02
2004-08-03
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C536S025300
Reexamination Certificate
active
06770760
ABSTRACT:
BACKGROUND OF THE INVENTION
Nucleic acid bulges refer to regions of unpaired bases in a double-stranded nucleic acid molecule. These bulges have been known to take part in many important biological processes.
For example, ENA bulges form crucial motifs for specific nucleic acid-protein recognition and binding. It has been known that the human immunodeficiency virus transactivator protein Tat binds to a three-pyrimidine bulge in the response element TAR. See, e.g., Weeks et al.,
Science
249, 1281-1285 (1990). Nucleic acid bulges also produce frameshift mutations which can change the product of the protein translation and result in various disorders. According to one report, Myerowitz et al.,
J. Biol. Chem.
263, 18587-18589 (1988), approximately 70% of Ashkenazi Tay-Sachs disease is caused by a four-base pair insertion mutation in the HEX A gene encoding the &agr;-subunit of hexosaminidase A. Another disease, cystic fibrosis, is also caused by frameshift mutation. A three-base pair deletion (AF508) is commonly found among cystic fibrosis patients. Rommens et al.,
Am. J. Hum. Genet.
46, 395-396 (1990).
Comparative gel electrophoresis assay has been used to detect the presence of bulges in nucleic acids. This assay differentiates nucleic acids with and without bulges by their different mobility in gel. However, it can only provide information as to whether a nucleic acid contains a bulge. Thus, there exists a need for a detection method which can provide additional information, e.g., the location of a bulge in a nucleic acid.
SUMMARY OF THE INVENTION
One aspect of this invention relates to a metal complex of formula (I):
Each of R
1
, R
2
, R
3
, R
4
, R
5
, R
6
, R
7
and R
8
, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of R
2
and R
3
, and R
6
and R
7
, independently, optionally join together to form a cyclic moiety which is fused with the two pyridyl rings to which R
2
and R
3
, or R
6
and R
7
are bonded. The cyclic moiety, if present, is optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl, alkylcarbonylamino, alkylaminocarbonyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of L
1
and L
2
, independently, is —C(R
a
) (R
b
)—, —O—, —S—, or —N(R
c
)— and each of R
a
, R
b
, and R
c
, independently, is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. M is a Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion, and the oxidation state of M is 0, 1, 2, 3, or 4. Each of X
1
and X
2
, independently, is a labile ligand.
Examples of a metal complex of formula (I) include cobalt(II)(hexaazacyclophane)(trifluoroacetate)
2
, cobalt (II) (hexaazacyclophane) (H
2
O) (trifluoroacetate) ruthenium(II) (hexaazacyclophane) (trifluoroacetate)
2
, and manganese(II)(hexaazacyclophane)(trifluoroacetate)
2
.
Another aspect of this invention relates to a method of specifically cleaving a nucleic acid bulge. The method comprising contacting the bulge with a metal complex of formula (I), supra, where M is a Fe, Co, Ni, Ru, Rh, Mn, Os, Ag, Cr, Zn, Cd, Hg, Re, Ir, Pt, or Pd ion. In one embodiment, the method is performed in the presence of an oxidant, e.g., hydrogen peroxide, or in a medium having a pH values which ranges from 4-9.
In this disclosure, a nucleic acid bulge is a region in a double-stranded nucleic acid molecule (DNA or RNA), the region having at least one unpaired nucleotide and being flanked by two paired nucleotides. The nucleic acid bulge can contain 1-5 unpaired nucleotides (e.g., 1-3). Using nucleic acid substrate A in
FIG. 1
as an example, the nucleic acid bulge present therein contains three unpaired nucleotides, i.e., T
6
, C
7
, and T
8
. This three-base bulge is flanked by two paired nucleotides, i.e., A
5
-T
23
and G
9
-C
22
. In contrast, the C
13
-A
18
hairpin loop, which is also present in substrate A, is not a bulge as the unpaired nucleotides are only connected to one paired nucleotide, i.e., C
12
-G
19
. A nucleic acid bulge can also contain two nucleotides. See the bulge present in substrate D which is formed of two unpaired nucleotides, i.e., C
6
and T
7
.
A salt of a metal complex of formula (I) is also within the scope of this invention. Note that a metal complex of formula (I) can be positively charged. A salt of such a metal complex can be formed with an anionic counterion. Examples of counterions include fluoride, chloride, bromide, iodide, sulfate, sulfite, phosphate, acetate, oxalate, and succinate.
As described above, each of R
2
and R
3
, and R
6
and R
7
, independently, can join together to form a cyclic moiety. The cyclic moiety can contain 5 or 6 ring members and can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. For example, when the cyclic moiety formed by joining R
2
and R
3
is a benzene, it fuses with the two pyridine rings co which R
2
and R
3
are bonded, and the benzene ring and the two pyridine rings together form phenanthroline.
As used herein, alkyl is a straight or branched hydrocarbon chain containing 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and hexyl.
By “cycloalkyl” is meant a cyclic alkyl group containing 3 to 8 carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl include piperidine, piperazine, tetrahydropyran, tetrahydrofuran, and morpholine.
In this disclosure, aryl is an aromatic group containing 6-12 ring atoms and can contain fused rings, which may be saturated, unsaturated, or aromatic. Examples of an aryl group include phenyl and naphthyl. Heteroaryl is aryl containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heteroaryl include pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, and benzothiazolyl.
Note that an amino group can be unsubstituted, mono-substituted, or di-substituted. It can be substituted with groups such as alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Halo refers to fluoro, chloro, bromo, or iodo.
A labile ligand (i.e., X
1
or X
2
) refers to a group which coordinates with less affinity to the metal ion (i.e., M) of a complex of formula (I) relative to the four pyridyl nitrogen atoms. Such ligand can therefore undergo rapid equilibrium with other labile ligands. Examples of a labile ligand include H
2
O, Cl, trifluoroacetate, or pyridine.
A metal complex of formula (I) possesses unexpectedly high specificity toward nucleic acid containing a bulge structure. As described above, a nucleic acid with such a structure is associated with various disorders. A metal complex of formula (I) can therefore be used in a diagnostic kit for detecting nucleic acid bulge-associated disorders.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
REFERENCES:
patent: 6348588 (2002-02-01), Cheng et al.
Chang et al., “Metal Ion Dependent Binding and Nuclease Activity of Hexaazacyclophane”, Journal of the Chinese Chemical Society 43:73-75, 1996.
Kappen et al., “Bulge-Specific Cleavage in Transactivation Response Region RNA and Its DNA Analogue by Neocarzinostatin Chromophore”, Biochemistry 34:5997-6002, 1995.
Kappen et al., “Characterization of a Covalent Monoadduct of Neocarzinostatin Chromophore at a DNA Bulge”, Biochemistry 36:14861-14867, 1997.
Kappen et al., “DNA Conformation-Induced Activation of an Enediyne for Site-Specific Cleavage”, Science 261:1319-1321, 1993.
Kappen
Cheng Chien-Chung
Wang Wen Jwu
Academia Sinica
Fish & Richardson P.C.
Marschel Ardin H.
LandOfFree
Nucleic acid bulge-detecting agent does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid bulge-detecting agent, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid bulge-detecting agent will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3284471