Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-19
2001-09-11
Marschel, Ardin H. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C422S131000, C435S006120, C536S025320
Reexamination Certificate
active
06288221
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides metal-containing purines, pyrimidines, nucleosides, nucleotides and oligonucleotides; including phosphoramidite and photolabile derivatives thereof, including methods of making and methods of using same. The present invention also provides a method for detection of nucleic acid sequences via electrochemical or photochemical means. The present invention provides an improved method of making labeled nucleic acid polymers.
BACKGROUND
Modifying a nucleoside, nucleotide or oligonucleotide with a specific chemical functionality, such as a photo-, redox- or chemically-active metal complex, is of wide-spread interest for analytical applications (sequencing, hybridization assays), therapeutic uses (anticancer, antiviral pharmaceuticals) and mechanistic studies (electron transfer, structure-function). U. Englisch, D. D. Gauss, Angew.
Chem. Int. Ed. Engl
. 30 (1991) 613-629; G. H. Keller, M. M. Manak,
DNA Probes
, Stockton Press, New York 1993; P. G. Sammes, and G. Yahioglu,
Natural Product Reports
(1996) 1-28. Synthetic strategies toward these supra-molecular bioassemblies focus primarily on post-modification of the synthesized nucleic acid single strand or complementary duplex. A number of researchers, (C. J. Murphy, M. R. Arkin, J. K. Barton,
Science
262 (1993) 1025-1029; R. E. Holmlin, P. J. Dandliker, J. K. Barton,
Angew, Chem. Int. Ed. Engl
. 36 (1997) 2714-2730 and references therein; D. Magda, R. A. Miller, J. L. Sessler, B. L. Iverson,
J. Am. Chem. Soc
. 119, (1994) 7439-7440; J. Telser, K. A. Cruickshank. K. S. Schanze, T. L. Netzel.
J. Am Chem. Soc
. 111 (1989) 7221-7226; W. Bannwarth, D. Schmidt. R. L. Stallard. C. Hornung. R. Knorr, F. Müller,
Helv. Chim. Acta
71 (1988) 2085-2099), have used this strategy to link a metal complex, usually as the activated succinimide ester, to the terminus of the nucleic acid single strand previously modified to contain an alkyl amine. In another approach, an amino- or diimine-modified (e.g., phenanthroline) nucleoside is synthesized, and subsequently reacted with a metal to form the desired complex. G. B. Dreyer, P. B. Dervin,
Biochemistry
82 (1985) 968-972; C. B. Chen. D. S. Sigman,
J. Am. Chem. Soc
. 110 (1988) 6570-6572; M. Matsumura. M. Endo, M. Komiyama,
J. Chem. Soc., Chem. Commun
. (1994) 2019-2020; J. K. Bashkin. E. I. Frolova. U. Sampath,
J. Am. Chem. Soc
. 116 (1994) 5981-5982; T. J. Meade, J. F. Kayyem.
Angew. Chem. Int. Ed. Engl
. 34 (1995) 352-354. An alternative strategy that offers clear synthetic advantages over the previous systems is to use DNA/RNA solid-phase synthetic methodologies for the site-specific labeling of an oligonucleotide with a transition metal complex. A number of investigations are currently exploring this approach. J. Schliepe, U. Berghoff, B. Lippert, D. Cech,
Angew. Chem. Int. Ed. Engl
. 35 (1996) 646-648; R. Manchanda. S. U. Dunham, S. J. Lippard.
J. Am. Chem. Soc
. 118 (1996) 5144-5145; W. Bannwarth. D. Schmidt,
Tetrahedron Letters
30 (1989) 1513-1516; E. Meggers. D. Kusch. B. Giese,
Helvetica Chim. Acta
80 (1997) 640-652; D. J. Hurley. Y. Tor.
J. Am. Chem. Soc
. 120 (1998) 2194-2195).
The present invention provides novel metal-containing purines, pyrimidines, nucleosides and nucleotides, and derivatives thereof, including those which are useful in nucleic acid synthesis, and identification, such as in detection and sequencing. The present invention also provides a new method of labeling purines, pyrimidines, nucleosides, nucleotides and their derivatives during automated or manual synthesis of nucleic acid polymers, such as oligonucleotides, DNA or RNA molecules.
SUMMARY OF THE INVENTION
It is an object of the invention to provide metal-containing purines, pyrimidines, nucleosides, nucleotides, and oligonucleotides, including derivatives and intermediates thereof.
Another object of the present invention is to provide a novel method of synthesizing labeled nucleic acid polymers, such as oligonucleotides, DNA or RNA molecules, preferably by automated methods, which preferably, include use of known iodonucleoside derivatives.
It is another object of the invention to provide a method of making the purine, pyrimidine, nucleoside and nucleotide derivatives of the present invention.
It is yet another object of the invention to provide a method of conducting a polymerase chain reaction or other primer-directed reaction to make and/or detect a nucleic acid of interest in an amplification product of the reaction by incorporating in the reaction product a metal-containing derivative or iodonucleosides, (which may be subsequently labeled) according to the present invention.
It is also an object of the invention to provide a method of detecting a nucleic acid analyte, such as DNA or RNA or dideoxy residues of same, of interest in the product of a polymerase chain reaction or other primer-directed reaction by incorporating at least one purine, pyrimidine, nucleoside or nucleotide derivative of the present invention in the reaction.
Yet another object of the present invention is to provide a solid-phase method of making a nucleic acid polymer, such as an oligonucleotide, which incorporates the metal-containing derivative or halonucleoside, such as iodonucleoside, derivatives of the present invention.
An object of the present invention is to provide a solid surface, including, but not limited to, a bead, plate, or electrode, with a metal-containing derivative of the present invention; and a method for using same.
Specifically, the present invention provides ferrocene; bis(2,2′-bipyridine)(4′-methyl-2,2′-bipyridine-4-carbornyl propargyl amine) ruthenium (11); and bis(2,2′-bipyridine) (4′-methyl-2,2′-bipyridine-4-carbonyl propargyl amine) osmium (II), and corresponding Rh, Cr and Co, nucleoside derivatives. Dimethoxytrityl; 2-cyanoethyl-N,N-diisopropyl-phosphoramidite; phosphotriester; phosphoramidite; and 1-(2-nitro-4-,5-methylene dioxyphenyl) ethyl-choloroformate derivatives are also provided.
The metal complexes described in the present invention are linked to the nucleoside via an amide bond through either the 5, 3 or 2 carbon of the sugar (ribose, deoxyribose or dideoxyribose, for example) or the 5 carbon of a pyrimidine or the 8 carbon of a purine. For example, the amide bond will allow for delocalization of the excited electron on the metal diimine and this will facilitate the electron transfer into the nucleic acid. Preferred amide bonds include alkynylamines. This is a preferred bonding motif, however other bonding configurations are possible such as, for example, ethers and esters.
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Beilstein Amy E.
Grinstaff Mark W.
Khan Shoeb I.
Duke University
Marschel Ardin H.
Nixon & Vanderhye P.C.
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