Base analogues

Details Base analogues Base analogues

2000-04-18

2002-09-03

Shah, Mukund J. (Department: 1624)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S269000, C514S470000, C514S471000, C514S414000, C514S023000, C536S022100, C544S299000, C544S301000, C544S302000, C544S304000

Reexamination Certificate

active

06444682

ABSTRACT:

This invention concerns base analogues which may be used to make nucleoside analogues and nucleotide analogues which may be incorporated into nucleic acids and nucleic acid analogues e.g. PNA. Some of these analogues are base-specific and may be incorporated into DNA or RNA or PNA in the place of a single native base i.e. A, T, G, or C. Other analogues have the potential for base-pairing with more than one native base or base analogue.
The present invention provides a compound having the structure
where X═O or NH or S
Y═N or CHR
6
or CR
6
or CO
W═N or NR
6
or CHR
6
or CR
6
or S or CO
n=1 or 2 or 3
each R
6
is independently H or alkyl or alkenyl or alkoxy or aryl or a reporter moiety,
where necessary (i.e. when Y and/or W is N or CR
6
) a double bond is present between Y and W or W and W, and
Q is H or a sugar or a sugar analogue,
provided that
i) when n is 2 and X is NH and W is CHR
6
or CR
6
, and Y is CO, then at least one reporter moiety is present,
ii) when n is 1 and X is NH and W is N or NR
6
, then at least one reporter moiety is present,
iii) when n is 1 and X is 0 and Y is CHR
6
or CR
6
and W is CHR
6
or CR
6
, then at least one R
6
is a reporter moiety which is a reactive group or signal moiety or solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms,
iv) when n is 1 and X is NH and Y is CHR
6
or CR
6
and W is CHR
6
or CR
6
, then a least one reporter moiety is present,
v) when W is S, then n is 2 and W
n
is —CHR
6
—S— or ═CR
6
—S—,
vi) when n is 2 and X is NH and Y is CHR
6
or CR
6
, then at least one R
6
is a reporter moiety which is a reactive group or signal moiety or solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms.
Q may be
 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, SH or NH
2
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, or a reporter moiety,
or Q consists of one of the following modified sugar structures
or Q is a nucleic acid backbone consisting of sugar-phosphate repeats or modified sugar-phosphate repeats (e.g. LNA) (Koshkin et al, 1998, Tetrahedron 54, 3607-30) or a backbone analogue such as peptide or polyamide nucleic acid (PNA). (Nielsen et al, 1991, Science 254, 1497-1500).
When Q is H, these compounds are base analogues. When Q is a sugar or sugar analogue or a modified sugar, these compounds are nucleotide analogues or nucleoside analogues. When Q is a nucleic acid backbone or a backbone analogue, these compounds are herein called nucleic acids or polynucleotides.
Preferred general structures covered by the invention are
In the context of this invention, a nucleotide is a naturally occurring compound comprising a heterocyclic base and a backbone including a phosphate. A nucleoside is a corresponding compound in which a backbone phosphate may or may not be present. Nucleotide analogues and nucleoside analogues are analogous compounds having different bases and/or different backbones. A nucleoside analogue is a compound which is capable of forming part of a nucleic acid (DNA or RNA or PNA) chain, and is there capable of base-pairing with a base 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 more than one of the natural bases, e.g. with pyrimidines (T/C) or purines (A/G); or universal, by pairing with each of the natural bases with little discrimination; or it may pair with another analogue or itself.
In one preferred aspect of the invention, the base analogue is linked to a sugar moiety such as ribose or deoxyribose to form a nucleoside analogue. When the group R
5
is triphosphate, the nucleoside triphosphate analogues of the invention are capable of being incorporated by enzymatic means into nucleic acid chains.
Preferably n is 1 or 2, and W is N or NR
6
or CR
6
or CHR
6
.
In another preferred aspect, the nucleoside analogue or nucleotide analogue which contains a base analogue as defined is labelled with at least one 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 14-C or an iodine isotope. It may be an isotope detectable by mass spectrometry or NMR. It may be a signal moiety e.g. an enzyme, hapten, fluorophore, chromophore, 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. Linkage to a solid surface enables the use of nucleic acid fragments containing nucleoside analogues to be used in assays including bead based probe assays or assays involving arrays of nucleic acid samples or oligonucleotides which are interrogated with e.g. oligonucleotide or nucleic acid or even peptide or protein probes. 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.
To avoid risk of steric hindrance, a linker preferably has at least three chain atoms, e.g. —(CH
2
)
n
— where n is at least 3.
Two (or more) reporter moieties may be present, e.g. a signal moiety and a solid surface, or a hapten and a different signal moiety, or two fluorescent signal groups to act as donor and acceptor. Various formats of these arrangements may be useful for separation or detection purposes.
Purine and pyrimidine nucleoside derivatives labelled with reporter moieties are well known and well described in the literature. Labelled nucleoside derivatives have the advantage of being readily detectable during sequencing or other molecular biology techniques.
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 any that might be present on the base.
R
5
is OH, SH, NH
2
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 amide, 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 oligonucleotides is well known and described in the literature.
In the new base or 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. 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).
Nucleoside analogues of this invention are useful for labelling DNA or RNA or for incorporating in oligonucleotides or PNA

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