Heterocyclic compounds and their use for isolating nucleic...

Details Heterocyclic compounds and their use for isolating nucleic... Heterocyclic compounds and their use for isolating nucleic...

1999-07-02

2002-06-11

Gitomer, Ralph (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S026100, C536S023100, C536S024300, C514S049000, C514S051000

Reexamination Certificate

active

06403786

ABSTRACT:

The invention concerns heterocyclic compounds which can be used to label, detect and sequence nucleic acids.
Nucleic acids are of major importance in the living world as carriers and transmitters of genetic information. Since their discovery by F. Miescher they have aroused a wide scientific interest which has led to the elucidation of their function, structure and mechanism of action.
An important tool for explaining these connections and for solving the problems was and is the detection of nucleic acids and namely with regard to their specific detection as well as with regard to their sequence i.e. their primary structure.
The specific detectability of nucleic acids is based on the property of these molecules to interact, i.e. to hybridize, with other nucleic acids to form base pairs by means of hydrogen bridges. Nucleic acids (probes) labelled in a suitable manner, i.e. provided with indicator groups, can thus be used to detect complementary nucleic acids (target).
The determination of the primary structure (sequence), i.e. the sequence of the heterocyclic bases of a nucleic acid, is achieved by means of sequencing techniques. Knowledge of the sequence is in turn a basic requirement for a targeted and specific use of nucleic acids for molecular biological problems and working techniques.
The sequencing also ultimately utilizes the principle of specific hybridization of nucleic acids to one another. As mentioned above labelled nucleic acid fragments are also used for this. Hence a suitable labelling of nucleic acids is an essential prerequisite for any detection method.
At an early period radioactive labelling was mainly used with suitable isotopes such as
32
P or
35
S. However, the disadvantages of using radioactive reagents are obvious: such work requires special room facilities and licences as well as a controlled and elaborate disposal of the radioactive waste. The reagents for radioactive labelling are expensive. It is not possible to store such labelled probes for long periods due to the short half-life of the above-mentioned nuclides.
Therefore many attempts have been made in recent years to circumvent these serious disadvantages i.e. to get away from using a radioactive label. However, the high sensitivity of this type of label should be retained as far as possible.
Major advances have in fact already been achieved [see e.g. Nonradioactive Labeling and Detection of Biomolecules (Kessler, C., publ.) Springer Verlag Berlin, Heidelberg 1992].
An essential requirement for any detection of a nucleic acid is the prior labelling. As indicated above it is desirable to achieve this in a non-radioactive manner. Whereas radioactive labelling of nucleic acids is usually carried out by the enzymatically catalysed incorporation of appropriate radioactive nucleoside triphosphates, non-radioactive labelling has to be achieved by incorporating a suitable signal or reporter group.
Haptens (such as biotin or digoxigenin), enzymes (such as alkaline phosphatase or peroxidase) or fluorescent dyes (such as fluorescein or rhodamine) have, among others, mainly proven to be suitable as non-radioactive indicator molecules. These signal groups can be attached to or incorporated in nucleic acids by various methods.
A relatively simple procedure is for example to label the 5′ end of an oligonucleotide provided with a terminal amino group by means of activated indicator molecules of the above-mentioned type. However, this only allows the introduction of one or a few indicator molecules into only a low molecular oligomer whereas a denser labelling of longer chain, high molecular nucleic acids with the aim of achieving a high sensitivity usually has to be accomplished by incorporating nucleoside triphosphates provided with reporter groups by means of polymerases as in a de novo synthesis.
Such current methods are known to a person skilled in the art as nick translation [Rigby, P. W. et al., (1977), J.Mol.Biol. 113, 237] and random primed labeling [Feinberg, A. P. & Vogelstein, B. (1984) Anal.Biochem. 137, 266]. A further method is the so-called 3′-tailing reaction with the aid of the enzyme terminal transferase [e.g. Schmitz, G. et al (1991) Anal.Biochem. 192, 222].
The nucleoside triphosphates which have been previously used in these methods are almost exclusively appropriately modified derivatives of the heterocyclic bases adenine, guanine, cytosine and thymine in the deoxyribonucleotide series or adenine, guanine, cytosine and uracil in the ribonucleotide series. Such derivatives are described for example by Langer et al. in Proc.Natl.Acad.Sci. USA 78, 6635 (1981), Mühlegger et al. Biol.Chem. Hoppe-Seyler 371, 953 (1990) and in EP 0 063 879. In this case the building blocks which occur naturally in DNA and RNA are used in a labelled form i.e. provided with signal groups.
The main disadvantages of these N-nucleosides is that the N-glycosidic bond is sensitive to acidic pH conditions and they can be degraded by nucleases.
Furthermore individual C-nucleosides (see e.g. Suhadolnik, R. J. in “Nucleoside Antibiotics”, Wiley-Interscience, New York 1970) and their use in the therapeutic (antiviral or cancerostatic) field has also been known for a long time. In addition fluorescent C-nucleoside derivatives and their incorporation into DNA and RNA oligonucleotides has been described (WO 93/16094). The so-called intrinsic fluorescence of these nucleosides is, however, many times lower with regard to quantum yield than that of the special fluorophores such as fluorescein or corresponding rhodamine derivatives. A further disadvantage of the self-fluorescent C-nucleosides is their comparatively low excitation and emission wavelengths. As a result detection systems which are based on such derivatives only have a low sensitivity of detection and on the other hand influences of the measuring environment which interfere spectrally (such as biological material, autofluorescence of gel matrices etc.) have a very major effect. Hence the known nucleosides and nucleoside derivatives have a series of disadvantages which especially have an adverse effect on the detection of nucleic acids. Hence the object of the invention is to provide nucleoside derivatives modified with signal groups for the detection of nucleic acids which do not have the afore-mentioned disadvantages i.e. in particular are more stable and at the same time capable of being processed enzymatically and are suitable for the detection of nucleic acids at a practicable wavelength.
The object is achieved by heterocyclic compounds of the general formula I
in which
R
1
and R
2
can be the same or different and represent hydrogen, oxygen, halogen, hydroxy, thio or substituted thio, amino or substituted amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
R
3
and R
4
each represent hydrogen, hydroxy, thio or substituted thio, amino or substituted amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, a protecting group or a reporter group,
R
5
represents hydrogen, hydroxy, thio or substituted thio, amino or a substituted amino group, a reactive trivalent or pentavalent phosphorus group such as e.g. a phosphoramidite or H-phosphonate group, an ester or amide residue that can be cleaved in a suitable manner or a reporter group,
R
4
and R
5
together form a further bond between C-2′ and C-3′ or an acetal group,
R
6
represents hydrogen or a hydroxy, thio or substituted thio, amino or substituted amino group,
R
7
represents hydrogen, a monophosphate, diphosphate or triphosphate group or the alpha, beta or gamma thiophosphate analogue of this phosphoric acid ester or a protective group as well as possible tautomers and salts thereof.
X denotes methylene or methine substituted with halogen, hydroxy, thio or substituted thio, amino or substituted amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group, or oxygen and n=0 or 1, Z denotes nit

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