Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-10-17
2004-02-17
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S004100, C536S022100, C536S026100, C558S059000, C558S061000, C560S169000, C562S126000, C562S561000, C568S013000
Reexamination Certificate
active
06693187
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the fields of nucleic acid chemistry and oligonucleotide synthesis, and more particularly relates to novel phosphinoamidite carboxylates and analogs thereof in the synthesis of oligonucleotides having reduced internucleotide charge and enhanced nuclease resistance, i.e., phosphinocarboxylate oligonucleotides, phosphonocarboxylate oligonucleotides, and analogs thereof.
BACKGROUND
The derivatives of phosphoric acid have been shown to have a wide range of biological utility (Emsley and Hall (1976) in
The Chemistry of Phosphorus: Chapter
12
Biophosphorus Chemistry pp.
471-510 Harper and Row: London, England). In turn, molecules that mimic phosphoric acid and its derivatives have been shown to work as biological effectors and are often used as diagnostic and therapeutic agents (Uhlmann and Peyman (1990)
Chem. Rev.
90: 544). Examples of these derivatives are phosphonocarboxylates (Becker et al. (1977)
Antimicrob. Agents Chemother.
11: 919), phosphorothioates (Eckstein (1989)
Trends Biochem. Sci.
14: 97), phosphorodithioates (Nielsen et al. (1988)
Tetrahedron Lett.
29: 2911), methylphosphonates (Miller and Ts'o (1988)
Annu. Rep. Med. Chem.
23: 295), and phosphoramidates (Iyer et al. (1996)
Tetrahedron Lett.
37: 1543).
Phosphonocarboxylate mimics of phosphoric acid, specifically phosphonoformic acid and phosphonoacetic acid, have been shown to be especially useful as biological effectors and have been used as therapeutic agents (Shipkowitz et al. (1973)
Appl. Microbiol.
26: 264; Helgestrand et al. (1978)
Science
201: 819). The syntheses of phosphonoformic acid (Nylen (1924)
Chem Berichte.
57: 1023) and phosphonoacetic acid (Basinger et al. (1959)
J. Org. Chem.
24: 434) have relied upon the introduction of the carboxylate group onto the phosphorus moiety through an oxidative transformation such as a Michaelis-Arbuzov reaction (Arbuzov and Dunin (1914)
J. Chem. Soc.
653; Arbuzov (1964)
Pure Appl. Chem.
9: 307). The resulting phosphonocarboxylic acid products are in the oxidation state P(V). Once the phosphorus atom is in this pentacoordinate oxidation state the products are typically very stable. However, these stable products are difficult and sometimes impossible to utilize in performing high yielding chemical transformations, chemical couplings, or chemical derivatizations. As a result of the low chemical reactivity of these pentacoordinate phosphorus molecules, many biologically important molecules that exist as phosphoric acid derivatives have not been mimicked with phosphonocarboxylic acid derivatives (Hildebrand (1983), in
The Role of Phosphonates in Living Systems: Chapters
5 & 6, pp. 97-169, CRC Press In: Boca Raton, USA).
Two clear examples of biologically important molecules that exist naturally as phosphoric acid derivatives and have not been mimicked as phosphonocarboxylic acid derivatives are the polynucleotides DNA and RNA. Polynucleotides modified at the phosphodiester internucleotide linkage are of significant interest to the emerging fields of antisense therapeutics, nucleic acid diagnostics, and genomics. Phosphorus-containing chemical compounds and compositions that have been successfully utilized to enable the synthesis of polynucleotides have been frequently reviewed in the scientific literature (Verma et al. (1998)
Annu. Rev. Biochem.
67:99; Sekine et. al. (1998)
Nucleosides and Nucleotides
17:203; Iyer et al (1999)
Curr. Opin. Mol. Ther.
1:344). The successful chemical synthesis of polynucleotides or modified polynucleotides is a task especially dependent upon the ability to find and employ phosphorus-containing compounds that enable high yield chemical couplings and chemical transformations (Caruthers (1985)
Science
230:281; Caruthers et al., U.S. Pat. No. 4,415,732, issued Nov. 15, 1983). To enable the chemical synthesis of polynucleotides or modified polynucleotides, the phosphorus compounds used must be able to perform high yield coupling reactions that are general to the four nucleobases and specific for the desired polynucleotide products. High yield coupling efficiencies for the formation of internucleotide bonds are necessary in order to enable the synthesis of biologically relevant lengths of polynucleotides (Koster et al., U.S. Pat. No. 4,725,677 issued Feb. 16, 1988), wherein a “biologically relevant length” is a length that allows the polynucleotide to stably and specifically bind to other polynucleotides by hybridization through base-pairing interactions. Stable binding of polynucleotides to other polynucleotides via hybridization is also affected by temperature, salt concentration, nucleotide sequence, and other factors, as has been extensively discussed in the literature; see, e.g., Sanger (1984) in
Principles of Nucleic Acid Structure: Chapter
6, pp. 116-158 (Springer-Verlag: New York, USA).
The need for high yield coupling reactions in synthesizing polynucleotides of a biologically relevant length is due to the mathematical relationship between the final yield of the desired polynucleotide product and the efficiency for each individual coupling reaction giving rise to a new internucleotide bond. The final yield of the desired polynucleotide product is a multiplication product of all individual coupling and deprotection steps required in achieving that product. As a result, the yield of the final polynucleotide product decreases exponentially with a linear decrease in the coupling efficiency. That is, the effect of the coupling efficiency on the overall yield of product can be described by the equation Y=X
N
, where Y is the fractional overall yield, X is the fractional coupling efficiency, and N is the number of couplings. For the synthesis of a typical polynucleotide 20 nucleotides in length with 19 internucleotide linkages, 19 coupling reactions are involved and the overall yield is given by Y=X
19
. The table below illustrates the relationship between the coupling efficiency (X) and overall yield of polynucleotide product (Y).
X
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0.95
0.99
Y
1
−19
5
−14
1
−10
3
−3
2
−6
6
−5
1
−3
1
−2
0.14
0.38
0.82
As clearly illustrated by this example, and as well known by those skilled in the art, the synthesis of a 20-mer polynucleotide is not possible until the coupling efficiency achieved during synthesis approaches 90% or greater. Only at these coupling efficiencies can full-length polynucleotides be reproducibly isolated and purified from the reaction mixtures. As a further example for illustration, at an 80% coupling efficiency, the theoretical maximum amount of full-length product (Y), after 19 couplings, is 1.4%. However, the overall yield shown in the table above is a simplification that considers only the effect of the efficiency for the formation of the internucleotide bond. The actual overall yield of a polynucleotide product is additionally adversely affected by any inefficiency in deprotection reactions used during synthesis, post-synthesis, or from side-reactions leading to undesired products. The ability to isolate a full-length polynucleotide product, 20 nucleotides in length, from a polynucleotide synthesis that achieves an 80% per cycle coupling efficiency is precarious and rarely reproducible, and directly linked to the yield of the individual deprotection reactions following each coupling step. As a direct result of these requirements for high yield reactions, the chemical synthesis of polynucleotides has been accomplished by only very few methods; see Brown (1983) in
Protocols for Oligonucleotides and Analogs: Chapters
1, pp. 1-17 (Humana Press: Totowa, N.J., USA, Ed. S. Agrawal). Each of the methods that has enabled the chemical synthesis of polynucleotides has in turn been enabled by the development of high yielding coupling reactions at the phosphorus moiety, in concert with the development of high yielding deprotection reactions. The fact that so few methods have enabled polynucleotide synthesis is a direct result of the difficul
Eberle Shelley P.
Lievre Cornu LLC
McIntosh III Traviss C.
Reed Dianne E.
Reed & Eberle LLP
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