Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1999-05-03
2001-05-29
Riley, Jezia (Department: 1656)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S395000, C435S006120, C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024330, C536S024320
Reexamination Certificate
active
06239159
ABSTRACT:
Nucleic acids are manipulated in vitro in a wide variety of research and diagnostic techniques. The methods can involve the synthesis of nucleic acid probes by means of polymerase or terminal transferase enzymes for the purposes of labelling or determination of base sequence identity. Labelling often involves the incorporation of a nucleotide which is chemically labelled or which is of a particular chemical composition so as to make it detectable. Nucleic acid probes made in this way can be used to determine the presence of a nucleic acid target which has a complementary sequence by means of hybridisation of the probe to the target.
Another method for introducing chemically labelled or otherwise modified nucleotides into DNA involves chemical synthesis using nucleoside phosphoramidite or other precursors which are linked together in any desired sequence in oligonucleotide synthesisers, the final product being indistinguishable from DNA made by the use of polymerases.
In certain situations it is useful to be able to incorporate a base analogue into an oligo- or poly-nucleotide which does not have the base pairing specificity of the natural bases. Hypoxanthine has been proposed for this purpose in the past because it can form hydrogen bonds with all four natural bases though the strength of binding varies with the different bases. More recently 3-nitropyrrole (WO 94/06810) and 5-nitroindole (Loakes and Brown, Nucleic Acids Research, 1994, 22, 4039-43) have been proposed. They are unlike native bases in their structure, containing only one heteroatom each within the ring structure, nitropyrrole consisting of a single 5-membered ring, and each possessing a nitro grouping as the single exocyclic constituent. Both base analogues have been incorporated into oligonucleotides by chemical synthesis. Melting temperatures (Tm) have been determined for the modified oligonucleotide hybridised to its template. Whilst the Tm for the oligonucleotides was lower when a nitropyrrole or nitroindole was present than when the correct native base was included, the nitroindole and nitropyrrole did not discriminate strongly between different bases at the position opposite within the complementary strand. The synthesis of an imidazole-carboxamide nucleoside monomer for incorporation has also been described (Pochet et al, Bioorganic and Medicinal Chemistry Letters, 5, 1679, (1995)).
The present invention provides a nucleoside analogue of the formula:
where Z is O, S, Se, SO, NR
9
or CH
2
,
R
1
, R
2
, R
3
and R
4
are the same or different and each is H, OH, F, NH
2
, N
3
, O-hydrocarbyl or a reporter moiety,
R
5
is OH or mono, di- or tri-phosphate or -thiophosphate, or corresponding boranophosphate,
or one of R
2
and R
5
is a phosphoramidite or other group for incorporation in a polynucleotide chain,
and M is:
where X
1
, X
2
and X
3
are the same or different and each is C or N, when X
3
is N then there is no R
7
group,
R
6
and R
7
are the same or different and each is H, NO
2
, CO, COR
8
, OR
8
, CN, O, CON(R
8
)
2
, COOR
8
, SO
2
R
8
, SO
3
R
8
, SR
8
, NHCHO, (CH
2
)
n
N(R
8
)
2
, halogen, or a reporter moiety,
each of R
8
and R
9
is H or hydrocarbyl or a reporter moiety,
and n is 0, 1, 2, 3 or 4,
provided that when R
5
is not triphosphate, then the nucleoside analogue comprises a reporter moiety.
A nucleoside analogue is a compound which is capable of being incorporated, by enzymatic or chemical means, in a nucleic acid (DNA or RNA) chain, and is there capable of base-pairing with a nucleotide residue in a complementary chain or base stacking in the appropriate nucleic acid chain. A nucleoside analogue may be specific, by pairing with only one complementary nucleotide; or degenerate, by base pairing with two or three of the natural bases, e.g. with pyrimidines (T/C) or purines (A/G); or universal, by pairing with each of the natural bases without discrimination.
M is a moiety hereafter called a base analogue which may be, but is not necessarily, a base. For example, M may comprise a pyrrole or an indole ring structure.
In one preferred aspect of the invention, the group R
5
is triphosphate. When this is the case, the nucleoside triphosphate analogues of the invention are capable of being incorporated by enzymatic means into nucleic acid chains.
In another preferred aspect, the nucleoside analogue of the invention contains a reporter moiety. A reporter moiety may be any one of various things. It may be a radioisotope by means of which the nucleoside analogue is rendered easily detectable, for example 32-P or 33-P or 35-S incorporated in a phosphate or thiophosphate or phosphoramidite or H-phosphonate group, or alternatively 3-H or 125-I. It may be a stable isotope detectable by mass spectrometry. It may be a signal moiety e.g. an enzyme, hapten, fluorophore, chemiluminescent group, Raman label or electrochemical label. The reporter moiety may comprise a signal moiety and a linker group joining it to the remainder of the molecule, which linker group may be a chain of up to 30 carbon, nitrogen, oxygen and sulphur atoms, rigid or flexible, unsaturated or saturated as well known in the field. The reporter moiety may comprise a solid surface and a linker group joining it to the rest of the molecule. The reporter moiety may consist of a linker group with a terminal or other reactive group, e.g. NH
2
, OH, COOH, CONH
2
or SH, by which a signal moiety and/or solid surface may be attached, before or after incorporation of the nucleoside analogue in a nucleic acid chain.
Purine and pyrimidine nucleoside analogues labelled with reporter moieties are well known and well described in the literature. Labelled nucleoside analogues have the advantage of being readily detectable during sequencing or other nucleic acid manipulations.
In the moiety M, X
1
and X
3
are preferably C and X
2
is preferably N. Examples of preferred moieties M are:
In (i), pyrrole dicarboxamide, either carboxamide group could act as a linkage to a signal moiety or could be replaced by a reporter moiety. In (ii) and (iii), Rp designates a reporter moiety. In (iv), a reporter moiety may also be present at the 2, 4 or 5 position. In (v) a nitro group is present at the 4, 5 or 6 position, and a reporter moiety may also be present. Other examples of M are:
3-aminoethyl-5-nitroindole
3-aminomethyl-4-carboxamido pyrrole
R1 = sugar triphosphate or sugar phosphoramidite R2 = reporter/substituent
R
1
, R
2
, R
3
and R
4
may each be H, OH, F, NH
2
, N
3
, O-alkyl or a reporter moiety. Thus ribonucleosides, and deoxyribonucleosides and dideoxyribonucleosides are envisaged together with other nucleoside analogues. These sugar substituents may contain a reporter moiety in addition to one or two present in the base.
R
5
is OH or mono-, di- or tri-phosphate or -thiophosphate or corresponding boranophosphate. Alternatively, one of R
2
and R
5
may be a phosphoramidite or H-phosphonate or methylphosphonate or phosphorothioate, or an appropriate linkage to a solid surface e.g. hemisuccinate controlled pore glass, or other group for incorporation, hemisuccinate controlled pore glass, or other group for incorporation, generally by chemical means, in a polynucleotide chain. The use of phosphoramidites and related derivatives in synthesising oligonucleotides is well known and described in the literature.
Preferably the moiety M contains a reporter moiety. When a reporter moiety is present, R
5
is preferably triphosphate, or alternatively one of R
2
and R
5
is preferably a phosphoramidite residue. Alternatively when a reporter moiety is not present, the moiety M may itself be detectable, e.g. by binding an antibody or other specific binding reagent which carries an enzyme or fluorescent group.
In the new nucleoside analogues to which this invention is directed, at least one reporter moiety is preferably present in the base analogue or in the sugar moiety or a phosphate group. Reporter moieties may be introduced into the sugar moiety of a nucleoside analogue by literature methods (e.g. J. Chem. Soc. Chem. Commun. 199
Brown Daniel
Hamilton Alan
Loakes David
Simmonds Adrian
Smith Clifford
Amersham Pharmacia Biotech UK Limited
Marshall O'Toole Gerstein Murray & Borun
Riley Jezia
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