US 5,525,711 AGranted

Pteridine nucleotide analogs as fluorescent DNA probes

AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services

InventorsMary E. Hawkins, Wolfgang Pfleiderer, Michael D. Davis, Frank Balis

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Abstract

The invention provides novel pteridine nucleotides which are highly fluorescent under physiological conditions and which may be used in the chemical synthesis of fluorescent oligonucleotidcs. The invention further provides for fluorescent oligonucleotides comprising one or more pteridine nucleotides. In addition the invention provides for pteridine nucleotide triphosphates which may be used as the constituent monomers in DNA amplification procedures.

Claims85

Dependent claims

A compound having the formula shown below, with ring vertices 1 through 8 as shown:

##STR25## in which:
R.sup.11 is combined with R.sup.12 to form a single oxo oxygen joined by a double bond to ring vertex 4, or with R.sup.13 to form a double bond between ring vertices 3 and 4;
R.sup.12 when not combined with R.sup.11 is a member selected from the group consisting of NH.sub.2, and NH.sub.2 either mono- or disubstituted with a protecting group;
R.sup.13 when not combined with R.sup.11 is lower alkyl or H;
R.sup.14 is a member selected from the group consisting of H, lower alkyl and phenyl;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a double bond to ring vertex 2, or with R.sup.17 to form a double bond between ring vertices 1 and 2, such that ring vertices 2 and 4 collectively bear at most one oxo oxygen;
R.sup.16 when not combined with R.sup.15 is a member selected from the group consisting of H, phenyl, NH.sub.2, and NH.sub.2 mono- or disubstituted with a protecting group;
when R.sup.15 is not combined with R.sup.16, R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a double bond to ring vertex 7;
when R.sup.15 is combined with R.sup.16, R.sup.18 is combined with R.sup.20 to form a double bond between ring vertices 7 and 8, and R.sup.19 is a member selected from the group consisting of H and lower alkyl;
and
R.sup.17 when not combined with R.sup.15, and R.sup.20 when not combined with R.sup.18, are ##STR26## in which:
R.sup.21 is a member selected from the group consisting of H, a triphosphate, and protecting groups;
R.sup.22 is a member selected from the group consisting of H, OH and OH substituted with a protecting group;
and
R.sup.23 is a member selected from the group consisting of H, a phosphoramidite, an H-phosphonate, a methyl phosphonate, a phosphorothioate, a phosphotriester, a hemisuccinate, a hemisuccinate covalently bound to a solid support, a dicyclohexylcarbodiimide, and a dicyclohexylcarbodiimide covalently bound to a solid support;
when R.sup.13 is H and R.sup.23 is H, R.sup.21 is a tfiphosphate;
and
when R.sup.11 is combined with R.sup.13 to form a double bond between ring vertices 3 and 4 and R.sup.23 is H, R.sup.21 is a triphosphate.

15 more elements

Dependencies are inferred from the claim text.

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Abstract

A compound having the formula shown below, with ring vertices 1 through 8 as shown:

##STR25## in which:
R.sup.11 is combined with R.sup.12 to form a single oxo oxygen joined by a double bond to ring vertex 4, or with R.sup.13 to form a double bond between ring vertices 3 and 4;
R.sup.12 when not combined with R.sup.11 is a member selected from the group consisting of NH.sub.2, and NH.sub.2 either mono- or disubstituted with a protecting group;
R.sup.13 when not combined with R.sup.11 is lower alkyl or H;
R.sup.14 is a member selected from the group consisting of H, lower alkyl and phenyl;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a double bond to ring vertex 2, or with R.sup.17 to form a double bond between ring vertices 1 and 2, such that ring vertices 2 and 4 collectively bear at most one oxo oxygen;
R.sup.16 when not combined with R.sup.15 is a member selected from the group consisting of H, phenyl, NH.sub.2, and NH.sub.2 mono- or disubstituted with a protecting group;
when R.sup.15 is not combined with R.sup.16, R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a double bond to ring vertex 7;
when R.sup.15 is combined with R.sup.16, R.sup.18 is combined with R.sup.20 to form a double bond between ring vertices 7 and 8, and R.sup.19 is a member selected from the group consisting of H and lower alkyl;
and
R.sup.17 when not combined with R.sup.15, and R.sup.20 when not combined with R.sup.18, are ##STR26## in which:
R.sup.21 is a member selected from the group consisting of H, a triphosphate, and protecting groups;
R.sup.22 is a member selected from the group consisting of H, OH and OH substituted with a protecting group;
and
R.sup.23 is a member selected from the group consisting of H, a phosphoramidite, an H-phosphonate, a methyl phosphonate, a phosphorothioate, a phosphotriester, a hemisuccinate, a hemisuccinate covalently bound to a solid support, a dicyclohexylcarbodiimide, and a dicyclohexylcarbodiimide covalently bound to a solid support;
when R.sup.13 is H and R.sup.23 is H, R.sup.21 is a tfiphosphate;
and
when R.sup.11 is combined with R.sup.13 to form a double bond between ring vertices 3 and 4 and R.sup.23 is H, R.sup.21 is a triphosphate.

15 more elements