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Claims  |
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What is claimed is:
1. 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.
2. A compound in accordance with claim 1 in which R.sup.14 is a member
selected from the group consisting of H, CH.sub.3 and phenyl.
3. A compound in accordance with claim 1 in which R.sup.14 is a member
selected from the group consisting of H and CH.sub.3.
4. A compound in accordance with claim 1 in which 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 disubstituted with a protecting group.
5. A compound in accordance with claim 1 in which R.sup.16, when not
combined with R.sup.15, is a member selected from the group consisting of
H and phenyl.
6. A compound in accordance with claim 1 in which, when R.sup.18 is
combined with R.sup.20, R.sup.19 is a member selected from the group
consisting of H and CH.sub.3.
7. A compound in accordance with claim 1 in which R.sup.14 is a member
selected from the group consisting of H, CH.sub.3 and phenyl; R.sup.16,
when not combined with R.sup.15, is a member selected from the group
consisting of H, phenyl and NH.sub.2 ; and, when R.sup.18 is combined with
R.sup.20, R.sup.19 is a member selected from the group consisting of H and
CH.sub.3.
8. A compound in accordance with claim 1 in which R.sup.12 is NH.sub.2
either mono- or disubstituted by a protecting group selected from the
group consisting of benzoyl, isobutyryl, phthaloyl,
di-n-butylaminomethylidene, dimethylaminomethylidene,
p-nitrophenylethoxycarbonyl and dimethylaminomethylenamino.
9. A compound in accordance with claim 1 in which R.sup.12 is NH.sub.2
monosubstituted by a protecting group selected from the group consisting
of di-n-butylaminomethylidene, p-nitrophenylethoxycarbonyl, and
dimethylaminomethylenamino.
10. A compound in accordance with claim 1 in which R.sup.16 is NH.sub.2
either mono- or disubstituted by a protecting group selected from the
group consisting of benzoyl, isobutyryl, phthaloyl,
di-n-butylaminomethylidene, dimethylaminomethylidene,
p-nitrophenylethoxycarbonyl and dimethylaminomethylenamino.
11. A compound in accordance with claim 1 in which R.sup.16 is NH.sub.2
monosubstituted by a protecting group selected from the group consisting
of di-n-butylaminomethylidene, p-nitrophenylethoxycarbonyl, and
dimethylaminomethylenamino.
12. A compound in accordance with claim 1 in which R.sup.21 is a member
selected from the group consisting of H, trityl, monomethoxytrityl,
dimethoxytrityl, phthaloyl, di-n-butylaminomethylene, and
dimethylaminomethylidene.
13. A compound in accordance with claim 1 in which R.sup.21 is a member
selected from the group consisting of dimethoxytrityl,
di-n-butylaminomethylene, and dimethylaminomethylidene.
14. A compound in accordance with claim 1 in which R.sup.22 is a member
selected from the group consisting of H, OH and OH substituted with a
member selected fro m the group consisting of trityl, monomethoxytrityl,
dimethoxytrityl, tetrahydropyran- 1-yl, 4-methoxytetrahydropyran-4-yl,
1-(2-chloro-4-methyl)phenyl-4-methoxypiperidin-4-yl, t-butyldimethylsilyl,
p-nitrophenylethylsulfonyl, tetrahydropyranyl, 4-methoxytetrahydropyranyl,
2-nitrobenzyl, 9-phenylxanthen-9-yl and p-nitrophenylethyl.
15. A compound in accordance with claim 1 in which R.sup.22 is a member
selected from the group consisting of H and OH substituted with a member
selected from the group consisting of dimethoxytrityl,
tetrahydropyran-1-yl, t-butyldimethylsilyl, 2-nitrobenzyl, and
p-nitrophenylethyl.
16. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2, and NH.sub.2
mono- or disubstituted with a protecting group;;
R.sup.14 is H;
R.sup.15 is combined with R.sup.17 to form a double bond between ring
vertices 1 and 2;
R.sup.16 is phenyl;
R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a
double bond to ring vertex 7; and
R.sup.20 is
##STR27##
17. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2, and NH.sub.2
mono- or disubstituted with a protecting group;
R.sup.14 is phenyl;
R.sup.15 is combined with R.sup.17 to form a double bond between ring
vertices 1 and 2;
R.sup.16 is H;
R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a
double bond to ring vertex 7; and
R.sup.20 is
##STR28##
18. A compound in accordance with claim 1 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;
R.sup.13 is CH.sub.3 ;
R.sub.14 is H;
R.sup.15 is combined with R.sup.17 to form a double bond between ring
vertices 1 and 2;
R.sup.16 is NH.sub.2 ;
R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a
double bond to ring vertex 7; and
R.sup.20 is
##STR29##
19. A compound in accordance with claim 1 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;
R.sup.13 is H;
R.sup.14 is H;
R.sup.15 is combined with R.sup.17 to form a double bond between ring
vertices 1 and 2;
R.sup.16 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or disubstituted with a protecting group;
R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a
double bond to ring vertex 7; and
R.sup.20 is
##STR30##
20. A compound in accordance with claim 1 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;
R.sup.13 is H;
R.sup.14 is CH.sub.3.
R.sup.15 is combined with R.sup.17 to form a double bond between ring
vertices 1 and 2;
R.sup.16 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or disubstituted with a protecting group;
R.sup.18 is combined with R.sup.19 to form a single oxo oxygen joined by a
double bond to ring vertex 7; and
R.sup.20 is
##STR31##
21. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or di-substituted with a protecting group;
R.sup.14 is CH.sub.3 ;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a
double bond to ring vertex 2;
R.sup.17 is
##STR32##
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 CH.sub.3.
22. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or di-substituted with a protecting group;;
R.sup.14 is H;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a
double bond to ring vertex 2;
R.sup.17 is
##STR33##
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 CH.sub.3.
23. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or di-substituted with a protecting group;
R.sup.14 is CH.sub.3 ;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a
double bond to ring vertex 2;
R.sup.17 is
##STR34##
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 H.
24. A compound in accordance with claim 1 in which:
R.sup.11 is combined with R.sup.13 to form a double bond between ring
vertices 3 and 4;
R.sup.12 is selected from the group consisting of NH.sub.2 and NH.sub.2
mono- or di-substituted with a protecting group;
R.sup.14 is H;
R.sup.15 is combined with R.sup.16 to form a single oxo oxygen joined by a
double bond to ring vertex 2;
R.sup.17 is
##STR35##
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 H.
25. A compound in accordance with claim 16 in which R.sup.12 is NH.sub.2.
26. A compound in accordance with claim 16 in which:
R.sup.12 is mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
27. A compound in accordance with claim 26 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
28. A compound in accordance with claim 27 in which:
R.sup.12 is dimethylaminomethylenamino.
29. A compound in accordance with claim 26 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
30. A compound in accordance with claim 29 in which:
R.sup.12 is dimethylaminomethylenamino.
31. A compound in accordance with claim 17 in which R.sup.12 is NH.sub.2.
32. A compound in accordance with claim 17 in which:
R.sup.12 is mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
33. A compound in accordance with claim 32 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
34. A compound in accordance with claim 33 in which R.sup.12 is
dimethylaminomethylenamino.
35. A compound in accordance with claim 32 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
36. A compound in accordance with claim 35 in which R.sup.12 is
dimethylaminomethylenamino.
37. A compound in accordance with claim 18 in which R.sup.23 is a member
selected from the group consisting of H, H-phosphonate, phosphoramidite,
hemisuccinate, and hemisuccinate covalently bound to a solid support.
38. A compound in accordance with claim 37 in which:
R.sup.21 is H;
R.sup.22 is H; and
R.sup.23 is H.
39. A compound in accordance with claim 37 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
40. A compound in accordance with claim 37 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
41. A compound in accordance with claim 19 in which R.sup.16 is NH.sub.2.
42. A compound in accordance with claim 19 in which:
R.sup.16 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
43. A compound in accordance with claim 42 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.13 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
44. A compound in accordance with claim 43 in which R.sup.16 is
dimethylaminomethylenamino.
45. A compound in accordance with claim 42 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalenfly bound to controlled pore glass.
46. A compound in accordance with claim 45 in which R.sup.16 is
dimethylaminomethylenamino.
47. A compound in accordance with claim 20 in which R.sup.16 is NH.sub.2.
48. A compound in accordance with claim 20 in which:
R.sup.16 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
49. A compound in accordance with claim 48 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
50. A compound in accordance with claim 49 in which R.sup.16 is
dimethylaminomethylenamino.
51. A compound in accordance with claim 48 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
52. A compound in accordance with claim 51 in which R.sup.16 is
dimethylaminomethylenamino.
53. A compound in accordance with claim 21 in which R.sup.12 is NH.sub.2.
54. A compound in accordance with claim 21 in which:
R.sup.12 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
55. A compound in accordance with claim 54 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
56. A compound in accordance with claim 55 in which R.sup.12 is
p-nitrophenylethoxycarbonyl.
57. A compound in accordance with claim 54 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
58. A compound in accordance with claim 57 in which R.sup.12 is
p-nitrophenylethoxycarbonyl.
59. A compound in accordance with claim 22 in which R.sup.12 is NH.sub.2.
60. A compound in accordance with claim 22 in which:
R.sup.12 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
61. A compound in accordance with claim 60 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
62. A compound in accordance with claim 61 in which R.sup.12 is
p-nitrophenylethoxycarbonyl.
63. A compound in accordance with claim 60 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
64. A compound in accordance with claim 63 in which R.sup.12 is
p-nitrophenylethoxycarbonyl.
65. A compound in accordance with claim 23 in which R.sup.12 is NH.sub.2.
66. A compound in accordance with claim 23 in which:
R.sup.12 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
67. A compound in accordance with claim 66 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
68. A compound in accordance with claim 67 in which R .sup.12 is
p-nitrophenylethoxycarbonyl.
69. A compound in accordance with claim 66 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
70. A compound in accordance with claim 69 in which R.sup.12 is
p-nitrophenylethoxycarbonyl.
71. A compound in accordance with claim 24 in which R.sup.12 is NH.sub.2.
72. A compound in accordance with claim 24 in which:
R.sup.12 is NH.sub.2 mono- or di-substituted with a protecting group; and
R.sup.23 is a member selected from the group consisting of H-phosphonate,
phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a
solid support.
73. A compound in accordance with claim 72 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a .beta.-cyanoethyl, N-diisopropyl phosphoramidite.
74. A compound in accordance with claim 73 in which:
R.sup.12 is p-nitrophenylethoxycarbonyl.
75. A compound in accordance with claim 72 in which:
R.sup.21 is dimethoxytrityl;
R.sup.22 is H; and
R.sup.23 is a hemisuccinate covalently bound to controlled pore glass.
76. A compound in accordance with claim 75 in which:
R.sup.12 is p-nitrophenylethoxycarbonyl.
77. A compound in accordance with claim 16 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
78. A compound in accordance with claim 17 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
79. A compound in accordance with claim 18 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
80. A compound in accordance with claim 19 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
81. A compound in accordance with claim 20 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
82. A compound in accordance with claim 21 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
83. A compound in accordance with claim 22 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
84. A compound in accordance with claim 23 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H.
85. A compound in accordance with claim 24 in which:
R.sup.21 is a triphosphate;
R.sup.22 is H; and
R.sup.23 is H. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
Synthetic oligonucleotides find numerous uses in molecular biology as
probes for screening genomic and complementary DNA libraries, as primers
for DNA synthesis, sequencing, and amplification, and in the study of
DNA-protein interactions. In addition, oligonucleotide probes have proven
useful for assaying in vitro gene expression using techniques of in situ
hybridization.
Recent improvements in DNA sequencing methods, fluorescent labels, and
detection systems have dramatically increased the use of fluorescently
labeled oligonucleotides in all of these applications. Typically
oligonucleotides are labeled with a fluorescent marker, either directly
through a covalent linkage (e.g., a carbon linker), or indirectly whereby
the oligonucleotide is bound to a molecule such as biotin or dioxigenin,
which, is subsequently coupled to a fluorescently labeled binding moiety
(e.g., streptavidin or a labeled monoclonal antibody).
These fluorescent labeling systems, however, suffer the disadvantage that
the fluorescent complexes and their binding moieties are relatively large.
The presence of large fluorescent labels and associated linkers may alter
the mobility of the oligonucleotide, either through a gel as in
sequencing, or through various compartments of a cell.
In addition, the presence of these markers alters the interaction of the
oligonucleotide with other molecules either through chemical interactions
or through steric hinderance. Thus the presence of these markers makes it
difficult to study the interactions of DNA with other molecules such as
proteins. The study of protein-DNA interactions is of profound interest as
they involve some of the most fundamental mechanisms in biology. They
include, for example, the progression of a DNA polymerase or reverse
transcriptase along the length of the oligonucleotide, the activation of
gene transcription as in the AP1 or steroid hormone pathway, or the
insertion of viral DNA into the host genome as mediated by the HIV IN
enzyme. For these reasons, it is desirable to obtain a fluorescent moiety
analogous in structure to a pyrimidine or purine nucleotide and capable of
being incorporated into an oligonucleotide. Such a moiety would preferably
render the oligonucleotide molecule fluorescent without significantly
altering the size or chemical properties of the oligonucleotide.
Numerous analogs of nucleotides are known. Among them are furanosyl
pteridine derivatives. Methods of synthesizing these pteridine
derivatives, which are structurally analogous to purine nucleotides, are
well known. Indeed, a number of pteridine-derived analogs have been
synthesized in the hope of discovering new biologically active compounds.
Thus, Pfleiderer (U.S. Pat. No. 3, 798,210 and U.S. Pat. No. 3,792,036)
disclosed a number of pteridine-glycosides which possessed antibacterial
and antiviral properties. Pfleiderer, however, did not investigate the
fluorescent properties of these compounds.
Similarly, Schmidt et al., Chem. Ber. 106:1952-1975 (1973) describe the
ribosidation of a series of pteridine derivatives to produce structural
analogs of the nucleoside guanosine, while Harris et al., Liebigs. Ann.
Chem. 1457-1468 (1981), describe the synthesis of pteridine derivatives
structurally analogous to adenosine. Again, neither reference describes
measurements of the fluorescent properties of the nucleosides.
The synthesis of oligonucleotides incorporating lumazine derivatives has
been described by Bannwarth et al., Helvetica Chimica Acta. 74:1991-1999
(1991), Bannwarth et al., Helvetica Chimica Acta. 74:2000-2007 (1991) and
Bannwarth et al., (European Patent Application No. 0439036A2). Bannwarth
et al. utilized the lumazine derivative in conjunction with a
bathophenanthroline-ruthenium complex as an energy transfer system in
which the lumazine derivative acted as an energy donor and the ruthenium
complex acted as an energy receptor. Energy transfer occurred when the two
compounds were brought into proximity resulting in fluorescence. The
system provided a mechanism for studying the interaction of molecules
bearing the two groups. The references, however, did not describe the use
of a lumazine derivative alone in an oligonucleotide. In addition,
Bannwarth recognized that a major disadvantage of the lumazine derivative
was the ". . . relatively low extinction coefficient for the long
wave-length absorption of the lumazine chromophore (.epsilon.=8900
m.sup.-1 cm.sup.-1 at 324 nm pH 6.9)." Bannwarth et al., Helv. Chim.
Acta., 74:1991-1999 (1991).
The present invention overcomes the limitations of these prior art
compounds by providing a number of pteridine nucleotides which are
analogous in structure to purine nucleotides, highly fluorescent under
normal physiological conditions, and suitable for use in the chemical
synthesis of oligonucleotides.
SUMMARY OF THE INVENTION
The present invention provides for pteridine nucleotides of the form:
##STR1##
where 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 either NH.sub.2 or NH.sub.2 either mono- or
disubstituted with a protecting group; R.sup.13 when not combined with
R.sup.11 is a lower alkyl or H; R.sup.14 is either H, lower alkyl or
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; and 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 either H or a lower alkyl. R.sup.17 when not combined with
R.sup.15, and R.sup.20 when not combined with R.sup.18, are compounds of
formula:
##STR2##
where the symbol R.sup.21 represents a hydrogen, protecting groups, or a
triphosphate; the symbol R.sup.22 represents a hydrogen, a hydroxyl, or a
hydroxyl substituted with a protecting group; and R.sup.23 represents 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 triphosphate 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.
These compounds are highly fluorescent under normal physiological
conditions, and suitable for use in the chemical synthesis of
oligonucleotides. The invention further provides for oligonucleotides that
incorporate these pteridine nucleotides.
In addition, the invention provides for pteridine nucleotide triphosphates
that may be utilized in various DNA amplification processes. When used in
a DNA amplification process, the nucleotide triphosphates are directly
incorporated into the amplified DNA sequence rendering it fluorescent.
This provides for a rapid assay for the presence or absence of the
amplified product.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
As used herein, the term "lower alkyl" refers to a saturated hydrocarbon
radical which may be straight-chain or branched-chain (for example, ethyl,
isopropyl, t-amyl, or 2,5-dimethylhexyl). Preferred alkyl groups are those
containing one to six carbon atoms. All numerical ranges in this
specification and claims are intended to be inclusive of their upper and
lower limits.
The term "oligonucleotide" refers to a molecule comprised of two or more
deoxyribonucleotides, ribonucleotides, modified ribonucleotides, modified
dexoyribonucleotides, ribonucleotide analogs, deoxyribonucleotide analogs,
or pteridine derivatives of the present invention. The exact size of an
oligonucleotide depends on many factors and the ultimate function or use
of the oligonucleotide. Generally, chemically synthesized oligonucleotides
range in length from 2 to 200 bases, although, it is well known that
oligonucleotides may be ligated together to provide longer sequences. As
used herein, the term "oligonucleotide" also encompasses these longer
sequences. It is also recognized that double-stranded polynucleotides may
be created by hybridization with a complementary sequence or enzymatically
through primer extension. The term oligonucleotide as used in this
application encompasses both single and double-stranded oligonucleotides.
The term "solid support" refers to a solid material which is functionalized
to permit the coupling of a monomer used in polynucleotide synthesis. The
solid support is typically coupled to a nucleoside monomer through a
covalent linkage to the 3'-carbon on the furanose. Solid support materials
typically are unreactive during the polynucleotide synthesis and simply
provide a substratum to anchor the growing polynucleotide. Solid support
materials include, but are not limited to, polacryloylmorpholide, silica,
controlled pore glass (CPG), polystyrene, polystyrene/latex, and carboxyl
modified teflon.
The term "cleavage" in reference to solid phase oligonucleotide synthesis
refers to the breaking of the bond which binds an oligonucleotide to a
solid support. Typically, cleavage involves hydrolysis of a succinate
ester bond between the 3'-hydroxyl of an attached oligonucleotide and the
solid support.
The term "deprotection" refers to the removal of protecting groups from the
exocyclic amines of the heterocyclic bases of an oligonucleotide.
Typically, deprotection consists of hydrolysis of an amide moiety
consisting of an exocyclic amine and an amino protection group, e.g.
benzoyl, p-nitrophenoxycarbonyl, di-n-butylaminomethylidene, and
dimethyaminomethylenamino. The term deprotection is also used to refer to
the removal of protecting groups from the phosphate diesters
(internucleotide phosphates) of the oligonucleotide. When such protecting
groups are methoxy, "deprotection" as used herein may not encompass their
removal. Instead, additional treatment with a standard
thiophenol-containing reagent may be desired to produce a "thiolated"
oligonucleotide.
The term "pteridine nucleotide" or "pteridine monomer" is used herein to
refer to the furanosyl pteridine derivatives of the present invention with
a 3'-phosphate group. It is recognized that properly speaking the
furanosyl pteridine derivatives are not nucleotides as the pteridine is
neither a purine or a pyrimidine. However, because the furanosyl pteridine
derivatives are structurally analogous to purine nucleotides, and the
furanosyl pteridines of this invention are used in the same manner as
nucleotides both will be referred to as nucleotides. As used herein, the
pteridine nucleotide or pteridine monomer may be fully protected for use
in polynucleotide synthesis or it may be deprotected when used as a
triphosphate or when incorporated into an oligonucleotide.
The term "nucleotide monomer" as used herein refers to pteridine
nucleotides, the "standard" nucleotides; adenosine, guanosine, cytidine,
thymidine, and uracil, or derivatives of these nucleotides. Such
derivatives include, but are not limited to, inosine,
5-bromodeoxycytidine, 5-bromo-deoxyuridine, N.sup.6 -methyl-deoxyadenosine
and 5-methyl-deoxycytidine.
As used herein, the term "protecting group" refers to a group which is
joined to or substituted for a reactive group (e.g. a hydroxyl or an
amine) on a molecule. The protecting group is chosen to prevent reaction
of the particular radical during one or more steps of a chemical reaction.
Generally the particular protecting group is chosen so as to permit
removal at a later time to restore the reactive group without altering
other reactive groups present in the molecule. The choice of a protecting
group is a function of the particular radical to be protected and the
compounds to which it will be exposed. The selection of protecting groups
is well known to those of skill in the art. See, for example Greene et
al., Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons,
Inc. Somerset, N.J. (1991), which is herein incorporated by reference.
As used herein, the term "protected amine" refers to an amine which has
been reacted with an amino protecting group. An amino protecting group
prevents reaction of the amide function during either the synthesis of the
derivatized pteridine nucleoside or during the chemical synthesis of DNA
or RNA using that nucleotide. The amino protecting group can be removed at
a later time to restore the amino group without altering other reactive
groups present in the molecule. For example, the exocyclic amine may be
reacted with dimethylformamid-diethylacetal to form the
dimethylaminomethylenamino function. Amino protecting groups generally
include carbamates, benzyl radicals, imidates, and others known to those
of skill in the art. Preferred amino protecting groups include, but are
not limited to, p-nitrophenylethoxycarbonyl or dimethyaminomethylenamino.
The term "coupling" is generally used in DNA synthesis to refer to the
joining of one nucleotide monomer to another nucleotide monomer or to the
5' terminal of an oligonucleotide. The coupling is generally accomplished
by the formation of a phosphodiester linkage from the 3'- phosphate of one
nucleotide monomer to the 5'-hydroxyl of a second monomer or
oligonucleotide. Coupling is also used to refer to the joining of an
initial nucleoside to a solid support.
The term "capping" refers to a step in which unreacted 5'-hydroxyl groups
that fail to condense and successfully couple with the next derivatized
nucleotide are blocked. This insures that subsequent reactions proceed
only by propagating chains of the desired sequence. Typically capping
involves the acetylation of the 5'-hydroxyl functions. Usually this is
accomplished by acetic arthydride catalyzed by 4-dimethylaminopyridine
(DMAP). Other reagents, known to those of skill in the art are suitable.
The term "synthesis cycle" refers to the sequence of reactions necessary to
couple a nucleotide monomer to the 5' terminal of the oligonucleotide
being synthesized. Typically, a synthesis cycle involves removal of the
5'-hydroxyl blocking group on the terminus of the oligonucleotide,
reaction with the phosphite derivative of a nucleotide monomer to form a
phosphodiester bond, and then capping of molecules in which coupling was
unsuccessful.
The term "normal physiological conditions" is used herein to refer to
conditions that are typical inside a living organism or a cell. While it
is recognized that some organs provide extreme conditions, the
intra-organismal and intra-cellular environment normally varies around pH
7 (i.e. from pH 6.5 to pH 7.5), contains water as the predominant solvent,
and exists at a temperature above 0.degree. C. and below 50.degree. C.
This invention provides a number of pteridine nucleotides which are highly
fluorescent under normal physiological conditions and which may be
utilized in the chemical synthesis of oligonucleotides to produce
fluorescent oligonucleotides. These fluorescent oligonucleotides have many
uses including, for example, probes for screening genomic and
complementary DNA libraries, probes for in situ hybridization, primers for
DNA synthesis, sequencing, and amplification, and as model substrates to
investigate DNA-protein interactions.
In one embodiment, the pteridine nucleotides of this invention are suitable
for use in the chemical synthesis of oligonucleotides. In general, this
requires blocking the exocyclic amines on the pteridine, derivatizing the
phosphite moiety with a reactive group appropriate to the particular
synthetic chemistry contemplated, and blocking the 5' hydroxyl with a
protecting group that may be removed during synthesis to facilitate the
stepwise coupling of derivatized nucleotides to the 5' terminus of the
growing oligonucleotide. Where the sugar of the pteridine derivative is a
ribose, the reactive 2'-hydroxyl group must also be protected.
In a preferred embodiment, the invention provides for nucleotide monomers
of formula I.
##STR3##
These nucleotide monomers are pteridine derivatives with ring vertices 1
through 8 as shown, where 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 either NH.sub.2 or NH.sub.2 either
mono- or disubstituted with a protecting group; R.sup.13 when not combined
with R.sup.11 is a lower alkyl or H; R.sup.14 is either H, lower alkyl or
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; and 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 either H or a lower alkyl. R.sup.17 when not combined with
R.sup.15, and R.sup.20 when not combined with R.sup.18, are compounds of
formula II.
##STR4##
where the symbol R.sup.21 represents a hydrogen, protecting groups or a
triphosphate; the symbol R.sup.22 represents a hydrogen, a hydroxyl, or a
hydroxyl substituted with a protecting group; and R.sup.23 represents a
hydrogen, 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 triphosphate 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.
In another preferred embodiment R.sup.14 is hydrogen, a methyl or a phenyl,
more particularly a hydrogen or a methyl.
In still another preferred embodiment, R.sup.16, when not combined with
R.sup.15, is a hydrogen, a phenyl, an amino group, or NH.sub.2
disubstituted with a protecting group. More particularly, R.sup.16 is a
hydrogen and a phenyl.
In yet another preferred embodiment when R.sup.18 is combined with
R.sup.20, R.sup.19 is a hydrogen or a methyl.
In still yet another preferred embodiment, R.sup.14 is a hydrogen, a
methyl, or a phenyl, R.sup.16, when not combined with R.sup.15, is a
hydrogen, a phenyl or an amino, and, when R.sup.18 is combined with
R.sup.20, R.sup.19 is a hydrogen or a methyl.
Among the compounds of the present invention, nine embodiments are
particularly preferred. In a first preferred embodiment R.sup.11 is
combined with R.sup.13 to form a double bond between ring vertices 3 and
4; R.sup.12 is NH.sub.2 or NH.sub.2 mono- or disubstituted with a
protecting group; R.sup.14 is a hydrogen; R.sup.15 is combined with
R.sup.17 to form a double bond between ring vertices 1 and 2; R.sup.16 is
a phenyl; R.sup.18 is combined with R.sup.19 to form a single oxo oxygen
joined by a double bond to ring vertex 7; and R.sup.20 is formula II. This
embodiment is illustrated by formula III. Particularly preferred compounds
of this embodiment are illustrated by formula III when R.sup.12 is
NH.sub.2.
##STR5##
In a second preferred embodiment R.sup.11 is combined with R.sup.13 to form
a double bond between ring vertices 3 and 4; R.sup.12 is NH.sub.2 or
NH.sub.2 mono- or disubstituted with a protecting group; R.sup.14 is a
phenyl; R.sup.15 is combined with R.sup.17 to form a double bond between
ring vertices 1 and 2; R.sup.16 is a hydrogen; R.sup.18 is combined with
R.sup.19 to form a single oxo oxygen joined by a double bond to ring
vertex 7 and R.sup.20 is formula II. This embodiment is illustrated by
formula IV. Particularly preferred compounds of this embodiment are
illustrated by formula IV when R.sup.12 is NH.sub.2.
##STR6##
In a third preferred embodiment R.sup.11 is combined with R.sup.12 to form
a single oxo oxygen joined by a double bond to ring vertex 4; R.sup.13 is
CH.sub.3 ; R.sup.14 is H; R.sup.15 is combined with R.sup.17 to form a
double bond between ring vertices 1 and 2; R.sup.16 is NH.sub.2 ; R.sup.18
is combined with R.sup.19 to form a single oxo oxygen joined by a double
bond to ring vertex 7; and R.sup.20 is formula II. This embodiment is
illustrated by formula V. One particularly preferred communal of this
embodiment is the nucleoside illustrated by formula V when R.sup.23 of
formula II is H and more particularly when R.sup.21, R.sup.22, and
R.sup.23 of formula II are all H.
##STR7##
In a fourth preferred embodiment R.sup.11 is combined with R.sup.12 to form
a single oxo oxygen joined by a double bond to ring vertex 4; R.sup.13 is
a hydrogen; R.sup.14 is hydrogen; R.sup.15 is combined with R.sup.17 to
form a double bond between ring vertices 1 and 2; R.sup.16 is NH.sub.2 or
NH.sub.2 mono- or disubstituted with a protecting group; R.sup.18 is
combined with R.sup.19 to form a single oxo oxygen joined by a double bond
to ring vertex 7; and R.sup.20 is formula II. This embodiment is
illustrated by formula VI. Particularly preferred compounds of this
embodiment are illustrated by formula VI when R.sup.16 is NH.sub.2.
##STR8##
In a fifth preferred embodiment R.sup.11 is combined with R.sup.12 to form
a single oxo oxygen joined by a double bond to ring vertex 4; R.sup.13 is
a hydrogen; R.sup.14 is CH.sub.3 ; R.sup.15 is combined with R.sup.17 to
form a double bond between ring vertices 1 and 2; R.sup.16 is NH.sub.2 or
NH.sub.2 mono- or disubstituted with a protecting group; R.sup.18 is
combined with R.sup.19 to form a single oxo oxygen joined by a double bond
to ring vertex 7; and R.sup.20 is formula II. This embodiment is
illustrated by formula VH. Particularly preferred compounds of this
embodiment are illustrated by formula VII when R.sup.16 is NH.sub.2.
##STR9##
In a sixth preferred embodiment R.sup.11 is combined with R.sup.13 to form
a double bond between ring vertices 3 and 4; R.sup.12 is NH.sub.2 or
NH.sub.2 mono- | | |
