Double-stranded peptide nucleic acids

Details Double-stranded peptide nucleic acids Double-stranded peptide nucleic acids

1993-07-02

2001-05-08

Marschel, Ardin H. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S006120, C514S002600, C514S04400A, C530S333000, C530S402000

Reexamination Certificate

active

06228982

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to generally linear compounds or “strands” wherein naturally-occurring nucleobases or other nucleobase-binding moieties preferably are covalently bound to a polyamide backbone. In particular, the invention concerns compounds wherein two such strands coordinate through hydrogen bonds to form a DNA-like double strand.
BACKGROUND OF THE INVENTION
The transcription and processing of genomic duplex DNA is controlled by generally proteinaceous transcription factors that recognize and bind to specific DNA sequences. One strategy for the control of gene expression is to add to a cell double-stranded DNA or double-stranded DNA-like structures that will bind to the desired factor in preference to or in competition with genomic DNA, thereby inhibiting processing of the DNA into a protein. This modulates the protein's action within the cell and can lead to beneficial effects on cellular function. Naturally occurring or unmodified oligonucleotides are unpractical for such use because they have short in vivo half-lives and they are poor cell membrane penetrators.
These problems have resulted in an extensive search for improvements and alternatives. In order to improve half-life as well as membrane penetration, a large number of variations in polynucleotide backbones has been undertaken. These variations include the use of methylphosphonates, phosphorothioates, phosphordithioates, phosphoramidates, phosphate esters, bridged phosphoroamidates, bridged phosphorothioates, bridged methylenephosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various “plastic” DNAs, &agr;-anomeric bridges, and borane derivatives. The great majority of these backbone modifications lead to decreased stability for hybrids formed between the modified oligonucleotide and its complementary native oligonucleotide, as assayed by measuring T
m
values.
Consequently, there remains a need in the art for stable compounds that can form double-stranded, helical structures mimicking double-stranded DNA.
OBJECTS OF THE INVENTION
It is one object of the present invention to provide compounds that mimic the double-helical structure of DNA.
It is a further object of the invention to provide compounds wherein linear, polymeric strands coordinate through hydrogen bonds to form double helices.
It is another object to provide compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a non-sugar-phosphate backbone.
It is yet another object to provide therapeutic, diagnostic, and prophylactic methods that employ such compounds.
SUMMARY OF THE INVENTION
The present invention provides a novel class of compounds, known as peptide nucleic acids (PNAs), that can coordinate with one another or with single-stranded DNA to form double-stranded (i.e., duplex) structures. The compounds include homopolymeric PNA strands and heteropolymeric PNA strands (e.g., DNA/PNA strands), which coordinate through hydrogen bonding to form helical structures. Duplex structures can be formed, for example, between two complementary PNA or PNA/DNA strands or between two complementary regions within a single such strand.
In certain embodiments, each strand of the double-stranded compounds of the invention includes a sequence of ligands covalently bound by linking moieties and at least one of said linking moieties comprising an amide, thioamide, sulfinamide or sulfonamide linkage. The ligands on one strand hydrogen bond with ligands on the other strand and, together, assume a double helical structure. The compounds of the invention preferably comprise ligands linked to a polyamide backbone. Representative ligands include either the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine or thiouracil) or artificial bases (e.g., bromothymine, azaadenines or azaguanines, 5-propynylthymine, etc.) attached to a peptide backbone through a suitable linker. These ligands are linked to the polyamide backbone through aza nitrogen atoms or through amido and/or ureido tethers.
In certain preferred embodiments, the peptide nucleic acids of the invention have the general formula (I):
wherein:
n is at least 2,
each of L
1
-L
n
is independently selected from the group consisting of hydrogen, hydroxy, (C
1
-C
4
)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, at least one of L
1
-L
n
being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
each of C
1
-C
n
is (CR
6
R
7
)
y
where R
6
is hydrogen and R
7
is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R
6
and R
7
are independently selected from the group consisting of hydrogen, (C
2
-C
6
)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C
1
-C
6
)alkoxy, (C
l
-C
6
)alkylthio, NR
3
R
4
and SR
5
, where R
3
and R
4
are as defined above, and R
5
is hydrogen, (C
1
-C
6
)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C
1
-C
6
)alkyl, or R
6
and R
7
taken together complete an alicyclic or heterocyclic system;
each of D
1
-D
n
is (CR
6
R
7
)
z
where R
6
and R
7
are as defined above;
each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 2 but not more than 10;
each of G
1
-G
n−1
is —NR
3
CO—, —NR
3
CS—, —NR
3
SO— or —NR
3
SO
2
—, in either orientation, where R
3
is as defined above;
each pair of A
1
-A
n
and B
1
-B
n
are selected such that:
(a) A is a group of formula (IIa), (IIb) or (IIc) and B is N or R
3
N
+
; or
(b) A is a group of formula (IId) and B is CH;
where:
X is O, S, Se, NR
3
, CH
2
or C(CH
3
)
2
;
Y is a single bond, O, S or NR
4
;
each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;
each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
each R
1
and R
2
is independently selected from the group consisting of hydrogen, (C
1
-C
4
)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
each of G
1
-G
n−1
is —NR
3
CO—, —NR
3
CS—, —NR
3
SO— or —NR
3
SO
2
—, in either orientation, where R
3
is as defined above;
Q is —CO
2
H, —CONR′R″, —SO
3
H or —SO
2
NR′R″ or an activated derivative of —CO
2
H or —SO
3
H; and
I is —NHR′″R″″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, oligonucleosides and soluble and non-soluble polymers.
In certain embodiments, at least one A is a group of formula (IIc) and B is N or R
3
N
+
. In other embodiments, A is a group of formula (IIa) or (IIb), B is N or R
3
N
+
, and at least one of y or z is not 1 or 2.
Preferred peptide nucleic acids have general formula (IIIa)-(IIIc):
wherein:
each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;
each R
7′
is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;
n is an integer from 1 to 60;
each of k, l, and m is independently zero or an integer from 1 to 5;
p is zero or 1;
R
h
is OH, NH
2
or —NHLysNH
2
; and
R
i
is H or COCH
3
.
Particularly preferred are compounds having formula (IIIa)-(IIIc) wherein each L is independently selected from the g

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