Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-10-16
2001-01-16
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026230, C536S026260, C536S026800, C536S028520, C536S028550
Reexamination Certificate
active
06175001
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The field of this invention is nucleotide chemistry. More particularly, the present invention pertains to modified and functionalized pyrimidines and DNA molecules incorporating such nucleotides.
BACKGROUND OF THE INVENTION
Nucleic acid libraries provide tremendous opportunities for the selection of novel ligands and catalysts since the polymerase chain reaction, PCR, allows for the synthesis and selection of libraries containing more than 10
14
different molecules. There are now many examples of nucleic acids that have been selected to bind proteins and small molecules and to catalyze a limited set of reactions (S. E. Osborne, A. D. Ellington,
Chem. Rev.
(Washington, D.C.) 1997, 97, 349-370; M. Famulok, J. W. Szostak,
Angew. Chem.
1992, 104, 1001-11;
Angew. Chem.Int. Ed. Engl.
1992, 31, 979-88; L. Gold, B. Polisky, O. Uhlenbeck, M. Yarus,
Annu. Rev. Biochem.
1995, 64, 763-97; R. R. Breaker,
Chem. Rev.
(Washington, D.C.) 1997, 97, 371-390; R. R. Breaker,
Curr. Opin. Chem. Biol.
1997, 1, 26-31; J. R. Lorsch, J. W. Szostak,
Acc. Chem. Res.
1996, 29, 103-10; R. R. Breaker, G. F. Joyce,
Chem. Biol.
1994, 1, 223-9; B. Cuenoud, J. W. Szostak,
Nature
(London) 1995, 375, 611-14; R. R. Breaker, G. F. Joyce,
Chem. Biol.
1995, 2, 655-60; C. R. Geyer, D. Sen,
Chem. Biol.
1997, 4, 579-593; S. W. Santoro, J. G. F.,
Proc. Natl. Acad Sci. U.S.A.
1997, 94, 4262-4266; P. Burgstaller, M. Famulok, Angew. Chem. 1995,107,1303-06;
Angew. Chem. Int. Ed. Engl.
1995, 34, 1189-92; D. Faulhammer, M. Famulok,
Angew. Chem.
1996, 108, 2984-88;
Angew. Chem. Int. Ed. Engl.
1996,35,2837-2841; D. Faulhammer, M. Famulok,
J. Mol. Biol.
1997, 269, 188-202; Y. Li, D. Sen,
Nat. Struct. Biol.
1996, 3, 743-747; J. Burmeister, G. von Kiedrowski, A. D. Ellington,
Angew. Chem.
1997, 109, 1379-81;
Angew. Chem. Int. Ed. Engl.
1997,36, 1321-1324; N. Carni, L. A. Shultz, R. R. Breaker,
Chem. Biol.
1996, 3, 1039-1046; N. Carmi, H. R. Balkhi, R. R. Breaker,
Proc. Natl. Acad Sci. U.S.A.
1998, 95, 2233-2237).
The catalytic and mechanistic scope of nucleic acids is limited since the natural nucleotide monomers possess minimal functionality compared to the repertoire available to Nature's dominant catalytic biopolymers, proteins. In recognition of this shortcoming, much attention has been focused on the development of functionalized nucleotides suitable for in vitro selection with the hope of increasing the potential of nucleic acids for binding and catalysis (B. E. Eaton, W. A. Pieken,
Annu. Rev. Biochem.
1995, 64, 837-63; B. E. Eaton,
Curr. Opin. Chem. Biol.
1997, 1, 10-16; G. J. Crouch, B. E. Eaton,
Nucleosides Nucleotides
1994, 13 939-44; T. M. Dewey, A. Mundt, G. J. Crouch, M. C. Zyzniewski, B. E. Eaton,
J. Am. Chem. Soc.
1995, 117, 8474-5; T. M. Dewey, M. C. Zyzniewski, B. E. Eaton,
Nucleosides Nucleotides
1996, 15, 1611-1617; C. Tu, C. Keane, B. E. Eaton,
Nucleosides Nucleotides
1995, 14, 1631-8; P. A. Limbach, P. F. Crain, J. A. McCloskey,
Nucleic Acids Res.
1994, 22, 2183-96; H. Aurup, D. M. Williams, F. Eckstein,
Biochemistry
1992, 31, 9636-41). Functionalized nucleotide triphosphates have been shown to be substrates for RNA polymerases (T. M. Dewey, A. Mundt, G. J. Crouch, M. C. Zyzniewski, B. E. Eaton,
J. Am. Chem. Soc.
1995,117,8474-5; H. Aurup, D. M. Williams, F. Eckstein,
Biochemistry
1992, 31, 9636-41).and catalytic RNA's dependent on the modified base for their activity have been selected (T. W. Wiegand, R. C. Janssen, B. E. Eaton,
Chem. Biol.
1997, 4, 675-683; T. M. Tarasow, S. L. Tarasow, B. E. Eaton,
Nature
(London) 1997, 389, 54-57). Like RNA, DNA has also been selected to bind proteins and small molecules and more recently to catalyze reactions (S. E. Osborne, A. D. Ellington,
Chem. Rev.
(Washington, D.C.) 1997, 97, 349-370; M. Famulok, J. W. Szostak,
Angew. Chem.
1992,104, 1001-11;Angew.
Chem. Int. Ed. Engl.
1992, 31, 979-88; L. Gold, B. Polisky, 0. Uhlenbeck, M. Yarus,
Annu. Rev. Biochem.
1995, 64, 763-97; R. R. Breaker,
Chem. Rev.
(Washington, D.C.) 1997, 97, 371-390; R. R. Breaker,
Curr. Opin. Chem. Biol.
1997, 1, 26-31; J. R. Lorsch, J. W. Szostak,
Acc. Chem. Res.
1996, 29, 103-10; R. R. Breaker, G. F. Joyce,
Chem. Biol.
1994, 1, 223-9; B. Cuenoud, J. W. Szostak,
Nature
(London) 1995, 375, 611-14; R. R. Breaker, G. F. Joyce,
Chem. Biol.
1995, 2, 655-60; C. R. Geyer, D. Sen,
Chem. Biol.
1997, 4, 579-593; S. W. Santoro, J. G. F.,
Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 4262-4266; P. Burgstaller, 3M. Famulok,
Angew. Chem.
1995,107,1303-06;
Angew. Chem. Int. Ed. Engl.
1995, 34, 1189-92; D. Faulhammer, M. Famulok, Angew. Chem. 1996, 108, 2984-88;
Angew. Chem. Int. Ed. Engl.
1996, 35,2837-2841; D. Faulhammer, M. Famulok,
J Mol Biol.
1997, 269, 188-202; Y. Li, D. Sen,
Nat. Struct. Biol.
1996, 3, 743-747; J. Burmeister, G. von Kiedrowski, A. D. Ellington,
Angew. Chem.
1997, 109, 1379-81; Angew.
Chem. Int. Ed. Engl.
1997, 36, 1321-1324; N. Carmi, L. A. Shultz, R. R. Breaker,
Chem. Biol.
1996, 3, 103 9-1046; N. Carmi, H. R. Balkhi, R. R. Breaker,
Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 2233-2237).
While DNA possesses enhanced stability as compared to RNA, the lack of a 2′-hydroxyl group which provides for the enhanced stability of this molecule further reduces the functionality available to this molecule for chemistry. In contrast to the success achieved in identifying modified nucleotide triphosphates for RNA libraries, there is but a single example of a deoxynucleotide triphosphate, 5-(1-pentynyl)-2′ deoxyuridine triphosphate, that has been demonstrated to be a good substrate for a thermostable DNA polymerase and utilized in an in vitro DNA selection study (J. A. Latham, R. Johnson, J. J. Toole,
Nucleic Acids Res.
1994, 22, 2817-22). Indeed, difficulties in identifying modified deoxynucleotide triphophates substrates for the thermostable polymerases required for PCR have led recently to the development of novel strategies for in vitro selection without enzymatic amplification (J. Smith, E. V. Anslyn,
Angew. Chem.
1997, 109, 1956-58;
Angew. Chem. Int. Ed. Engl.
1997, 36, 1879-1881). These authors state that when modified oligomers are incorporated, the selection procedure is terminated after just one round of amplification since polymerases will not tolerate most modified mononucleotides. Therefore, the major impediment to the creation of novel functionally modified DNA catalysts and binding molecules is the determination of the substrate structures accepted by the thermostable polymerases.
The present invention provides a solution to this problem through the systematic synthesis of pyrimidine derivatives suitable for in vitro selections of modified DNAs.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a modified pyrimidine nucleotide of the structure, below
wherein X is NH
2
or ═O, where when X is NH
2
, (H) is absent and - - - is a chemical bond and when X is ═O, - - - is absent and (H) is a hydrogen bonded to N-1, and C:C represents a carbon-carbon double bond or a carbon-carbon triple bond. In a preferred embodiment, C:C is a carbon-carbon double bond. Each R is independently a cation or
wherein each R′ is independently absent or a cation and m is 1, 2 or 3. The cation of R and R′ is preferably a monovalent cation such as hydrogen or an alkali metal such as sodium (Na), potassium (K) or lithium (Li). Preferably, only one R is a cation. Thus, a compound of Structure II is preferably a 2′-deoxy-3′- or 5′-phosphate (mono-, di-, or tri-phosphate).
R
1
of Structure II is a functional group that mimics the structure and function of a naturally occurring amino acid residue. The functional group is preferably an amine or a carboxyl group. In accordance with this embodiment, R
1
is preferably
where R
2
is
R
3
is
and n is an integer from 0 to 6. Preferably n is 0, 1 or 2.
In another aspect, the present invention provides a DNA molecule comprising one or more compounds of St
Barbas Carlos F.
Kandasamy Sakthiel
Crane L. Eric
Geist Gary
Northrup Thomas E.
The Scripps Research Institute
LandOfFree
Functionalized pyrimidine nucleosides and nucleotides and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Functionalized pyrimidine nucleosides and nucleotides and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Functionalized pyrimidine nucleosides and nucleotides and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2458522