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US 8,013,147 B2Granted
Heterocyclic compounds and their use in the detection of nucleic acids
AssigneeRoche Molecular Systems, Inc.
InventorsKlaus Muhlegger, Herbert Von Der Eltz
Life of this patent
Estimating the term…
Abstract
The invention concerns compounds of the general formula (I) in which the residues R1 to R7 have the meanings given in the application as well as methods for their preparation. The compounds are in particular suitable as substrates for RNA or DNA polymerases and can thus be incorporated into RNA or DNA oligonucleotides and are especially suitable for labelling and detecting nucleic acids or for DNA sequencing.
Claims18
A nucleotide analog of the formula
- in which
- R1 is hydrogen, halogen, hydroxy, thio, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R3 and R4 each is independently hydrogen, hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, an ester, amide or a reporter group,
- R5 is hydrogen, hydroxy, thio, amino, a trivalent phosphorus group, a pentavalent phosphorus group, an ester, amide or a reporter group, or
- R4 and R5 together form a further bond between C-2′ and C-3′ or an acetal group,
- R6 is hydrogen, hydroxy, thio, or amino,
- R7 is hydrogen, a monophosphate, a diphosphate a triphosphate group, an alpha, beta or gamma thiophosphate analogue of a phosphate group, an ester or an amide group,
- X is methine substituted with halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- each reporter group is independently selected from the group consisting of a hapten and a fluorophore, optionally linked via a linker group;
- and salts thereof.
7 more elements
An isolated oligonucleotide comprising at least one nucleotide analog of the formula:
- wherein
- R1 is hydrogen, oxygen, halogen, hydroxy, thio, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R2 is oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R3 and R4 each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, an ester, an amide group or a reporter group,
- R5 is hydrogen, hydroxy, thio, amino, a trivalent phosphorus group, a pentavalent phosphorus group, an ester, an amide or a reporter group,
- R4 and R5 together form a further bond between C-2′ and C-3′ or an acetal group,
- R6 is hydrogen, a hydroxy, thio, or amino group,
- R7 is hydrogen, a monophosphate, a diphosphate a triphosphate group an alpha, beta or gamma thiophosphate analogue of a phosphate group, an ester or an amide group;
- X is methylene or methine substituted with halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- each reporter group is independently selected from the group consisting of a hapten and a fluorophore;
- Z is nitrogen or carbon,
- provided that if Z denotes nitrogen, m is 0, and if X is methylene, substituted methylene or substituted methine, Z is nitrogen and at least one of R5 and R7 indicates the point of attachment to the oligonucleotide.
9 more elements
A isolated nucleic acid comprising at least one nucleotide analog of the formula
- wherein
- R1 is hydrogen, oxygen, halogen, hydroxy, thio, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R2 is oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R3 and R4 each independently is hydrogen, hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, an ester, an amide group or a reporter group,
- R5 is hydrogen, hydroxy, thio, amino, a trivalent phosphorus group, a pentavalent phosphorus group, an ester, an amide or a reporter group,
- R4 and R5 together form a further bond between C-2′ and C-3′ or an acetal group,
- R6 is hydrogen, a hydroxy, thio, or amino group,
- R7 is hydrogen, a monophosphate, a diphosphate a triphosphate group an alpha, beta or gamma thiophosphate analogue of a phosphategroup, an ester or an amide group;
- X is methylene or methine substituted with halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- each reporter group is independently selected from the group consisting of a hapten and a fluorophore;
- Z is nitrogen or carbon,
- provided that if Z denotes nitrogen, m is 0, and if X is methylene, substituted methylene or substituted methine, Z is nitrogen and at least one of R5 and R7 indicates the point of attachment to the oligonucleotide.
9 more elements
A reaction mixture comprising:
- a) a nucleotide analog of the general formula
- in which:
- R1 and R2 are the same or different and represent hydrogen, oxygen, halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- R3 and R4 each represent hydrogen, hydroxy, thio, amino, lower alkyloxy, lower alkenoxy, lower alkinoxy, a protecting group or a reporter group,
- R5 represents hydrogen, hydroxy, thio, amino, a reactive trivalent or pentavalent phosphorus group, an ester or amide residue that can be cleaved or a reporter group, or
- R4 and R5 together form a further bond between C-2′ and C-3′ or an acetal group,
- R6 represents hydrogen or a hydroxy, thio, or amino group,
- R7 represents hydrogen, a monophosphate, diphosphate or triphosphate group or the alpha, beta or gamma thiophosphate analogue of said phosphate group or a protective group, and tautomers and salts thereof,
- X represents methine substituted with halogen, hydroxy, thio, amino, carboxy, lower alkyl, lower alkenyl, lower alkinyl, aryl, lower alkyloxy, aryloxy, aralkyl, aralkyloxy or a reporter group,
- each reporter group is independently selected from the group consisting of a hapten and a fluorophore;
- and
- Z represents nitrogen or carbon, provided that if Z represents nitrogen, m is zero (0) Z is nitrogen;
- and
- b) a polymerase.
11 more elements
A nucleotide analog selected from the group
- consisting of:
- 3-(4-Hydroxy-5-hydroxy-methyl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-(1,8-diamino-3,6-dioxa-octyl)-pyrazine;
- 3-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-trifluoro-acetamido-(3,6-dioxa)-octyl]-amino-pyrazine;
- and
- 3-(4-Hydroxy-5-triphosphoryl-tetrahydrofuran-2-yl)-2,6-dihydroxy-5-[N-tetramethyl-rhodaminyl-5,6-carboxamido-(3,6-dioxa)-octyl]-amino-pyrazine.
2 more elements
Dependencies are inferred from the claim text.