Base analogues

Details Base analogues Base analogues

2001-07-03

2003-12-16

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S023100, C536S025300

Reexamination Certificate

active

06664058

ABSTRACT:

INTRODUCTION
The present invention relates to novel compounds e.g. modified base analogues that can be used to label nucleic acids which can be used in a wide variety of molecular biology applications.
BACKGROUND
Nucleic acid molecules labelled with reporter groups have been used in many molecular biology techniques such as sequencing and hybridisation studies. The labelled nucleic acid molecules have been produced by a variety of methods. These methods have included labelled nucleoside, deoxynucleoside, or dideoxynucleoside triphosphates, labelled phosphoramidites and direct coupling of labels to nucleic acids (Renz, EP 120376). The labelled nucleotides and labelled nucleic acid molecules produced can be used in hybridisation studies of nucleic acid and nucleic acid sequencing. A wide variety of labels have been used in these techniques including radioactive isotopes, eg
3
H,
14
C,
32
P,
33
P and
35
S, hapten, biotin, mass tags or fluorescence.
There has been increasing interest in the use of modified base and nucleotide analogues in the labelling of nucleic acids. Some of these analogues are base specific and may be incorporated into nucleic acids in the place of a single natural base i.e. A, T, G or C. Other analogues have the potential to base pair with more than one natural base and hence be incorporated in the place of more than one natural base.
WO 97/28177 discloses nucleoside analogues containing the structure
Wherein X is O, S, Se, SO, CO or NR
7
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-hydorcarbyl or a reporter group,
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,
Z is O, S, Se, SO, NR
9
or CH
2
and R
6
, R
7
, R
8
, R
9
are the same or different and each is H, alkyl, aryl or a reporter group.
WO 99/06422 discloses base analogues of the structure
where X=O or NH or S
Y=N or CHR
6
or CR
6
W=N or NR
6
or CHR
6
or CR
6
n=1 or 2
each R
6
is independently H or O 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,
Q is a sugar or sugar analogue
SUMMARY OF PRESENT INVENTION
The present invention describes novel compounds of the formula
Wherein Q is H or a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue, Y=O, S, NR
10
, where R
10
is H, alkyl alkenyl, alkynyl, X is H, alkyl, alkenyl, alkynyl, aryl, heteroaryl or a combination thereof or, preferably, a reporter group.
DETAILED DESCRIPTION OF INVENTION
In a first aspect, the present invention provides novel compounds of the formula (I), Wherein Q is H or a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue, Y=O, S, NR
10
, where R
10
is H, alkyl alkenyl, alkynyl, X is H, alkyl, alkenyl, alkynyl, aryl, heteroaryl or a combination thereof or preferably a reporter group. The reporter group may be joined to the heterocycle via a suitable linker arm, which can be similar to the options already defined for X or may be larger. In this aspect, suitably X can comprise a chain of up to 30 atoms, more preferably up to 12 atoms. The reporter may contain more than 12 atoms. X may also contain a charged group, which imparts a net positive or negative charge to the nucleotide base.
Suitably, Q may be H or a group selected from
Where Z is O, S, Se, SO, NR
9
or CH
2
where R
9
is H, alkyl, alkenyl, alkynyl or a reporter, 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, NHR
11
where R
11
is alkyl, alkenyl, alkynyl, or a reporter group. Suitably, the hydrocarbyl group has up to 6 carbon atoms. R
11
and R
9
may comprise a chain of up to 30 atoms, preferably up to 12 atoms.
R
5
is OH, SH or NH
2
or mono-, di or tri-phosphate or-thiophosphate, or corresponding boranophosphate, or one of R
2
, R
4
, and R
5
is a phosphoramidite or other group for incorporation in a polynucleotide chain, or a reporter group;
or Q may be of one of the following modified sugar structures:
Acyclic sugars having structures (ii) or (iii)
wherein R
12
is C1-C4 alkyl, hydroxyC1-C4alkyl, or H, preferably methyl, hydroxymethyl or H, or sugars having structures (iv) to (vi)
R
14
=C1-C6 alkyl, hydroxy C1-C6 alkyl, C1-C6 alkylamine, C1-C6 carboxyalkyl or preferably a reporter moiety.
(vii) 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) or a polycationic ribonucleic acid guanidine (RNG), Bruice et al, 1995 PNAS 92 6097, or pentopyranosyl oligonucleotides (HNA), Eschenmosser A., 1999, Science, 284 2118-2124.
In one preferred embodiment, when Q is H, these compounds are base analogues. In a second preferred embodiment, Q is a sugar or sugar analogue or a modified sugar, e.g. a group having a structure according to (i) to (vi) and the compounds are nucleotide analogues or nucleoside analogues. When Q is a nucleic acid backbone or a backbone analogue, (vii), these compounds are herein after called nucleic acids or polynucleotides.
When Q is a group of structure (i) 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 group in addition to any that might be present on the base Preferably, R
1
=H, R
2
=H, OH, F, N
3
, NH
2
, NH(CH
2
)n R
13
or O—(CH
2
)
n
NH
2
where n is 0-12, R
4
=H, OH, N
3
, NH
2
, F, OR
3
, R
3
=H, OH or OR
13
where R
13
is alkyl, alkenyl, alkynyl or a reporter. More preferably at least one of R
1
and R
2
and at least one of R
3
and R
4
is H.
R
5
is OH, SH, NH
2
or mono, di- or tri-phosphate or thiotriphosphate or corresponding boranophosphate. When R
5
is triphosphate, such triphosphate nucleotides may be incorporated into a polynucleotide chain by using a suitable template- primer together with a DNA polymerase or reverse transcriptase and appropriate dNTPs and ddNTPs when necessary. NTPs can be used with suitable RNA polymerases. The compounds of the present invention may be incorporated into a PCR product using standard techniques or used in the production of cDNA from a suitable RNA template, primer dNTP mix and reverse transcriptase. Oligonucleotide and polynucleotide chains may also be extended by nucleotide analogues of the present invention by the use of terminal transferase.
Alternatively, one of R
2
, R
4
, 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 another preferred embodiment, the nucleoside analogue or nucleotide analogue which contains a base analogue as defined is labelled with at least one reporter group. Suitable reporter moieties may be selected from various types of reporter. The reporter group 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 group or moiety e.g. an enzyme, hapten, fluorophore, chromophore, chemiluminescent group, Raman label or electrochemical label. Particularly preferred reporters are fluorescent dyes such as fluorescein, rhodamine, bodipy and cyanines.
The

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