Nucleoside analogues

Details Nucleoside analogues Nucleoside analogues

1999-02-05

2001-11-06

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S023100, C536S027110, C536S027130, C536S027200, C435S006120

Reexamination Certificate

active

06313286

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. This invention describes new nucleoside analogues which are capable of forming base pairs with more than one nucleotide, and which carry a reporter group.
P Kong Thoo Lin and D M Brown reported (Nucleic Acids Research, 1989, Vol 17, pages 10373-10383) the synthesis of a monomer containing the degenerate base analogue P, (6H, 8H-3-4-dihydropyrimido[4,5-c][1.2]oxazin-7-one). Due to the ability of this base analogue to exist in amino and imino tautomers, it can base pair with both purine bases A and G. The authors found that oligomers containing one or more P bases formed DNA duplexes of comparable stability to the parent duplexes and which also showed sharp transitions on melting. Further evidence confirmed that the base pairs P/A and P/G were essentially of the Watson-Crick type.
The authors also discussed the potential use of this base analogue in hybridisation probes and primers, when the base can be put at positions of degeneracy thus both avoiding the need for multiple-chain primers (or probes) and significantly reducing the chain multiplicity. Indeed oligonucleotides containing several P bases were effective in dot blot hybridisation and DNA sequencing experiments.
In Nucleic Acids Research, 1992, Vol 20, No 19, pages 5149-5152, these authors also demonstrated the use of oligonucleotides containing the P base at the 3′-end and elsewhere as primers in polymerase chain reaction (PCR) experiments.
EP 0 235 301 describes pyridopyrimidine nucleotide derivatives which can form base pairs with guanine or adenine, and which are fluorescent in their own right. Excitation of, these derivatives is in the UV region (330-350 nm)
Purine and pyrimidine base nucleosides and nucleotides have been derivatised with reporter groups and are well known and widely used for labelling DNA or RNA and in other molecular biology applications. But these molecules are capable of base-pairing only with one of A, C, G and T and the nucleoside triphosphates are often poor enzyme substrates. There is a need for a nucleoside analogue whose triphosphate is a good enzyme substrate and which has a base analogue that is degenerate, by having the ability to base pair with two or three of the natural bases e.g. with both pyrimidines (T/C) or both purines (A/G), or universal, by forming base-pairs with each of the natural bases without discrimination. This invention makes use of the P base to address these needs.
The invention provides a nucleoside analogue of the formula
X is O, S, Se, SO, CO or N—R
10
,
the curved dotted line represents an optional link between R
6
and R
10
,
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,
Z is O, S, Se, SO, NR
9
or CH
2
,
and R
6
, R
7
, R
8
, R
9
and R
10
are the same or different and each is H or alkyl or aryl or a reporter moiety,
n is 0 or 1,
provided that at least one reporter moiety is present,
wherein a reporter moiety comprises a linker group, together with a signal moiety or a solid surface or a reactive group by which a signal moiety or a solid surface may be attached to the nucleoside analogue.
A nucleoside analogue is a molecule which is capable of being incorporated, either chemically or enzymatically, into an oligomeric or polymeric nucleic acid (DNA or RNA) chain, and when so incorporated of forming a base pair with a nucleotide in a complementary chain or base stacking in the appropriate nucleic acid chain.
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 a solid surface may be attached, before or after incorporation of the nucleoside analogue in a nucleic acid chain. Such reporter groups are well known and well described in the literature.
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 the 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, generally by chemical means, in a polynucleotide chain. The use of phosphoramidites and related derivatives in synthesising ofigonucleotides is well known and described in the literature. From nucleosides (R
5
is OH) it is readily possible to make the corresponding nucleotides (R
5
is triphosphate) by literature methods.
In the new nucleoside analogues to which this invention is directed, at least one reporter moiety is present preferably 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. 1990, 1547-8; J. Med. Chem., 1988, 31. 2040-8). Reporters in the form of isotopic labels may be introduced into phosphate groups by literature methods (Analytical Biochemistry, 214, 338-340, 1993; WO 95/15395).
The base analogue with which this invention is concerned is a fused ring structure in which one ring contains an optional double bond shown in the diagram as a dashed line. When a double bond is present, the ring is flat and no chirality problem arises. When a single bond is present, the introduction of a reporter moiety R
6
or R
8
creates a chiral centre. It may be helpful if the chirality is chosen so that the reporter sticks out of, and not back into, the nucleic acid helix.
The nucleoside analogues of this invention are usef

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