Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-09-15
2001-01-16
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S007100, C435S091100, C435S091200, C536S022100, C536S025300, C536S025340, C536S026260, C536S026710, C536S026800, C536S027130, C536S027200, C536S028100
Reexamination Certificate
active
06174998
ABSTRACT:
The invention concerns C-nucleosides and derivatives thereof as well as their use for labelling, detecting and sequencing nucleic acids.
Nucleic acids are of central importance in living nature as carriers or transmitters of genetic information. Therefore since their discovery by F. Miescher they have stimulated a broad scientific interest which has led to the elucidation of their function, structure and mechanism of action. The increasing knowledge of these basic mechanisms in molecular biology has made it possible in recent years to make new combinations of genes. This technology opens for example new opportunities in medical diagnosis and therapy and in plant breeding.
An important tool for elucidating these relationships and to solve problems was and is the detection of nucleic acids 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 or hybridize with other nucleic acids by forming base pairs via 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 carried out by means of sequencing techniques. This knowledge of the sequence is in turn a prerequisite for a targetted and specific use of nucleic acids in problems and methods of molecular biology. In the end sequencing also utilizes the specific hybridization among nucleic acids. Labelled nucleic acid fragments are also used for this as mentioned above.
Consequently the suitable labelling of nucleic acids is an indispensable prerequisite for any detection method.
Radioactive labelling with suitable isotopes such as
32
P or
35
S was already used for this at an early stage. The disadvantages of using radioactive reagents are, however, obvious: such work requires specially equipped facilities and permits as well as a controlled and complicated disposal of the radioactive waste. Furthermore the reagents for radioactive labelling are expensive. It is not possible to store such labelled samples for long periods due to the short half-lives of the above nuclides.
In recent years there have therefore been many attempts to circumvent these serious disadvantages i.e. to get away from radioactive labelling. In doing so the high sensitivity of this type of label should be retained as far as possible.
Major advances have in fact already been achieved [see for example Nonradioactive Labeling and Detection of Biomolecules (Kessler, C., pub.) Springer Verlag, Berlin, Heidelberg 1992].
An essential prerequisite for any detection of a nucleic acid is that it should be previously labelled and—as explained above—if possible in a non-radioactive manner. Whereas the radioactive labelling of nucleic acids is usually carried out by enzymatically catalysed incorporation of corresponding radioactive nucleoside triphosphates, non-radioactive labelling must be achieved by incorporating a suitable signal or reporter group.
Non-radioactive indicator molecules that have proven to be suitable among others are above all haptens (such as biotin or digoxigenin), enzymes (such as alkaline phosphatase or peroxidase) or fluorescent dyes (such as fluorescein or rhodamine). These signal groups can be attached to or incorporated into 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 function by means of activated indicator molecules of the above-mentioned type. However, this only enables one or a few indicator molecules to be introduced into only a low molecular oligomer whereas a denser labelling of longer chained, high molecular nucleic acids with the goal of achieving a higher sensitivity must usually be achieved by incorporating nucleoside triphosphates provided with reporter groups by means of polymerases along the lines of a de novo synthesis.
Corresponding 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 labelling [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.
The nucleoside triphosphates which have been used up to now 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 for example described by Langer et al., Proc. Natl. Acad. Sci. USA 78, 6635 (1981); Muhlegger et al., Biol. Chem. Hoppe-Seyler 371, 953 (1990) and in EP 0 063 879. In this case the building blocks that occur naturally in DNA and RNA are used in a labelled form i.e. in a form provided with signal groups. The main disadvantages of the N-nucleosides are the sensitivity of the N-glycosidic bond towards acidic pH conditions and degradability by nucleases.
Furthermore individual C-nucleosides (see e.g. Suhadolnik, R. J. in Nucleoside Antibiotics, Wiley-Interscience, New York 1970) and their therapeutic use (antiviral or cancerostatic) has 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 inherent fluorescence of these C-nucleosides is, however, many-fold less with regard to quantum yield than of special fluorophores such as fluorescein or corresponding rhodamine derivatives. A further disadvantage of the autofluorescent C-nucleosides is their relatively low excitation and emission wavelengths. As a consequence detection systems which are based on such derivatives only have a low detection sensitivity and on the other hand spectrally interfering influences of the measurement environment (such as biological material, autofluorescence of gel matrices etc.) become very apparent.
The known nucleosides and nucleoside derivatives thus have a number of disadvantages which in particular have adverse effects 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 said disadvantages i.e. are in particular more stable and at the same time able to be processed enzymatically and are suitable for the detection of nucleic acids at a more practical wavelength.
The object is achieved by pyrrolo-[3,2-d]pyrimidine, pyrazolo-[4,3-d]pyrimidine and pyrimidine-furanosides of the general formulae I-V:
in which R
1
, R
2
, R
3
, R
4
can be same or different and represent hydrogen, 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
5
and R
6
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
7
represents hydrogen, hydroxy, thio or substituted thio, amino or substituted amino, a phosphoramidite or H-phosphonate group, an ester or amide residue that can be cleaved in a suitable manner or a reporter group,
R
6
and R
7
together form a further bond between C-2′ and C-3′ or an acetal group,
R
8
represents hydrogen or a hydroxy, thio or substituted thio, amino or substituted amino group,
R
9
represents hydrogen, a mono-, di- or triphosphate group or the alpha, beta or gamma-thiophosphate analogues of these phosphoric acid esters or a protecting group,
as well as possible tautomers and salts thereof.
Any detectable groups come into consideration as a reporter group such as in particular haptens, a fluoropho
M{umlaut over (u)}hlegger Klaus
Rosemeyer Helmet
Seela Frank
von der Eltz Herbert
Arent Fox Kintner & Plotkin & Kahn, PLLC
Riley Jezia
Roche Diagnostics GmbH
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