Pyrimidine derivatives for labeled binding partners

Details Pyrimidine derivatives for labeled binding partners Pyrimidine derivatives for labeled binding partners

1999-09-21

2002-07-02

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S025300, C536S025310, C536S025320, C536S025330, C536S025340, C536S025400, C435S006120, C435S087000, C435S090000, C514S256000, C514S267000, C544S242000, C544S245000, C544S249000

Reexamination Certificate

active

06414127

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the field of labels, particularly labels for diagnostic or analytical use. In particular, it relates to oligonucleotides that are modified to enhance the binding affinity or the binding specificity of the oligonucleotides for complementary sequences and that in addition optionally bear a readily detectable characteristic.
Sequence specific binding of oligonucleotides both to single stranded RNA and DNA and to duplex DNA is widely known. This phenomenon has been harnessed for a great variety of diagnostic, therapeutic and analytical, e.g., sequence determination or gene mapping, purposes. Previously, one objective of research in this field has been to increase the affinity of such oligonucleotides for their complementary sequences. For example, workers have described oligonucleotides containing 5-substituted pyrimidine bases that substantially increase the Tm for oligonucleotide binding to complementary bases (International Publication No. WO 93/10820).
Publications have described the use of fluorescent cytosine derivatives to prepare labeled DNA probes. See Inoue et al., Jpn. Kokai JP 62059293, (1987). In addition, fluorescent labeled nucleotides have been employed in DNA sequencing. See Prober et al., “Science” 238:336-341 (1987).
1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors are disclosed by Raeymaekers et al. (EP 541,153).
U.S. Pat. No. 5,502,177, discloses phenoxazine polycycle-containing oligonucleotides and monomers for preparing the oligonucleotides.
OBJECTS OF THE INVENTION
The invention compositions or methods accomplish one or more of the following objects.
An object of this invention is to increase the affinity of oligonucleotides for their complementary sequences.
An object of this invention is to increase the specificity of oligonucleotides for their complementary sequences.
Another object of this invention is to provide detectable labels for use in diagnostic assays.
Another object is to enhance diagnostic assays that use oligonucleotides.
Another object is to improve the therapeutic efficacy of oligonucleotides.
Another object is to improve the potency of oligonucleotides as antisense reagents that affect gene expression by altering intracellular metabolism of complementary RNA sequences encoding a target gene(s).
Another object is to provide chemical intermediates and synthesis methods to prepare the invention compositions.
These and other objects of the invention will be apparent when one considers the disclosure as a whole.
SUMMARY OF THE INVENTION
In accordance with the objects, the invention provides compounds having the structure (1)
and tautomers, solvates and salts thereof, wherein
R
1
is a binding partner, a protecting group, a linker or —H;
R
2
is A(Z)
X1
, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R
2
is not amine, protected O amine, nitro or cyano;
R
27
is independently —CH═, —N═, —C(C
1-8
alkyl)═ or —C(halogen)═, but no adjacent R
27
are both —N═, or two adjacent R
27
are taken together to form a ring having the structure,
where R
a
is independently —CH═, —N═, —C(C
1-8
alkyl)═ or —C(halogen)═, but no adjacent R
a
are both —N═;
R
34
is —O—, —S— or —N(CH
3
)—; and
X1 is 1, 2 or 3.
When the binding partner R
1
is an oligonucleotide, embodiments of the compounds of this invention include oligonucleotides of structure (2), (2A), (2B), or (2C)
wherein
D is —OH, protected —OH, an oligonucleotide coupling group or a solid support;
D
1
is an oligonucleotide coupling group, —OH, protected —OH or a solid support, wherein D
1
is bonded to one 2′ or 3′ position in the oligonucleotide of structure (2) and the adjacent 2′ or 3′ position in structure (2) is substituted with R
21
, provided that D and D
1
are not both an oligonucleotide coupling group or they are not both a solid support;
D
2
is —CO
2
R
5
, —C(O)N(R
5
)
2
, —SO
3
R
5
, —SO
2
N(R
5
)
2
or an activated derivative of —CO
2
H or —SO
3
H;
D
3
is a protecting group, —H or —(CH
2
)
2-6
—N(R
5
)
2
;
R
4
is independently a phosphodiester linkage or a phosphodiester substitute linkage, wherein R
4
is bonded to one 2′ or 3′ position in the structure (2) oligonucleotide and the adjacent 2′ or 3′ position in structure (2) is substituted with R
21
;
R
5
is independently —H or a protecting group;
R
21
is independently —H, —OH, halogen or a moiety that enhances the oligonucleotide against nuclease cleavage;
R
37
is independently —O—, —CH
2
— or —CF
2
—;
n is an integer from 0 to 98; and
B independently is a purine or pyrimidine base or a protected derivative thereof, provided that at least one B is a base of structure (3)
Embodiments include compositions useful as intermediates in making the structure (1) compounds, including intermediates having structure (4)
and tautomers, solvates and salts thereof wherein,
R
24
is a halogen; and
R
25
is —SH, —OH, ═S or ═O.
In a further embodiment, the invention includes contacting a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, with a sample suspected to contain a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes detecting the presence, absence or amount of a complex comprising a structure (2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7, and a nucleic acid having a base sequence that is at least substantially complementary to the structure (2), (2A), (2B), or (2C) oligonucleotide.
In a further embodiment, the invention includes converting a structure (4) compound to a compound of structure (1) where R
34
is —O— or —S— and the R
2
atom or moiety alpha to the ring containing R
27
is —O—, —S— or —CH
2
—, by displacing R
24
.
In a further embodiment, the invention includes converting a structure (4A) compound (a structure (1) compound where R
2
is replaced with —NH
2
); to a compound of structure (1), by reductive alkylation of the —NH
2
group to yield a structure (1) compound where the R
2
moiety alpha to the ring containing R
27
is —NH—.
DETAILED DESCRIPTION OF THE INVENTION
The invention comprises all combinations formed by independently selecting individual Markush group members and assembling them in accordance with the teachings herein. The invention optionally excludes any feature or substance found in, or obvious over, the prior art.
Structural formulas are designated as parenthetical numerals. We intend that designation of aromaticity with respect to carbocycles and heterocycles herein includes any highly resonant unsaturated ring structure. Alternatively, placement of double bonds, where indicated, represents one potential structure for the depicted compound but we intend this depiction to include other resonant states of the compound as well as protonated and charged species, only one of which a structure may show.
The invention includes invention compounds in unpurified, substantially purified and purified forms. It includes invention compounds that are present with any additional component(s) such as a solvent, reactant or by-product that is present during invention compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of an invention compound.
Halo and halogen mean F, Cl, Br or I.
Alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof. Alkyl includes all isomers, e.g., stereoisomers, positional isomers, diastereomers and regioisomers. Alkyl moieties that are unsaturated will typically contain 1, 2, 3 or more —CH═CH— or —C≡C— groups, usually one such group.
Substituted alkyl means unbranched, branched or cyclic hydrocarbons that are saturated or unsaturated, or combinations thereof, where the hydrocarbon contains a heteroatom li

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