Tricyclic base analogues

Details Tricyclic base analogues Tricyclic base analogues

2000-02-01

2003-07-29

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S026230, C536S026260, C536S026600, C536S027100, C435S007200, C435S091200

Reexamination Certificate

active

06600028

ABSTRACT:

This invention concerns base analogues which may be used to make nucleoside analogues and nucleotide analogues which may be incorporated into nucleic acids. Some of these analogues are base-specific and may be incorporated into DNA or RNA 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 2′-deoxyribosides of such analogues as (1) (P: Kong Thoo Lin and Brown, 1989, Nucleic Acids Research, 17, 10373-83), N6-methoxyadenine and N6-methoxy-2,6-diaminopurine have been shown to be extremely useful in mixed sequence oligonucleotide primers used in PCR and DNA sequencing. In addition, the base pairing characteristics of the 2′-deoxynucleoside 5′-triphosphate of P have been exploited in a PCR-based random mutagenesis procedure (Zaccolo et al, 1996, J. Mol. Biol., 255, 589-603):
Synthesis of a nucleoside with a tricyclic base analogue where the third ring is between the 6 and 7 positions (purine nomenclature) was described by Schram and Townsend (1971) Tetrahedron Lett 49,. 4757-4760. However, this has a blocked hydrogen bonding face and would not be expected to participate in base pairing observed in native nucleic acids or, as a triphosphate, in polymerase mediated incorporation reactions.
The present invention provides a compound having the structure(2)
where
W is an alkylene or alkenylene chain of 0-5 carbon atoms any of which may carry a substituent R
8
,
X is O or N or NR
12
or CR
10
,
X′ is O or S or N,
provided that when X′ is O or S, X is C,
Y is CH or N,
R
6
is H or NH
2
or SMe or SO
2
Me or NHNH
2
,
each of R
7
and R
8
is independently H or F or alkyl or alkenyl or aryl or acyl or a reporter moiety,
each of R
9
and R
12
is independently H or alkyl or alkenyl or aryl or acyl or a reporter moiety,
R
10
is H or ═O or F or alkyl or alkenyl or aryl or acyl or a reporter moiety,
the dotted line indicates one optional double bond,
Q is H or a sugar moiety or a sugar analogue including but not limited to the structure
 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).
Preferably:
W=CH
2
X=O
X′=N
Y=CH or N
R
6
=H or NH
2
R
7
=H or reporter moiety.
The dotted line in the structure (2) shows that either —X—CHR
7
—W— or —X═CR
7
—W— or —X—CR
7
═W— is present. Of course, when X is O the second of these structures is not possible.
Depending on the identity of W, the ring containing X′ and X contains from 6 to 11 members.
Alkyl, alkenyl, aryl and acyl groups herein preferably contain 1-20 carbon atoms.
Any unfilled valencies are to be understood as being filled by H.
The 2-deoxynucleoside analogue termed as P (Kong, Thoo Lin and Brown, 1989, Nucleic Acids Research 17, 10373-83) can exist in two tautomeric forms as indicated below:
Base analogues of this invention where X′=N and X=O or N or CR
10
also exhibit similar types of tautomerism, e.g.
The exact ratio of these tautomers can be affected by the X substituent. The changes in tautomeric ratio can have subtle effects on the hybridisation properties of the analogue. For convention only one tautomer is drawn within this patent though it is implicit that both can be present.
When Q is H, these compounds are base analogues. When Q is a sugar moiety 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.
In the context of the this invention, a nucleotide is a naturally occurring compound comprising a base and a sugar 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) 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 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; 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.
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, electrochemical label or signal compound adapted for detection by mass spectrometry. 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 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, before or after hybridisation.
Two (or more) reporter groups may be present, e.g. a signal group and a solid surface, or a hapten and a different signal group, or two fluorescent signal groups to act as donor and acceptor. Various formats of these arrangements may be useful for separation 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 i

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