Oligonucleotides having chiral phosphorus linkages

专利摘要:
Sequence-specific oligonucleotides are provided having substantially pure chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp phosphotriester, and chiral Rp phosphotriester linkages. The novel oligonucleotides are prepared via a stereospecific SN2 nucleophilic attack of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate anion on the 3′ position of a xylonucleotide. The reaction proceeds via inversion at the 3′ position of the xylo reactant species, resulting in the incorporation of phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate linked ribofuranosyl sugar moieties into the oligonucleotide.
公开号:US20010008936A1
申请号:US09/784,917
申请日:2001-02-16
公开日:2001-07-19
发明作者:Phillip Cook
申请人:Isis Pharmaceuticals Inc；
IPC主号:A61K48-00

专利说明:
[0001] This application is a continuation-in-part of U.S. application Ser. No. 91/00243, filed Jan. 11, 1991, which is a continuation-in-part of application Ser. No. 463,358 filed Jan. 11, 1990 and of application Ser. No. 566,977, filed Aug. 13, 1990. The entire disclosures of both applications, which are assigned to the assignee of this invention, are incorporated herein by reference. [0001] FIELD OF THE INVENTION
[0002] This invention is directed to sequence-specific oligonucleotides having chiral phosphorus linkages and to a novel chemical synthesis of these and other oligonucleotides. The invention includes chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate-linked oligonucleotides. The invention further includes chiral phosphorothioate, chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate-linked oligonucleotides that contain at least one modified nucleoside unit. The novel chemical synthesis provides such chiral phosphorothioate, chiral alkylphosphonate, chiral phosphotriester, and chiral phosphoramidate oligonucleotides as well as “natural” or “wild type” phosphodiester oligonucleotides. [0002] BACKGROUND OF THE INVENTION
[0003] Messenger RNA (mRNA) directs protein synthesis. As a therapeutic strategy, antisense therapy strives to disrupt the synthesis of target proteins by using a sequence-specific oligonucleotide to form a stable heteroduplex with its corresponding mRNA. Such antisense oligonucleotides generally have been natural phosphodiester oligonucleotides. [0003]
[0004] As contrasted to natural phosphodiester oligonucleotides, the use of phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides in antisense therapy provides certain distinguishing features. Each of the phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus linkages can exist as diastereomers. Certain of these phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides have a greater resistance to nucleases. Some have solubilities similar to the solubility of natural phosphodiester oligonucleotides. Other have solubilities different from that of the natural phosphodiester oligonucleotides. Some are generally more chemically or thermodynamically stable than the natural phosphodiester oligonucleotides. At least the phosphorothioates have oligonucleotide-RNA heteroduplexes that can serve as substrates for endogenous RNase H. [0004]
[0005] The phosphorothioate oligonucleotides, like the natural phosphodiester oligonucleotides, are soluble in aqueous media. In contrast, methylphosphonate, phosphotriester, and phosphoramidate oligonucleotides, which lack a charge on the phosphorus group, can penetrate cell membranes to a greater extent and, thus, facilitate cellular uptake. The internucleotide linkage in methylphosphonate oligonucleotides is more base-labile than that of the natural phosphodiester internucleotide linkage, while the internucleotide linkage of the phosphorothioate oligonucleotides is more stable than the natural phosphodiester oligonucleotide linkage. [0005]
[0006] The resistance of phosphorothioate oligonucleotides to nucleases has been demonstrated by their long half-life in the presence of various nucleases relative to natural phosphodiester oligonucleotides. This resistance to nucleolytic degradation in vitro also applies to in vivo degradation by endogenous nucleases. This in vivo stability has -been attributed to the inability of 3′-5′ plasma exonucleases to degrade such oligonucleotides. Phosphotriester and methylphosphonate oligonucleotides also are resistant to nuclease degradation, while phosphoramidate oligonucleotides show some sequence dependency. [0006]
[0007] Since they exist as diastereomers, phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides synthesized using known, automated techniques result in racemic mixtures of Rp and Sp diastereomers at the individual phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages. Thus, a 15-mer oligonucleotide containing 14 asymmetric linkages has 2[0007] ¹⁴, i.e. 16,384, possible stereoisomers. Accordingly, it is possible that only a small percentage of the oligonucleotides in a racemic mixture will hybridize to a target mRNA or DNA with sufficient affinity to prove useful in antisense or probe technology.
[0008] Miller, P. S., McParland, K. B., Jayaraman, K., and Ts'o, P. O. P (1981), [0008] Biochemistry, 20:1874, found that small di-, tri- and tetramethylphosphonate and phosphotriester oligonucleotides hybridize to unmodified strands with greater affinity than natural phosphodiester oligonucleotides. Similar increased hybridization was noted for small phosphotriester and phosphoramidate oligonucleotides; Koole, L. H., van Genderen, M. H. P., Reiners, R. G., and Buck, H. M. (1987), Proc. K. Ned. Adad. Wet., 90:41; Letsinger, R. L., Bach, S. A., and Eadie, J. S. (1986), Nucleic Acids Res., 14:3487; and Jager, A., Levy, M. J., and Hecht, S. M. (1988), Biochemistry, 27:7237. The effects of the racemic diastereomers on hybridization becomes even more complex as chain length increases.
[0009] Bryant, F. R. and Benkovic, S. J. (1979), [0009] Biochemistry, 18:2825 studied the effects of diesterase on the diastereomers of ATP. Published patent application PCT/US88/03634 discloses dimers and trimers of 2′-5′-linked diastereomeric adenosine units. Niewiarowski, W., Lesnikowski, Z. J., Wilk, A., Guga, P., Okruszek, A., Uznanski, B., and Stec, W. (1987), Acta Biochimica Polonia, 34:217, synthesized diastereomeric dimers of thymidine, as did Fujii, M., ozaki, K., Sekine, M., and Hata, T. (1987), Tetrahedron, 43:3395.
[0010] Stec, W.J., Zon, G., and Uznanski, B. (1985), [0010] J. Chromatography, 326:263, have reported the synthesis of certain racemic mixtures of phosphorothioate or methyphosphonate oligonucleotides. However, they were only able to resolve the diastereomers of certain small oligomers having one or two diastereomeric phosphorus linkages.
[0011] In a preliminary report, J. W. Stec, Oligonucleotides as antisense inhibitors of gene expression: Therapeutic implications, meeting abstracts, Jun. 18-21, 1989, noted that a non-sequence-specific thymidine homopolymer octomer—i.e. a (dT)[0011] ₈-mer, having “all-except-one” Rp configuration methylphosphonate linkages—formed a thermodynamically more stable hybrid with a 15-mer deoxyadenosine homopolymer—i.e. a d(A)₁₅-mer—than did a similar thymidine homopolymer having “all-except-one” Sp configuration methylphosphonate linkages. The hybrid between the “all-except-one” Rp (dT)₈-mer and the d(A)₁₅-mer had a Tm of 38° C. while the Tm of the “all-except-one” Sp (dT)₈-mer and the d(A)₁₅-mer was <0° C. The hybrid between a (dT)₈-mer having natural phosphodiester linkages, i.e. octathymidylic acid, and the d(A)₁₅-mer was reported to have a Tm of 14° C. The “all-except-one” thymidine homopolymer octamers were formed from two thymidine methylphosphonate tetrameric diastereomers linked by a natural phosphodiester linkage.
[0012] To date, it has not been possible to chemically synthesize an oligonucleotide having more than two adjacent, chirally pure phosphorous linkages. Indeed, even in homopolymers it has been possible to produce only three such adjacent chiral linkages. For an oligonucleotide to be useful as an antisense compound, many nucleotides must be present. While not wishing to be bound by any particular theory, it is presently believed that generally at least about 10 or more nucleotides are necessary for an oligonucleotide to be of optimal use as an antisense compound. Because it has not been possible to resolve more than two or three adjacent phosphorus linkages, the effects of induced chirality in the phosphorus linkages of chemically synthesized antisense oligonucleotides has not been well assessed heretofore. [0012]
[0013] Except as noted above, the sequence-specific phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides obtained utilizing known automated synthetic techniques have been racemic mixtures. Indeed, it was recently stated in a review article that: “It is not yet possible to synthesize by chemical means diastereomerically pure chains of the length necessary for antisense inhibition,” see J. Goodchild (1990) [0013] Bioconjugate Chemistry, 1:165.
[0014] The use of enzymatic methods to synthesize oligonucleotides having chiral phosphorous linkages has also been investigated. Burgers, P. M. J. and Eckstein, F. (1979), [0014] J. Biological Chemistry, 254:6889; and Gupta, A., DeBrosse, C., and Benkovic, S. J. (1982) J. Bio. Chem., 256:7689 enzymatically synthesized diastereomeric polydeoxyadenylic acid having phosphorothioate linkages. Brody, R. S. and Frey, P. S. (1981), Biochemistry, 20:1245; Eckstein, F. and Jovin, T. M. (1983), Biochemistry, 2:4546; Brody, R. S., Adler, S., Modrich, P., Stec, W. J., Leznikowski, Z. J., and Frey, P. A. (1982) Biochemistry, 21: 2570-2572; and Romaniuk, P. J. and Eckstein, F. (1982) J. Biol. Chem., 257:7684-7688 all enzymatically synthesized poly TpA and poly ApT phosphorothioates while Burgers, P. M. J. and Eckstein, F. (1978) Proc. Natl. Acad. Sci. USA, 75: 4798-4800 enzymatically synthesized poly UpA phosphorothioates. Cruse, W. B. T., Salisbury, T., Brown, T., Cosstick, R. Eckstein, F., and Kennard, O. (1986), J. Mol. Biol., 192:891, linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer. Most recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R., and Watanabe, K. (1991) Nucleic Acids Research, 19:547, enzymatically synthesized RNA's having from several hundred to ten thousand nucleotides incorporating Rp diastereomeric phosphorothioate linkages. Enzymatic synthesis, however, depends on the availability of suitable polymerases that may or may not be available, especially for modified nucleoside precursors.
[0015] Thus, while phosphorothioate, alkylphosphonate, phosphoamidate, and phosphotriester oligonucleotides have useful characteristics, little is known concerning the effects of differing chirality at the phosphorus linkages. It would therefore be of great advantage to provide oligonucleotides having phosphorous linkages of controlled stereochemistry. [0015] OBJECTS OF THE INVENTION
[0016] Accordingly, it is one object of this invention to provide sequence-specific oligonucleotides having chirally pure phosphorothioate, alkylphosphonate, phosphotriester or phosphoramidate linkages. [0016]
[0017] It is a further object to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides comprising substantially all Rp or all Sp linkages. [0017]
[0018] It is another object to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides that have antisense hybridizability against DNA and RNA sequences. [0018]
[0019] It is still another object of this invention to provide phosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotides for use in antisense diagnostics and therapeutics. [0019]
[0020] A further object is to provide research and diagnostic methods and materials for assaying bodily states in animals, especially diseased states. [0020]
[0021] Another object is to provide therapeutic and research methods and materials for the treatment of diseases through modulation of the activity of DNA and RNA. [0021]
[0022] It is yet another object to provide new methods for synthesizing sequence-specific oligonucleotides having chirally pure phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages. [0022] SUMMARY OF THE INVENTION
[0023] The present invention provides stereoselective methods for preparing sequence-specific oligonucleotides having chiral phosphorous linkages. In certain preferred embodiments, these methods comprise the steps of: [0023]
[0024] (a) selecting a first synthon having structure (1): [0024]
[0025] wherein: [0025]
[0026] Q is 0 or CH[0026] ₂;
[0027] R[0027] _A and R_B are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
[0028] R[0028] _D is O, S, methyl, alkoxy, thioalkoxy, amino or substituted amino;
[0029] R[0029] _E is O or S;
[0030] R[0030] _X is H, OH, or a sugar derivatizing group;
[0031] B[0031] _X is a naturally occurring or synthetic nucleoside base or blocked nucleoside base; and
[0032] Y is a stable blocking group, a solid state support, a nucleotide on a solid state support, or an oligonucleotide on a solid state support; [0032]
[0033] (b) selecting a second synthon having structure (2): [0033]
[0034] wherein: [0034]
[0035] R[0035] _F is a labile blocking group; and
[0036] L is a leaving group or together L and Bx are a 2-3′ or 6-3′ pyrimidine or 8-3′ purine cyclonucleoside; [0036]
[0037] (c) adding the second synthon to the first synthon in the presence of a base to effect nucleophilic attack of the 5′-phosphate of the first synthon at the 3′-position of the second synthon to yield a new first synthon having structure (3): [0037]
[0038] via a stereospecific inversion of configuration at the 3′ position of the second synthon; and [0038]  
[0039] (d) treating the new first synthon with a reagent to remove the labile blocking group RF. [0039]
[0040] Additional nucleotides are added to the new first synthon by repeating steps (b), (c), and (d) for each additional nucleotide. Preferably, R[0040] _F is an acid-labile blocking group and said new first synthon in step (d) is treated with an acidic reagent to remove said acid-labile RF blocking group.
[0041] The present invention also provides sequence-specific oligonucleotides comprising a plurality of nucleotides linked bychiral phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides linkages wherein at least one of the nucleosides is a non-naturally occurring nucleoside. Preferably, the nucleosides are connected via linkages selected from the group consisting of chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rp phosphotriester linkages. In one embodiment of the invention each of the linkages of the oligonucleotide is a substantially pure chiral phosphorous linkage. In other embodiments less than all of the phosphate linkages are substantially pure chiral phosphorous linkages. In further embodiments, the oligonucleotides of the invention form at least a portion of a targeted RNA or DNA sequence. [0041]
[0042] The present invention also provides oligonucleotides comprising nucleoside units joined together by either all Sp phosphotriester linkages, all Rp phosphotriester linkages, all Sp phosphoramidate linkages, or all Rp phosphoramidate linkages. Also provided are oligonucleotides having at least 10 nucleoside units joined together by either all Sp alkylphosphonate linkages or all Rp alkylphosphonate linkages. Preferably such alkylphosphonate linkages are methylphosphonate linkages. Each of these oligonucleotides can form at least a portion of a targeted RNA or DNA sequence. [0042]
[0043] In preferred embodiments of the invention, the oligonucleotides include non-naturally occurring nucleoside units incorporated into the oligonucleotide chain. Such nucleoside units preferably have structure (4) or structure (5): [0043]
[0044] wherein Q, R[0044] _B, R_G, and B_X are defined as above and R_C is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N3, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂, OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, poly-alkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide.
[0045] In preferred embodiments, B[0045] _X is a pyrimidinyl-l or purinyl-9 moiety such as, for example, adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
[0046] In further preferred embodiments, the modified nucleosides include nucleosides having structures (6)-(1l): [0046]
[0047] wherein: [0047]
[0048] G and K are, independently, C or N; [0048]
[0049] J is N or CR[0049] ₂R₃;
[0050] R[0050] ₁ is OH or NH₂;
[0051] R[0051] ₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
[0052] R[0052] ₄ and R₅ are, independently, H, OH, NH₂, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic-properties of an oligonucleotide;
[0053] R[0053] ₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂, C(NH)NH₂, C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
[0054] X is a sugar or a sugar analog moiety where said sugar analog moiety is a sugar substituted with at least one substituent comprising an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide. [0054]
[0055] The present invention also provides compounds which are useful in forming the oligonucleotides of invention. Such compounds have structure (12): [0055]
[0056] wherein Q, R[0056] _A, R_D, R_E, R_X, L, and B_X are defined as above and R_F is H or a labile blocking group.
[0057] The oligonucleotides of the invention are useful to increase the thermodynamic stability of heterodupiexes with target RNA and DNA. Certain of the oligonucleotides of the invention are useful to elicit RNase H activity as a termination event. Certain other oligonucleotides are useful to increase nuclease resistance. The oligonucleotides of the invention are also useful to test for antisense activity using reporter genes in suitable assays and to test antisense activity against selected cellular target mRNA's in cultured cells. [0057] DETAILED DESCRIPTION OF THE INVENTION
[0058] As will be recognized, adjacent nucleosides of a naturally occurring or wild type oligonucleotide are joined together by phosphodiester linkages, i.e. diesters of phosphoric acid. The natural phosphodiester linkages in oligonucleotides are at the same time both non-chiral and prochiral sites. Substitution of one of the oxygen atoms of the phosphate moiety of a nucleotide with another atom yields an asymmetric center on the phosphorus atom. Since a nucleotide unit already contains a first asymmetrical center within its sugar moiety, further asymmetry at the phosphorus atom of the nucleotide yields a diasymmetric nucleotide. Such a diasymmetric nucleotide is a chiral nucleotide having Sp and Rp diastereomers. [0058]
[0059] Substitution of one of the oxygen atoms of the phosphate moiety of a nucleotide with a sulfur atom yields Sp and Rp diastereomeric phosphorothioate analogs. Similarly, substitution of a phosphate oxygen atom by an alkyl moiety yields diastereomeric alkylphosphonate analogs. Substitution with an alkoxy group yields diastereomeric Sp and Rp phosphotriesters. Substitution with a thioalkoxy group yields a mixed triester—a phosphodiesterthioester. Substitution with an amine or a substituted amine (including heterocyclic amines) yields diastereomeric Sp and Rp phosphoramidates. [0059]
[0060] It will be appreciated that the terms “phosphate” and “phosphate anion” as employed in connection with the present invention include nucleotides and oligonucleotides derived by replacement of one of the oxygen atoms of a naturally occurring phosphate moiety with a heteroatom, an alkyl group or an alkyoxy group. Thus, the terms “phosphate” or “phosphate anion” include naturally occurring nucleotides, phosphodiesters of naturally occurring oligonucleotides, as well as phosphorothioate, alkylphosphonate, phosphotriester, and phosphoamidate oligonucleotides. [0060]
[0061] Since there exist numerous phosphodiester linkages in an oligonucleotide, substitution of an oxygen atom by another atom such as, for example, sulfur, nitrogen, or carbon in one or more of the phosphate moieties yields a racemic mixture unless such substitution occurs in a stereospecific manner. As a practical matter, see Stec, W. J., Zon, G., and Uznanski, B. (1985), [0061] J. Chromatography, 326:263, above. Separation of the diastereomers of racemic mixtures of non-stereospecific synthesized oligonucleotides is only possible when there are a minimum of diasymmetric sites, for example, two diasymmetric sites. Since the diasymmetric substituent group at each diastereomeric phosphorus atom could have steric, ionic or other effects on conformation, binding, and the like at each such site, sequence-specific oligonucleotides having all Sp or all Rp chiral phosphorus linkages are desirable.
[0062] In accordance with this invention, sequence-specific oligonucleotides are provided comprising substantially pure chiral phosphate linkages such as, for example, phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkages. In contrast to prior art synthetic oligonucleotides, at least certain of the chiral phosphorous linkages of the present oligonucleotides are not racemic in nature but, rather, possess relatively high enantiomeric purity. As will be recognized by those skilled in the art, enantiomeric purity—also known as chiral purity—is manifested for a chemical compound by the predominance of one enantiomer over the other. Thus, an oligonucleotide can be said to possess a substantially pure chiral phosphate linkage where, for example, the Sp form of that linkage greatly predominates over the Rp form. In accordance with the present invention, at least certain of the chiral phosphate linkages present in an oligonucleotide should have chiral purity greater than about 75%. Preferably such linkages have chiral purity greater than about 90%, more preferably greater than about 95%, even more preferably about 100%. Chiral purity may be determined by any of the many methods known in the art, including but not limited to x-ray diffraction, optical rotary dispersion, and circular dichroism. [0062]
[0063] The oligonucleotides of the invention are expected to exhibit one or more efficacious properties such as, for example, hybridization with targeted RNA's and DNA's, cellular absorption and transport, or improved enzymatic interaction. At the same time, it is expected that these improvements to the basic oligonucleotide sequences will not significantly diminish existing properties of the basic oligonucleotide sequence. Thus, the present improvements are likely to lead to improved drugs, diagnostics, and research reagents. [0063]
[0064] Further improvements likely can be effected by making one or more substitutions or modifications to the base or the sugar moieties of the individual nucleosides employed to prepare the chiral oligonucleotides of the invention. Such substitutions or modifications generally comprise derivation at a site on the nucleoside base or at a site on the nucleoside sugar, provided such derivation does not interfere with the stereoselective syntheses of the present invention by, for example, blocking nucleophilic attack of the 5′-phosphate of a first synthon at the 3′-position of a second synthon. In certain embodiments, one or more of the nucleosides of the chiral oligonucleotides of the invention include a naturally occurring nucleoside unit which has been substituted or modified. These non-naturally occurring or “modified” nucleoside units preferably have either structure (4) or structure (5): [0064]
[0065] wherein: [0065]
[0066] Q is O or CHR[0066] _G;
[0067] R[0067] _A and R_B are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
[0068] R[0068] _C is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂, OCH═CH₂, CH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide;
[0069] R[0069] _G is H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
[0070] B[0070] _X is a nucleoside base, a blocked nucleoside base, a nucleoside base analog, or a blocked nucleoside base analog.
[0071] In preferred embodiments B[0071] _X is a pyrimidinyl-1 or purinyl-9 moiety as for instance adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine. Preferably, B_X is selected such that a modified nucleoside has one of the structures (6)-(11):
[0072] wherein: [0072]
[0073] G and K are, independently, C or N; [0073]
[0074] J is N or CR[0074] ₂R₂;
[0075] R[0075] ₁ is OH or NH₂;
[0076] R[0076] ₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
[0077] R[0077] ₄ and R₅ are, independently, H, OH, NH₂, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;
[0078] R[0078] ₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂, C(NH)NH₂, C(O)O-alkyl, CSNH,, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and
[0079] X is a sugar or a sugar analog moiety where said sugar analog moiety is a sugar substituted with at least one substituent comprising an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide. It is preferred that X have the general structure (4) or (5). [0079]
[0080] For the purposes of this invention, improving pharmacodynamic properties means improving oligonucleotide uptake, enhanced oligonucleotide resistance to degradation, and/or strengthened sequence-specific hybridization with RNA and improving pharmacokinetic properties means improved oligonucleotide uptake, distribution, metabolism or excretion. RNA cleaving moieties are chemical compounds or residues which are able to cleave an RNA strand in either a random or, preferably, a sequence-specific fashion. [0080]
[0081] Exemplary base moieties of the invention are any of the natural pyrimidinyl-1- or purinyl-9-bases including uracil, thymine, cytosine, adenine, guanine, 5-alkylcytosines such as 5-methylcytosine, hypoxanthine, 2-aminoadenine, and other modified bases as depicted in the formulas above. Exemplary sugars include ribofuranosyl, 2′-deoxyribofuranosyl, their corresponding five membered ring carbocyclic analogs as well as other modified sugars depicted in the formulas above. Particularly preferred modified sugars include 2′-fluoro and 2′-O-methyl-2′-deoxyribofuranosyl, i.e. 2′-fluoro and 2′-O-methyl-β-D-erythro-pentofuranosyl. [0081]
[0082] Alkyl groups of the invention include but are not limited to C[0082] ₁-C₁₂ straight and branched chained alkyls such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, isopropyl, 2-butyl, isobutyl, 2-methylbutyl, isopentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl. Alkenyl groups include but are not limited to unsaturated moieties derived from the above alkyl groups including but not limited to vinyl, allyl, crotyl, propargyl. Aryl groups include but are not limited to phenyl, tolyl, benzyl, naphthyl, anthracyl, phenanthryl, and xylyl. Halogens include fluorine, chlorine, bromine, and iodine. Suitable heterocyclic groups include but are not limited to imidazole, tetrazole, triazole, pyrrolidine, piperidine, piperazine, and morpholine. Carbocyclic groups include 3, 4, 5, and 6-membered substituted and unsubstituted alkyl and alkenyl carbocyclic rings. Amines include amines of all of the above alkyl, alkenyl, and aryl groups including primary and secondary amines and “tmasked amines” such as phthalimide. Amines are also meant to include polyalkylamino compounds and aminoalkylamines such as aminopropylamine and further heterocyclo-alkylamines such as imidazol-1, 2 or 4-yl-propylamine. Substituent groups for the above include but are not limited to other alkyl, haloalkyl, alkenyl, alkoxy, thioalkoxy, haloalkoxy, and aryl groups as well as halogen, hydroxyl, amino, azido, carboxy, cyano, nitro, mercapto, sulfides, sulfones, and sulfoxides. Other suitable substituent groups also include rhodamines, coumarins, acridones, pyrenes, stilbenes, oxazolo-pryidocarbazoles, anthraquinones, phenanthridines, phenazines, azidobenzenes, psoralens, porphyrins, cholesterols, and other “conjugate” groups.
[0083] Methods of synthesizing such modified nucleosides are set forth in copending applications for United States Letters Patent, assigned to the assignee of this invention, and entitled Compositions and Methods for Modulating RNA Activity, serial number 463,358, filed Jan. 11, 1990; Sugar Modified Oligonucleotides That Detect And Modulate Gene Expression, Ser. No. 566,977, filed Aug. 13, 1990; and Compositions and Methods for Modulating RNA Activity, U.S. Ser. No. 91/00243, filed Jan. 11, 1991, the entire disclosures of which are incorporated herein by reference. [0083]
[0084] The chirally pure phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides of the invention can be evaluated for their ability to act as inhibitors of RNA translation in vivo. Various therapeutic areas can be targeted for such antisense potential. These therapeutic areas include but are not limited to herpes virus (HSV), the TAR and tat regions of HIV, the codon regions of [0084] Candida albicans chitin synthetase and Candida albicans β tubulin, papilloma virus (HPV), the ras oncogene and protooncogene, ICAM-1 (intercellular adhesion molecule-1) cytokine, and 5′-lipoxygenase. A targeted region for HSV includes GTC CGC GTC CAT GTC GGC. A targeted region for HIV includes GCT CCC AGG CTC AGA TCT. A targeted region for Candida albicans includes TGT CGA TAA TAT TAC CA. A targeted region for human papillomavirus, e.g. virus types HPV-11 and HPV-18, includes TTG CTT CCA TCT TCC TCG TC. A targeted region for ras includes TCC GTC ATC GCT CCT CAG GG. A targeted region for ICAM-1 includes TGG GAG CCA TAG CGA GGC and the sequence CGA CTA TGC AAG TAC is a useful target sequence for 5-lipoxygenase. In each of the above sequences the individual nucleotide units of the oligonucleotides are listed in a 5′ to 3′ sense from left to right.
[0085] The phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides of the invention may be used in therapeutics, as diagnostics, and for research, as specified in the following copending applications for United States Letters Patent assigned to the assignee of this invention: Compositions and Methods for Modulating RNA Activity, Ser. No. 463,358, filed Jan. 11, 1990; Compositions and Methods for Detecting and Modulating RNA Activity, Ser. No. 463,358, filed Jan. 11, 1990; Antisense Oligonucleotide Inhibitors of Papilloma Virus, Ser. No. 445,196 Filed Dec. 4, 1989; Oligonucleotide Therapies for Modulating the Effects of Herpesvirus, Ser. No. 485,297, filed Feb. 26, 1990; Reagents and Methods for Modulating Gene Expression Through RNA Mimicry Ser. No. 497,090, filed Mar. 21, 1990; oligonucleotide Modulation of Lipid Metabolism, Ser. No. 516,969, filed Apr. 30, 1990; Oligonucleotides for Modulating the Effects of Cytomegalovirus Infections, Ser. No. 568,366, filed Aug. 16, 1990; Antisense Inhibitors of the Human Immunodeficiency Virus, Ser. No. 521,907, filed May 11, 1990; Nuclease Resistant Pyrimidine Modified Oligonucleotides for Modulation of Gene Expression, Ser. No. 558,806, filed Jul. 27, 90; Novel Polyamine Conjugated Oligonucleotides, Ser. No. 558,663, filed Jul. 27, 1990; Modulation of Gene Expression Through Interference with RNA Secondary Structure, Ser. No. 518,929, filed May 4, 1990; Oligonucleotide Modulation of Cell Adhesion, Ser. No. 567,286, filed Aug. 14, 1990; Inhibition of Influenza Viruses, Ser. No. 567,287, filed Aug. 14, 1990; Inhibition of Candida, Ser. No. 568,672, filed Aug. 16, 1990; and Antisense oligonucleotide Inhibitors of Papillomavirus, Ser. No. PCT/US90/07067, filed Dec. 3, 1990. These patents disclose a number of means whereby improved modulation of RNA and DNA activity may be accomplished through oligonucleotide interaction. To the extent that the specific sequences disclosed therein may be used in conjunction with the present invention, the disclosures of the foregoing United States patent applications are incorporated herein by reference. [0085]
[0086] The oligonucleotides of the invention preferably are prepared via the process shown in Scheme 1, wherein a selected nucleotide is coupled to another nucleotide or to a growing oligonucleotide chain via a nucleophilic displacement reaction. As will be recognized, this process is also applicable to the preparation of oligonucleotides comprising non-chiral phosphodiester linkages. In Scheme 1, R[0086] _F is a phosphate blocking group, B_X is a suitable heterocyclic nucleoside base (blocked or unblocked), R_X is a sugar derivatizing group and Q is an oxygen or methylene group. Together R_D and R_E are the necessary oxygen, sulfur, nitrogen, substituted nitrogen, alkoxy or thioalkoxy groups that form the phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linking groups. For Scheme 1, the Y group of the above formulas is depicted as a CPG (Controlled Pore Glass) group. Other Y groups also may be used.
[0087] In Scheme 1, a first nucleotide (13) is attached to a solid state CPG support via its 3′-hydroxyl group in a standard manner, yielding compound (14). Compound (14), which forms a first synthon, is treated with an appropriate base, producing anionic compound (15). Compound (15) is reacted with a first unit of compound (2)—a xylofuranosyl nucleotide bearing blocking group R[0087] _F on its phosphate functionality and leaving group L at its 3′ position. Compound (2) is a second synthon. The B_X moiety of structure (2) may be the same as or different than the B_X moiety of compound (15), depending on the sequence of the desired oligonucleotide. The anion of compound (15) displaces the leaving group at the 3′ position of compound (2). The displacement proceeds via inversion at the 3′ position of the second synthon, forming compound (16) with n=1.
[0088] The resulting phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkage of compound (16) extends from the 5′ position of the first synthon (compound (14)) to the 3′ position of the second synthon (compound (2)). The inversion at the 3′ position of the second synthon results in the final configuration at the 3′ nucleotide derived from the second synthon being a normal ribofuranosyl sugar conformation. Compound (16) on the solid state CPG support is now washed to free it of any unreacted compound (2). [0088]
[0089] The second synthon carries a phosphate blocking group R[0089] _F on its phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus group. After coupling of the second synthon to the first synthon to yield compound (16) wherein n=1 and washing, the phosphate blocking group R_F is removed with an acid, yielding compound (17) wherein n=1. Compound (17), which represents a new first synthon, is now treated with base to generate a further anionic, compound (18) with n=1. Compound (18) is suitable for nucleophilic attack on a further unit of compound (2) (the second synthon) to form a new compound (16) wherein n=2. In this further unit having compound (2), the B_X moiety may be the same or different from the B_X moiety of either of the nucleotides of compound (16) wherein n=1, depending on the desired sequence.
[0090] Compound (16) wherein n=2 is washed and then treated with acid to deblock the R[0090] _F blocking group, yielding a further new first synthon, compound (17) wherein n=2. This new first synthon, is now ready to be further cycled by treatment with base to yield compound (18) wherein n=2, which is now reacted with a further unit having compound (2) to yield a further unit of having structure (16) wherein n=3. Again, B_X may be the same or different than previously B_X moieties. The cycle is repeated for as many times as necessary to introduced further nucleotides of the desired sequence via compound (2).
[0091] If it is desired to have the 5′ terminal end of the final oligonucleotide as a phosphate group, then the last compound (17) is appropriately removed from the CPG support. If it is desired to have the 5′ terminal end as a hydroxyl group, then the penultimate nucleotide is added as compound (22), it is converted to compounds (16), (17), and (18) and the resulting compound (18) is reacted with a xylofuranosyl nucleoside, compound (19). Compound (19), like compound (2), includes a leaving group L within its structure. Reaction of compound (19) with compound (18) yields oligonucleotide, compound (20), which is released from the CPG support with concentrated ammonium ion to yield the desired oligonucleotide, compound (2′). The ammonium ion treatment will also remove any base blocking groups as is standard in automated oligonucleotide synthesis. [0091]
[0092] In summary, as shown in Scheme 1, a phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate 5′ nucleotide (or the 5′-terminal nucleotide of a growing oligonucleotide) functions as a first synthon. This is converted to an anion with a base. This anion displaces a leaving group at the 3′ position of a xylofuranosyl nucleotide. The xylofuranosyl nucleotide comprises a second synthon. The displacement proceeds via inversion at the 3′ position of the second synthon with the resulting phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate linkage that is formed extending from the 5′ position of the first synthon to the 3′ position of the second synthon. The inversion at the 3′ position of the second synthon results in the final configuration at the 3′ nucleotide derived from the second synthon being a normal ribofuranosyl sugar conformation. It has a 3′ to 4′ trans orientations (a ribofuranosyl sugar conformation) that is identical to natural or wild type oligonucleotides. [0092]
[0093] The second synthon carries a phosphate blocking group on its phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate phosphorus group. After coupling of the second synthon to the first synthon, this phosphate blocking group is removed, generating a new first synthon having an anion at its 5′ phosphate suitable for nucleophilic attack on a further second synthon. Thus, after coupling of the first and second synthon, the newly joined first and second synthons now form a new first synthon. The oligonucleotide is elongated nucleotide by nucleotide via the nucleophilic attack of a phosphate anion at the 5′ end of the growing oligonucleotide chain on the leaving group at the 3′ position of the soon-to-be-added xylofuranosyl configured second synthon nucleotide. [0093]
[0094] It is presently preferred that the phosphate blocking group be a base stable, acid labile group. Such a phosphate blocking group maintains the phosphate moiety of the second synthon in a protected form that cannot react with the leaving group of the second synthon. This inhibits polymerization of the second synthon during the coupling reaction. [0094]
[0095] The nucleophilic coupling of the first and second synthons is a stereoselective coupling process that maintains the stereospecific configuration about the phosphorus atom of the first synthon. Thus the particular Sp or Rp diastereomeric configuration of a resolved phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate moiety at the 5′ end of a starting second synthon nucleotide or the 5′ terminal end of a growing first synthon oligonucleotide is maintained. While the sugar portion of the second synthon undergoes inversion about its 3′ position as a result of the coupling process, the phosphate portion of the second synthon retains its stereospecific configuration. After coupling of the second synthon to the first, the phosphate moiety (for example, the phosphorothioate, alkylphosphate, phosphoamidate or phosphotriester moiety) of the second synthon retains its original stereochemistry. That is, an Rp diastereomeric second synthon retains the Rp configuration of its phosphate moiety, while an Sp diastereomeric second synthon retains the Sp configuration of its phosphate moiety. [0095]
[0096] For example, to form an Rp chiral phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotide a first Rp diastereomeric nucleotide is chosen as the first synthon. It is coupled with an Rp diastereomeric second synthon nucleotide. The resulting dinucleotide maintains the Rp orientation at the inter-nucleotide linkage between the first and second nucleotides. When a third nucleotide is next coupled to the first two, the Rp diastereomeric phosphate moiety from the second nucleotide forms the inter-nucleotide linkage between the second and third nucleotides and is maintained in the Rp orientation. If the third nucleotide was also an Rp diastereomeric nucleotide then when a fourth nucleotide is added to the growing oligonucleotide chain, the inter-nucleotide linkage between nucleotides three and four is also an Rp diastereomeric linkage. If each added “second synthon” is also an Rp diastereomer, then the resulting oligonucleotide will contain only Rp inter-nucleotide linkages. If an oligonucleotide having Sp inter-nucleotide linkages is desired, then the first nucleotide and each of the added subsequent nucleotides are selected as Sp diastereomeric nucleotides. [0096]
[0097] The first synthon can be a first nucleotide or a growing oligonucleotide chain. If it is desired that each of the nucleotides of the oligonucleotide be ribofuranoside configured nucleotides, then the first nucleotide is selected as a ribofuranoside configured nucleotide. Each added second synthon, while added as a xylofuranoside configured nucleotide, after inversion is converted to a ribofuranoside configured nucleotide. [0097]
[0098] The 3′ position of the first nucleotide is either blocked if a solution reaction is practiced or is coupled to a solid state support if a solid state reaction (as for instance one utilizing a DNA synthesizer) is practiced. Each additional nucleotide of the oligonucleotide is then derived from a xylofuranosyl nucleotide, i.e. a second synthon. Because the first nucleotide of the oligonucleotide can be a “standard” ribofuranosyl nucleotide coupled via its 3′ hydroxyl to a solid state support, the standard solid state supports known in the art, such as controlled pore glass (CPG) supports, can be utilized and the second synthons added as reagents to the growing oligonucleotide on a standard DNA synthesizer such as, for example, an Applied Biosystems Inc. 380B Nucleic Acid Synthesizer. However, unlike standard DNA synthesizer techniques, nucleotide coupling is not achieved using activated phosphoamidate chemistry. Instead, the above-noted nucleophilic displacement reaction of a phosphate anion on a 3′ leaving group of a xylofuranosyl nucleotide is utilized as the coupling reaction. Even when chiral phosphoramidate phosphate linkages are being incorporated into the sequence-specific chiral oligonucleotides of the invention, the phosphoramidate groups of individual nucleotides are not directly used to effect the coupling reaction between nucleotides. Rather, as with phosphorothioates, alkylphosphonates, and phosphotriesters, an anion is generated at the phosphate moiety. It is this anion—not an activated amidate species—that is the activated species for effecting coupling. [0098]
[0099] Once the first nucleotide is loaded on a solid state support utilizing standard techniques, the anion necessary for nucleophilic attack is generated via treatment of the first nucleotide, i.e. the first synthon, with a base. Suitable bases include but are not limited to sodium hydride, methylmagnesium chloride, and t-butylmagnesium chloride. These are used in a suitable solvent such as acetonitrile, tetrahydrofuran or dioxane. [0099]
[0100] The second synthon is added either concurrently with the base or subsequent to it. After coupling, the growing oligonucleotide is washed with a solvent and then treated with a reagent to effect deblocking of the phosphate blocking group of the second synthon. If a preferred acid-labile blocking group is used to block the phosphate of the second synthon, deblocking is easily effected by treating the growing oligonucleotide on the solid state support with an appropriate acid. [0100]
[0101] Suitable acid-labile blocking groups for the phosphates of the second synthon include but are not limited to t-butyl, dimethoxytrityl (DMT) or tetrahydropyranyl groups. Suitable acids for deblocking the second synthon phosphate blocking group include but are not limited to acetic acid, trichloroacetic acid, and trifluoromethane sulfonic acid. Such acids are suitably soluble in solvents such as tetrahydrofuran, acetonitrile, dioxane, and the like. [0101]
[0102] Following treatment with an appropriate deblocking reagent to effect deblocking of the phosphate protecting group, the growing oligonucleotide is then washed with an appropriate solvent such as tetrahydrofuran, acetonitrile or dioxane. The oligonucleotide is now ready for the addition of a further nucleotide via treatment with base to generate an anion on the 5′ terminal phosphate followed by the addition of a further second synthon. Alternatively, the anion can be generated concurrently with addition of a further second synthon. Suitable leaving groups for inclusion at the 3′ position of the xylofuranosyl second synthon include but are not limited to the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate. A more preferred group includes chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2, 2-trifluoroethanesulfonyl, imidazolesulfonyl, and 2,4,6-trichlorophenyl. These leaving groups are subject to SN[0102] ₂ displacement reactions with inversion about the 3′ position of the sugar to provide the required 3′-4′ trans ribofuranosyl configuration after inversion. The ionized oxygen atom of the phosphate moiety of the first synthon displaces these leaving groups to effect the coupling of the first synthon to the second synthon.
[0103] A further class of leaving groups include certain sugar-base cyclonucleosides. These include 2,3′ or 6,3′-cyclopyrimidines and 8,3′-cyclopurine nucleosides. These nucleosides are alternately known as anhydro nucleosides. Since the sugar-heterocycle bond of such cyclonucleosides is “syn” with the heterocycle base, nucleophilic addition with inversion at this site also yields the desired ribofuranoside configuration of the added nucleotide after addition of the second synthon to the first synthon. The linking atom between the 3′ position of the sugar and the 2 or 6 position of a pyrimidine base or the 3′ position of the sugar and the 8 position of the purine base can be oxygen, sulfur or nitrogen. [0103]
[0104] Since a basic environment is created during coupling of the first synthon to the second synthon and an acidic environment (utilizing the preferred acid-labile phosphate blocking group) is created during deblocking of the phosphate blocking group from the nucleotide derived from the second synthon, if blocking groups are utilized on the base or sugar portions of the nucleotides such base or sugar blocking groups must be stable to both acidic and basis conditions. Suitable blocking groups for the heterocyclic base or the sugar are selected to be stable to these conditions. One type of blocking groups that can be used are acidbase stable, hydrogenolysis-sensitive blocking groups; that is, blocking groups which can be removed with molecular hydrogen but not with acid or base. A benzyl blocking group is such a suitable hydrogenolysis-sensitive blocking group. [0104]
[0105] Other heterocycle base or sugar blocking groups are those that require more pronounced acid or base treatment to de-block than may be experienced during the basic activation of the nucleophilic displacement reaction of the second synthon blocking group or the acidic removal of the phosphate blocking group. Two such blocking groups are the benzoyl and isobutyryl groups. Both of these require strong basic conditions for their removal. These basic conditions are more stringent than that required to generate the phosphate anion for the nucleophilic displacement reaction. This allows the use of such benzoyl or isobutyryl blocking groups for the 6-amino group of adenine, the 2-amino group of guanine, and the 4-amino group of cytosine. Suitable precursor molecules for the second synthons include the xylo derivatives of the common nucleosides. Certain of these “xylo nucleosides” are commercially available and others are known in the nucleoside literature. [0105]
[0106] Xylo nucleosides include but are not limited to xylo derivatives of adenosine, guanosine, inosine, uridine, cytidine, thymidine, 5-methylcytidine, and 2-aminoadenosine, i.e. 9-(β-D-xylofuranosyl)adenine, 9-(β-D-xylofuranosyl)guanine, 9-(β-D-xylofuranosyl)hypoxanthine, 1-(β-D-xylofuranosyl)uracil, 1-(β-D-xylofuranosyl)cytosine, 1-(β-D-xylofuranosyl)thymine, 5-methyl-i-(β-D-xylofuranosyl)cytosine, and 2-amino-9-(β-D-xylofuranosyl)adenine. They also include the xylo equivalents of the common 2′-deoxy nucleosides such as 9-(β-D-2′-deoxy-threo-pentofuranosyl)adenine, 9-(β-D-2′-deoxy-threo-pentofuranosyl)guanine, 9-(β-D-2′-deoxy-threo-pentofuranosyl)hypoxanthine, 1-(β-D-2′-deoxy-threo-pentofuranosyl)uracil, 1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine, 1-(β-D-2′-deoxy-threo-pentofuranosyl)thymine, 5-methyl-1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine, and 2-amino-9-(β-D-2′-deoxy-threo-pentofuranosyl)adenine. [0106]
[0107] Other preferred nucleosides that are suitable precursors for the second synthon include but are not limited to 2′-fluoro, 2′-methoxy, 2′-O-allyl, 2′-methyl, 2′-ethyl, 2′-propyl, 2′-chloro, 2′-iodo, 2′-bromo, 2′-amino, 2′-azido, 2′-0-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-nonyl, 2′-O-pentyl, 2′-O-benzyl, 2′-O-butyl, 2′-O-(propylphthalimide), 2′-S-methyl, 2′-s-ethyl, 2′-aminononyl, 2′-aralkyl, and 2′-alkylheterocyclo such as propylimidazoyl derivatives of the above 2′-deoxy-threo-pentofuranosyl nucleosides. Representatives of this group include but are not limited to 9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)adenine, 9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)guanine, 9-(β-D-2′-deoxy-2-fluoro-threo-pentofuranosyl)hypoxanthine, 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)uracil, 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)cytosine, 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl) thymine, 5-methyl-1-(β-D-2′-deoxy-2,′-f luoro-threo-pentofuranosyl) cytosine, 2-amino-9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl) adenine, 9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl) adenine, 9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl) guanine, 9-(β-D-2′-deoxy-2-methoxy-threo-pentofuranosyl) hypoxanthine, 1-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl) uracil, 1-(J-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)cytosine, 1-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl) thymine, 5-methyl-1-(S-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl) adenine, 9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl) adenine, deoxy-2′-O-allyl-threo-pentofuranosyl)guanine, 9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl) hypoxanthine, 1-(D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl) uracil, 1-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl) cytosine,1-(β-D-2′-deoxy-2′-O-ailyl-threo-pentofuranosyl) thymine, 5-methyl-1-(β-D-2′-deoxy-2-O-allyl-threo-pentofuranosyl) cytosine,2-amino -9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl) adenine, 9-(β-D-2′-deoxy-2′-methyl-threo-pentofuranosyl)adenine, 9-(β-D-2′-deoxy-2′-chloro-threo-pentofuranosyl) guanine, 9-(β-D-2′-deoxy-2-amino-threo-pentofuranosyl) hypoxanthine, 1-(β-D-2′-deoxy-2′-O-nonyl-threo-pentofuranosyl) uracil, 1-((-D-2′-deoxy-2′-O-benzyl-threo-pentofuranosyl)cytosine, 1-(β-D-2′-deoxy-2′-bromo-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2′-deoxy-2′-O-butyl-threo-pentofuranosyl)scytosine, and 2-amino-9-[β-D-2′-deoxy-2′-O-(propylphthalimide)-threo-pentofuranosyl) adenine. The 2′-deoxy-2′-f luoro-threo-pentofuranosyl, and 2′-deoxy-2′-methoxy-threo-pentofuranosyl nucleosides are particularly preferred in that the 2′-fluoro and 2′-methoxy groups give improved nuclease resistance to oligonucleotide bearing these substituents on their respective nucleotides. [0107]
[0108] Further preferred nucleosides that are suitable precursors for the second synthon include but are not limited to thexylofuranosyl or 2′-deoxy-threo-pentofuranosyl derivatives of 3-deaza purine and 2-substituted amino purine nucleosides including but not limited to 3-deaza-2′-deoxyguanosine, 3-deaza-3-nonyl-2′-deoxyguanosine, 3-deaza-3-allyl-2′-deoxyguanosine, 3-deaza-3-benzyl-2′-deoxyguanosine, 3-deaza-3-nonyl-2′-deoxyguanosine, N2-[imidazol-1-yl-(propyl)]-2′-deoxyguanosine, and 2-amino-N2-[imidazol-1-yl(propyl)]adenosine. [0108]
[0109] Another preferred group of nucleoside precursors for the second synthon include the carbocyclic nucleosides, i.e. nucleosides having a methylene group in place of the pentofuranosyl ring oxygen atom. Such carbocyclic compounds may exhibit increased stability towards chemical manipulation during activation of the xylo nucleosides for nucleophilic attack. [0109]
[0110] The xylo nucleoside or derivatized xylo nucleoside is reacted with a suitable phosphorylating agent to phosphorylate the second synthon precursor. Various phosphorylation reactions are known in the art such as those described in Nucleotide Analogs, by Karl Heinz Scheit, John Wiley & Sons, 1980, Chapter Four—Nucleotides with Modified Phosphate Groups and Chapter Six—Methods of Phosphorylation; Conjugates Of Oligonucleotides and Modified Oligonucleotides: A Review Of Their Synthesis and Properties, Goodchild, J. (1990), [0110] Bioconjugate Chemistry, 1:165; and Antisense Oligonucleotides: A New Therapeutic Principle, Uhlmann, E. and Peyman, A. (1990), Chemical Reviews, 90:543.
[0111] Preferred phosphorylating agents include phosphoryl chlorides. Suitable phosphoryl chlorides include but are not limited to thiophosphoryl chloride, t-butoxyphosphoryl chloride, t-butoxy(methyl)phosphoryl chloride, t-butoxy-(methyl)thiophosphoryl chloride, t-butoxy(methoxy)phosphoryl chloride. Other phosphoryl chlorides may include t-butoxy(N-morpholino)phosphoryl chloride, t-butoxy(ethoxy-ethylamino)phosphoryl chloride, t-butoxy(methy-thioxy)phosphoryl chloride, and the like. Such phosphorylating agents are utilized to yield the corresponding phosphorothioate, phosphoramidate, phosphotriester, alkylphosphonates, and phosphodiester xylo nucleotides. [0111]
[0112] Even enzymatic phosphorylation is possible, as for example the phosphorylation of 9-(β-D-xylofuranosyl)guanine by nucleoside phosphotransferase from Pseudomonas trifolii as per the procedure of Suzaki, S., Yamazaki, A. Kamimura, A., Mitsugi, K., and Kumashior, I. (1970), [0112] Chem. Pharm. Bull. (Tokyo), 18:172.
[0113] 1-(β-D-Xylofuranosyl)uracil 5′-phosphate was identified but not separated from its 3′ isomer as reported by Holy, A. and Sorm, F. (1969), [0113] Coll. Czech. Chem. Commun., 34:1929. Also, 9-(2′-O-benzyl-β-D-xylofuranosyl)adenine 5′-phosphate was obtained as an intermediate by Hubert-Habart, M. and Goodman, L. (1969), Chem. Commun., 740. Removable of the benzyl blocking group would give the desired unblocked nucleotide.
[0114] Additionally, the alkylphosphonates can be prepared by the method of Holy, A. (1967), [0114] Coll. Czech. Chem. Commun., 32:3713. Phosphorothioates have also been prepared by treatment of the corresponding nucleoside with trisimidazolyl-1-phosphinesulfide followed by acid hydrolysis with aqueous acetic acid, Eckstein, F. (1966 & 1970), J. Am. Chem. Soc., 88:4292 & 92:4718, respectively. A more preferred method is by selective thiophosphorylation by thiophosphorylchloride in triethylphosphate, Murray, A. W. and Atkinson, M. R. (1968), Biochemistry, 7:4023.
[0115] The appropriate phosphorylated xylo nucleotide is then activated for nucleophilic displacement at its 3′ position by reacting the 3′-hydroxyl group of the xylo compound with an appropriate anhydride, chloride, bromide, acyloxonium ion, or through an anhydro or cyclo nucleoside or the like to convert the 3′-hydroxyl group of the xylo nucleoside to an appropriate leaving group. [0115]
[0116] In a further method of synthesis, treatment of 2′,3′-anhydroadenosine with sodium ethylmercaptide gives 9-(3-deoxy-3-(ethylthio)-β-D-xylofuranosyl]adenine. Treatment of this compound with a first synthon nucleophile may generate a terminal 2-ethylthio arabinofuranosyl nucleoside that could be desulfurized to yield the corresponding 2′-deoxynucleoside. [0116]
[0117] If during phosphorylation or conversion of the xylo 3′-hydroxyl to a 3′-activated leaving group stereospecific diastereomers are not obtained, after completion of the phosphorylation or conversion of the 3′-hydroxyl to an activated leaving group, the Rp and Sp diastereomers of these compounds will then be isolated by HPLC. This will yield pure diastereomers in a stereospecific form ready for use as the second synthons of Scheme 1. [0117]
[0118] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. [0118] EXAMPLE 1
[0119] Xyloadenosine [0119]
[0120] Method A—Condensation Reaction [0120]
[0121] Adenine is condensed with 1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside in the presence of TiCl[0121] ₄ as the condensation catalyst in a polar solvent utilizing the method of Szarek, W. A., Ritchie, R. G. S., and Vyas, D. M. (1978), Carbohydr. Res., 62:89.
[0122] Method B—Alkylation Reaction [0122]
[0123] 8-Mercaptoadenine is alkylated with 5-deoxy-5-iodo-1,2-O-isopropylidine-xylofuranose followed by treatment with acetic acid/acetic anhydride/sulfuric acid and then ammonia. This yields an 8-5′-anhydro intermediate nucleoside that is oxidized with aqueous N-bromosuccinimide to give the sulfoxide. This is blocked with benzoic anhydride and after a Pummerer rearrangement can be desulfurized with Rainey nickel to give 9-β-D-xylofuranosyladenine as per the procedure of Mizuno, Y., Knaeko, C., Oikawa, Y., Ikeda, T, and Itoh, T. (1972), [0123] J. Am. Chem. Soc., 94:4737. EXAMPLE 2
[0124] 3-Deaza-9-(β-D-xylofuranosyl)guanine [0124]
[0125] In a manner similar to Example 1, Method A, 3-deazaguanine is condensed with 1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside to yield the title compound. [0125] EXAMPLE 3
[0126] N[0126] ⁶ Benzoyl-9-(2′-Deoxy-2′-fluoro-threo-pentofuranosyl)adenine
[0127] In a manner similar to Example 1, Method A, N[0127] ⁵-benzoyladenine is condensed with 1,3,5-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield the title compound. EXAMPLE 4
[0128] 1-(2′-Deoxy-2′-methoxy-β-D-xylofuranosyl)uridine [0128]
[0129] In a manner similar to Example 1, Method A, uracil is condensed with 1,3,5-tri-O-acetyl-2-deoxy-2-methoxy-D-threo-pentofuranoside to yield the title compound. [0129] EXAMPLE 5
[0130] 1-(2′-Deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)cytosine [0130]
[0131] In a manner similar to Example 1, Method A, cytosine is condensed with 1,3,5-tri-O-acetyl-2-deoxy-2-O-allyl-D-threo-pentofuranoside to yield the title compound. [0131] EXAMPLE 6
[0132] Xyloguanosine [0132]
[0133] Method A [0133]
[0134] In a manner similar to Example 1, Method A, guanine is condensed with 1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside to yield the title compound. [0134]
[0135] Method B [0135]
[0136] The chloromercury derivative of 2-acetamido-6-chloropurine is condensed with 2,3,5-tri-O-acetyl-β-D-ribofuranosylpurine utilizing the method of Lee et. al. (1971), [0136] J. Med. Chem., 14:819. The condensation product was treated with ammonia to yield 2-amino-6-chloro-9-(β-D-xylofuranosyl)purine. Further treatment with sodium hydroxyethylmercaptide gives the title compound. EXAMPLE 7
[0137] 2-Amino-6-mercapto-9-(β-D-xylofuranosyl)purine [0137]
[0138] 2-Amino-6-chloro-9-(β-D-xylofuranosyl)purine as prepared by the Example 6, Method B, is treated with sodium hydrosulfide to give the title compound. [0138] EXAMPLE 8
[0139] 9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl)guanine [0139]
[0140] In a manner similar to Example 1, Method A, guanine is condensed with 1,3,5-tri-O-acetyl-2-deoxy-2-methyl-D-threo-pentofuranoside to yield the title compound. [0140] EXAMPLE 9
[0141] 2-Amino-xyloadenosine [0141]
[0142] 2-amino-8-mercaptoadenine is treated in the manner as per Example 6, Method B to yield the title compound. [0142] EXAMPLE 10
[0143] Carbocyclic Xyloadenosine [0143]
[0144] 5-Amino-4,6-dichloropyrimidine is treated with (+)-4α-amino-2α,3β-dihydroxy-1α-cyclopentanemethanol to give a pyrimidine intermediate that is aminated and ring closed to yield the carbocyclic analog of xylofuranosyladenine as per the procedure of Vince, R. and Daluge, S. (1972), [0144] J. Med. Chem., 15:171. EXAMPLE 11
[0145] Carbocyclic Xyloinosine [0145]
[0146] 5-Amino-6-chloro-pyrimidyl-4-one when treated with (±)-4α-amino-2α,3β-dihydroxy-1α-cyclopentanemethanol will give a pyrimidine intermediate that is then aminated and ring closed to yield the carbocyclic analog of xylofuranosylinosine as per the procedure of Example 8. [0146] EXAMPLE 12
[0147] O[0147] ²,3′-Cyclothymidine
[0148] Method A [0148]
[0149] 3′-O-Mesylthymidine is treated with boiling water and the pH is adjusted to pH 4-5 according to the procedure of Miller, N. and Fox, J. J. (1964), [0149] J. Org. Chem., 29:1771 to yield the title compound. This same compound can also prepared from 3′-deoxy-3′-iodothymidine by treatment with silver acetate in acetonitrile.
[0150] Method B [0150]
[0151] O[0151] ²,3′-Cyclothymidine and other 2′-deoxynucleosides are prepared by the treatment of the appropriate nucleoside with (2-chloro-1,1,3-trifluoroethyl)diethylamine in dimethylformamide according to the procedure of Kowollik, G., Gaertner, K., and Langen, P. (1969), Tetrahedron Lett., 3863. EXAMPLE 13
[0152] O[0152] ²,3′-Cyclouridine
[0153] 3′-O-tosyluridine is treated with t-butoxide according to the procedure of Letters, R. and Michelson, A. M. (1961), [0153] J. Chem. Soc., 1410. This compound is also prepared by treatment of 3′-O-mesyl-2′,5′-di-O-trityluridine with sodium benzoate in dimethylformamide followed by detritlation with hydrochloric in chloroform. EXAMPLE 14
[0154] S[0154] ², 3-Cyclo-2-thiothymidine
[0155] Method A [0155]
[0156] 3′-O-mesyl-O[0156] ²,5′-cyclothymidine is subjected to methanolysis followed by sulfhydryl ion attack. The S²,3′-cyclo linkage is then opened up with base to yield 2′,3′-dideoxy-3′-mercapto-1-(β-D-xylofuranosyl) thymidine as per the procedure of Wempen, I. and Fox, J. J. (1969), J. Org. Chem., 34:1020.
[0157] Method B [0157]
[0158] S[0158] ²,3′-Cyclo-2-thiouridine is also prepared from 2-thio-uridine by the method of Doerr, I. L. and Fox, J. J. (1967), J. Am. Chem., 89:1760. EXAMPLE 15
[0159] N[0159] ⁶,5′-Cyclothymidine
[0160] 5′-O-Trityl-3′-O-mesylthymidine is treated with sodium azide to yield N[0160] ⁶,5′-cyclothymidine as one of the products. 5′-O-trityl-3′-O-mesylthymidine is also cyclizable to O²,3′-cyclothymidine. EXAMPLE 16 8,3′-Cycloadenosine
[0161] The anhydro ring from the 3′ position of the sugar to the 8 position of the purine ring is formed by treatment of 5′-O-acetyl-8-bromo-2′ (or 3′)-O-p-toluenesulfonyladenosine with thiourea to yield the 8,3′-thiocyclonucleoside (as well as the corresponding 8,2′) product as per the procedure of Ikehara, M. and Kaneko, M. (1970), Tetrahedron, 26:4251. [0161] EXAMPLE 17
[0162] 8,3′-Cycloguanosine [0162]
[0163] The title compound is prepared as per Example 16 utilizing 8-bromoguanosine. Both this compound and the compound of Example 16 can be oxidized to their corresponding sulfoxides via tert-butyl hypochlorite in methanol or treated with chlorine in methanolic hydrogen chloride to yield the 3′-sulfo-8-chloro analog in a procedure analogous with that of Mizuno, Y., Kaneko, O., and Oikawa, Y. (1974), [0163] J. Orzg. Chem., 39:1440. EXAMPLE 18
[0164] 1-(3′-Bromo-3′-deoxy-N[0164] ⁴,2′,5′-O-triacetylxylofuranosyl)cytosine
[0165] The title compound is prepared by treating N[0165] ⁴-acetylcytidine with acetyl bromide according to the procedure of Marumoto, R. and Honjo, M. (1974), Chem. Pharm. Bull., 22:128. EXAMPLE 19
[0166] 9-(β-Deoxy-3-fluoro-β-D-xylofuranosyl)adenine [0166]
[0167] Treatment of 9-(2,3-anhydro-5-O-benzoyl-β-D-ribofuranosyl)-N,N-dibenzoyl (or N-pivaloyl)adenine with tetraethylammonium fluoride in hot acetonitrile followed by deacylation with sodium methoxide yields 9-(3-deoxy-3-fluoro-β-D-xylofuranosyl)adenine as per the procedure of Lichtenthaler, F. W., Emig, P., and Bommer, D. (1969), [0167] Chem. Ber., 102:964. EXAMPLE 20
[0168] 9-(β-Deoxy-3-fluoro-β-D-xylofuranosyl)guanine [0168]
[0169] In a like manner to Example 18 the corresponding guanosine compound will be prepared from the corresponding 2,3-anhydro guanosine. [0169] EXAMPLE 21
[0170] 9-(3′-Chloro-3′-deoxy-β-D-xylofuranosyl)hypoxanthine [0170]
[0171] 5′-O-Acetylinosine is treated with triphenylphosphine and carbon tetrachloride to yield the title compound according to the procedure of Haga, K., Yoshikawa, M., and Kato, T. (1970), [0171] Bull. Chem. Soc. Jpn., 43:3992. EXAMPLE 22
[0172] 9-(2-O-Acetyl-3-chloro-3-deoxy-5-O-pivaloyl-β-D-xylofuranosyl)-6-pivalamidopurine [0172]
[0173] The title compound is prepared via an intermediate 2′,3′-O-acyloxonium ion utilized to introduce a halogen atom at the 3′ position and convert the ribo configuration of a nucleoside into the corresponding 3′-halo-3′-deoxy xylo nucleoside. The acyloxonium ion is generated in situ by treatment of 2′,3′-O-methoxyethylidineadenosine with pivaloyl chloride in hot pyridine. Attack by chloride gives the title compound. Hypoxanthine and guanine nucleoside react in a similar manner. Sodium iodide will be used to generate the corresponding 3′-iodides according to the procedure of Robins, M. J., Fouron, Y., and Mengel, R. (1974), [0173] J. Org. Chem., 39:1564. EXAMPLE 23
[0174] 9-(2,5,-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine [0174]
[0175] This compound is prepared by a in situ acyloxonium ion generated by treating 3′,5′-di-O-acetyladenosine with boron trifluoride etherate followed by phosphorus tribromide according to the procedure of Kondo, K., Adachi, T., and Inoue, I. (1977), [0175] J. Org. Chem., 42:3967. The title compound can also be formed by treating adenosine with tetraacetoxysilane and phosphorus tribromide in the presence of boron trifluoride etherate. EXAMPLE 24
[0176] 1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)uracil [0176]
[0177] In a manner similar to Example 3, uracil is condensed with 1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield the title compound. [0177] EXAMPLE 25
[0178] 1-(1-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl) guanine [0178]
[0179] In a manner similar to Example 3, guanine is condensed with 1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield the title compound. [0179] EXAMPLE 26
[0180] 1-7(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)cytosine [0180]
[0181] In a manner similar to Example 3, cytosine is condensed with 1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield the title compound. [0181] EXAMPLE 27
[0182] O[0182] ²,3′-Cyclo-2′-deoxycytidine
[0183] The title compound is prepared by heating the 3′-O-sulfamate as per the procedure of Schuman, D., Robins, M. J., and Robins, R. K. (1970), [0183] J. Am. Chem. Soc., 92:3434. EXAMPLE 28
[0184] Sp and Rp Xyloadenosine 5′-Monophosphate [0184]
[0185] N[0185] ⁶-Benzoyl-xyloadenosine is phosphorylated with phosphoryl chloride in pyridine and acetonitrile at 0 C. The reaction will be quenched with ice water, rendered basic and added to an activated charcoal column. After elution with ethanol/water/concentrated ammonium hydroxide the solvent is evaporated to dryness and the residue dissolved in water and passed through an ion exchange column. The benzoyl blocking group is removed in concentrated ammonium hydroxide followed by separation of the diastereomers by HPLC to yield the title compound. EXAMPLE 29
[0186] Sp and Rp 1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)uracil 5′-t-butoxy(methyl)phosphonate [0186]
[0187] 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)thymine will be phosphorylated with t-butoxy(methyl)phosphoryl chloride in trimethylphosphate at 0° C. for 3 hrs. The solution is added to cold anhydrous ether. The racemic precipitate is taken up in acetonitrile and the Sp and Rp diastereomers of the title compound separated by HPLC utilizing a gradient of acetonitrile and triethylammonium acetate buffer. [0187] EXAMPLE 30
[0188] Sp and Rp 1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)cytosine 5′-t-butoxy(methyl)phosphonate [0188]
[0189] 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl) cytosine will be phosphorylated and purified as per the procedure of Example 29 to give the diastereomers of the title compound. [0189] EXAMPLE 31
[0190] Sp and Rp N[0190] ⁵-Benzoyl-9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)adenine 5,-t-butoxy(methyl)phosphonate
[0191] N[0191] ⁶-Benzoyl-9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)adenine will be phosphorylated and purified as per the procedure of Example 29 to give the diastereomers of the title compound. EXAMPLE 32
[0192] Sp and Rp 9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)guanine 5′-t-butoxy(methyl)phosphonate [0192]
[0193] 9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl) guanine will be phosphorylated and purified as per the procedure of Example 29 to give the diastereomers of the title compound. [0193] EXAMPLE 33
[0194] Sp and Rp Xylofuranosyluracil 5′-t-butoxyphosphbrothioate [0194]
[0195] Xylofuranosyluracil will be phosphorothioated with t-butoxythiophosphorylchloride in triethylphosphate utilizing the method of Murray, A. W. and Atkinson, M. R. (1968), [0195] Biochemistry, 7:4023. The diastereomers of the title compound are separated by HPLC. EXAMPLE 34
[0196] Sp and Rp 9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl)guanine 5′-Methylphosphonate [0196]
[0197] 9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl) guanine will-be alkylphosphonated utilizing the procedure of Holy, A. (1967), [0197] Coll. Czech. Chem. Commun., 32:3713. The racemic phosphorylation product is separated into its Sp and Rp diastereomers using HPLC chromatography to yield the title compound. EXAMPLE 35
[0198] Sp and Rp 9-(2′-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine 5′-Phosphormorpholidate [0198]
[0199] 9-(2′-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine is phosphorylated according to the procedure of Example 28. The resulting 5′-phosphate intermediate will be phosphormorpholidated by treatment with activated with dicyclohexylcarbodiimide in the presence of morpholine according to the procedure of Moffatt, J. G. and Khorana, H. G. (1961), [0199] J. Am. Chem. Soc., 83:3752 to yield the racemic title compound. The diastereomers of the product are separated by HPLC. EXAMPLE 36
[0200] Sp and Rp 9-(2′-Deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)cytosine 5′-Phosphate [0200]
[0201] 9-(2′-Deoxy-2′-allyl-β-D-threo-pentofuranosyl) cytosine will be phosphorylated according to the procedure of Example 28 to yield the racemic title compound. The diastereomers are separated by HPLC. [0201] EXAMPLE 37
[0202] Sp and Rp 9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl)-uracil 5′-Phosphate [0202]
[0203] 9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl)uracil will be phosphorylated according to the procedure of Example 28 to yield the racemic title compound. The diastereomers are separated by HPLC. [0203] EXAMPLE 38
[0204] Sp and Rp 3-Deaza-9-(xylofuranosyl)guanine 5′-Phosphate [0204]
[0205] 3-Deaza-9-(xylofuranosyl)guanine will be phosphorylated according to the procedure of Example 28 to yield the racemic title compound. The diastereomers are separated by HPLC. [0205] EXAMPLE 39
[0206] Sp and Rp Xyloguanosine 5′-Phosphorothioate [0206]
[0207] Xyloguanosine will be phosphorothioated with thiophosphoryl chloride according to the procedure of Example 28 to yield the racemic title compound. The diastereomers are separated by HPLC. [0207] EXAMPLE 40
[0208] Sp and Rp Carbocyclic Xyloadenosine 5′-Phosphate [0208]
[0209] In a like manner to Example 28, carbocyclic xyloadenosine will be treated with phosphoryl chloride to yield the racemic title compound. The diastereomers will be separated by HPLC. [0209] EXAMPLE 41
[0210] Activated 3′-Deoxy-3′-Active Leaving Group Phosphorylated Nucleosides [0210]
[0211] The 3′-halo nucleotides can be treated with methoxide to give an unstable 2′,3′-anhydro intermediate that slowly forms the corresponding 3,3′-cyclonucleoside. The cyclonucleoside in turn can undergo nucleophilic attack to yield other 3′-deoxy-3′-substituted derivatives, as for instance, the tosyl, triflate, trichloroacetimidate or other active species. [0211] EXAMPLE 42
[0212] 6-Benzoyl-9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)adenine 5′-Rp t-Butoxy(methyl)phosphonate [0212]
[0213] N[0213] ⁶-Benzoyl-9-(2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl) denine 5′-Rp t-butoxy(methyl)phosphonate will be treated with p-toluenesulfonlychloride in pyridine as per the procedure of Reist, E. J., Bartuska, V. J., Calkins, D. F., and Goodman, L. (1965), J. Org. Chem., 30:3401 to yield the title compound. EXAMPLE 43
[0214] 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-methoxy-β-D-threo-pentofuranosyl)uracil 5′-Rp t-Butoxy(methyl)phosphonate [0214]
[0215] 9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl) uridine 5′-Rp t-butoxy(methyl)phosphonate will be treated with p-toluenesulfonlychloride in pyridine according to the procedure of Example 42 to yield the title compound. [0215] EXAMPLE 44
[0216] 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)uracil 5′-Rp t-Butoxy(methyl)phosphonate [0216]
[0217] 9-(2′-Deoxy-2′-fluoro-β-D-threo-pentofuranosyl)uridine 5′-Rp t-butoxy(methyl)phosphonate will be treated with p-toluenesulfonlychloride in pyridine according to the procedure of Example 42 to yield the title compound. [0217] EXAMPLE 45
[0218] 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl) cytosine 5′-Rp t-Butoxy(methyl)phosphonate [0218]
[0219] 9-(2′-Deoxy-2′-f luoro-β-D-threo-pentofuranosyl) cytosine 5′-Rp t-butoxy(methyl)phosphonate will be treated with p-toluenesulfonlychloride in pyridine according to the procedure of Example 42 to yield the title compound. [0219] EXAMPLE 46
[0220] 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)guanine 5′-Sp Phosphate [0220]
[0221] 9-(2′-Deoxy-2′-fluoro-β-D-threo-pentofuranosyl)guanine 5′-Sp phosphate will be treated with p-toluenesulfonly chloride in pyridine according to the procedure of Example 42 to yield the title compound. [0221] EXAMPLE 47
[0222] 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)thymine 5′-Rp Phosphate [0222]
[0223] 9-(2′-deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)thymine 5′-Rp phosphate will be treated with p-toluenesulfonyl chloride in pyridine according to the procedure of Example 42 to yield the title compound. [0223] EXAMPLE 48
[0224] 3′-Deoxy-3′-trifluoromethanesulfonylxyloguanosine 5′-Sp Phosphorothioate [0224]
[0225] Xyloguanosine 5′-Sp phosphorothioate will be treated with trif luoromethane sulfonic acid anhydride in the presence of a sodium hydride to yield the title compound. [0225] EXAMPLE 49
[0226] Carbocyclic′-Deoxy-3′-trifluoromethanesulfonylxyloadenosine 5′-Rp Phosphate [0226]
[0227] In a like manner to Example 48, carbocyclic xyloadenosine 5′-Rp phosphate will be treated with trifluoromethane sulfonic acid to yield the title compound. [0227] EXAMPLE 50
[0228] S[0228] ²,3′-Cyclo-2-thiothymidine
[0229] S[0229] ²,3-Cyclo-2-thiothymidine is prepared from 3′-O-mesyl-O²,5′-cyclothymidine via methanolysis followed by sulfhydryl ion attack. The S²,3′-cyclo linkage is then opened up with base to yield 2′,3′-dideoxy-3′-mercapto-1-(β-D-xylofuranosyl)thymidine, Wempen, I. and Fox, J. J. (1969), J. Org. Chem., 34:1020. The 3′ position will then be activated to nucleophilic attack via an active leaving group such as conversion of the mercapto to a tosyl leaving group. In a like manner S²,3′-Cyclo-2-thiouridine prepared from 2-thio-uridine by the method of Doerr, I. L. and Fox, J. J. (1967), J. Am. Chem., 89:1760, can be ring opened and then derivatized with an activated leaving group such as a tosylate. EXAMPLE 51
[0230] Synthesis of 2′-Deoxy-2′-fluoro substituted CGA CTA TGC AAC TAC Rp Methylphosphonate Linked Oligonucleotide [0230]
[0231] 1-(2′-Fluoro-2′-deoxy-β-D-ribofuranosyl)cytosine 5′-Rp methylphosphonate will be attached via its 3′ hydroxyl to CPG beads in a standard manner as practiced in automated nucleic acid synthesis. This nucleotide forms the first synthon 1 [0231]
[0232] (a) Activation of Synthon 1 [0232]
[0233] The beads are washed with acetonitrile and treated with 1.1 equivalents of sodium hydride in acetonitrile to form an anion on the methylphosphonate moiety. [0233]
[0234] (b) Addition of Synthon 2 and Coupling of Synthons 1 and 2 [0234]
[0235] 2.0 Equivalents of N[0235] ⁶-Benzoyl-9-(3′-deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)adenine 5′-Rp t-butoxy(methyl)phosphonate in acetonitrile is added with stirring. After completion of the nucleophilic reaction and formation of the cytosine-adenine dimer as judged by tlc, the beads are filtered and washed with acetonitrile.
[0236] (c) Removal of t-Butoxy Blocking Group [0236]
[0237] The beads are re-suspended in acetonitrile and 1.5 equivalents of trichloracetic acid is added. The reaction is stirred to remove the t-butoxy blocking group on the terminal 5′-methylphosphonate group of the adenosine nucleotide, followed by washing with acetonitrile. [0237]
[0238] (d) Cycling [0238]
[0239] The reaction is cycled to step (a), followed by addition of the next synthon 2 nucleotide at step (b), and deblocking at step (c). The reaction is further cycled for each nucleotide making up the specific sequence of the oligonucleotide. After the addition of the penultimate nucleotide its phosphate moiety is activated at step (a) and the final nucleosidic unit is added at step (b) as a xylofuranosyl nucleoside. The oligonucleotide is concurrently deblocked and removed from the CPG beads by treatment with concentrate ammonium hydroxide. [0239]

权利要求:
Claims (39)
[1" id="US-20010008936-A1-CLM-00001] 1. A method for preparing an oligonucleotide comprising a predetermined sequence of nucleoside units, comprising the steps of:
(a) selecting a first synthon having structure:
[2" id="US-20010008936-A1-CLM-00002] 2. The method of
claim 1 wherein L is selected from the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate
[3" id="US-20010008936-A1-CLM-00003] 3. The method of
claim 1 wherein L is selected from the group consisting of chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and 2,4,6-trichlorophenyl.
[4" id="US-20010008936-A1-CLM-00004] 4. The method of
claim 1 wherein said base is selected from the group consisting of sodium hydride, methylmagnesium chloride, t-butylmagnesium chloride or methylmagnesium fluoride.
[5" id="US-20010008936-A1-CLM-00005] 5. The method of
claim 1 wherein R_F is an acid labile blocking group.
[6" id="US-20010008936-A1-CLM-00006] 6. The method of
claim 1 wherein Y is a hydrogenolysis sensitive blocking group.
[7" id="US-20010008936-A1-CLM-00007] 7. The method of
claim 1 wherein R_F is selected from the group consisting of t-butyl, dimethoxytrityl or tetrahydropyranyl.
[8" id="US-20010008936-A1-CLM-00008] 8. The method of
claim 1 wherein Y is a nucleotide bound to a solid state support, an oligonucleotide bound to a solid state support, or a solid state support.
[9" id="US-20010008936-A1-CLM-00009] 9. The method of
claim 1 wherein the new first synthon has the structure:
[10" id="US-20010008936-A1-CLM-00010] 10. The method of
claim 1 further comprising repeating said steps (b), (c), and (d) a plurality of times.
[11" id="US-20010008936-A1-CLM-00011] 11. The method of
claim 1 further comprising contacting said first synthon with said base in a solvent selected frown the group consisting of acetonitrile, tetrahydrofuran and dioxane.
[12" id="US-20010008936-A1-CLM-00012] 12. The method of
claim 1 wherein said reagent used to remove blocking group R_F is an acid selected from the group consisting of trichloroacetic acid, acetic acid or trifluoromethane sulfonic acid.
[13" id="US-20010008936-A1-CLM-00013] 13. The product of the process of
claim 1 .
[14" id="US-20010008936-A1-CLM-00014] 14. An oligonucleotide comprising a plurality of nucleoside units linked together via phosphate linkages, wherein:
at least one of the nucleoside units is a non-naturally occurring nucleoside unit; and
at least two of the nucleoside units are linked via chiral phosphate linkages.
[15" id="US-20010008936-A1-CLM-00015] 15. The oligonucleotide of
claim 14 wherein said chiral phosphate linkages are selected from the group consisting of chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rp phosphotriester.
[16" id="US-20010008936-A1-CLM-00016] 16. The oligonucleotide of
claim 14 wherein each of the phosphate linkages is a chiral phosphate linkage.
[17" id="US-20010008936-A1-CLM-00017] 17. The oligonucleotide of
claim 14 wherein:
at least one of the nucleoside units has one of the structures:
[18" id="US-20010008936-A1-CLM-00018] 18. The oligonucleotide of
claim 17 wherein B_X is a pyrimidinyl-1 or purinyl-9 moiety.
[19" id="US-20010008936-A1-CLM-00019] 19. The oligonucleotide of
claim 17 wherein B_X is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
[20" id="US-20010008936-A1-CLM-00020] 20. The oligonucleotide of
claim 14 wherein at least one of the modified nucleotides has one of the structures:
[21" id="US-20010008936-A1-CLM-00021] 21. The oligonucleotide of
claim 20 wherein X has one of the structures:
[22" id="US-20010008936-A1-CLM-00022] 22. The oligonucleotide of
claim 14 wherein the Qligonucleotide forms at least a. portion of a RNA or DNA sequence.
[23" id="US-20010008936-A1-CLM-00023] 23. An oligonucleotide comprising a plurality of nucleoside units linked together by all S_P phosphotriester linkages or by all R_P phosphotriester linkages.
[24" id="US-20010008936-A1-CLM-00024] 24. The oligonucleotide of
claim 23 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.
[25" id="US-20010008936-A1-CLM-00025] 25. An oligonucleotide comprising a plurality of linked of nucleoside units linked together by all S_P phosphoramidate linkages or by all R_P phosphoamidate linkages.
[26" id="US-20010008936-A1-CLM-00026] 26. The oligonucleotide of
claim 25 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.
[27" id="US-20010008936-A1-CLM-00027] 27. An oligonucleotide comprising at least 10 nucleoside units linked together by all S_P alkylphosphonate linkages or by all R_P alkylphosphonate linkages.
[28" id="US-20010008936-A1-CLM-00028] 28. The oligonucleotide of
claim 27 wherein said alkylphosphonate linkages are methylphosphonate linkages.
[29" id="US-20010008936-A1-CLM-00029] 29. The oligonucleotide of
claim 27 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.
[30" id="US-20010008936-A1-CLM-00030] 30. A composition of matter having the structure:
[31" id="US-20010008936-A1-CLM-00031] 31. The composition of matter of
claim 30 wherein L is selected from the group consisting of halogen, alkylsulfonyl, substitutedalkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate
[32" id="US-20010008936-A1-CLM-00032] 32. The composition of matter of
claim 30 wherein L is selected from the group consisting of chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and 2,4,6-trichlorophenyl.
[33" id="US-20010008936-A1-CLM-00033] 33. The composition of matter of
claim 30 wherein R_X is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂, OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group for improving the pharmacodynamic properties of an oligonucleotide or a group for improving the pharmacokinetic properties of an oligonucleotide.
[34" id="US-20010008936-A1-CLM-00034] 34. The composition of matter of
claim 30 wherein R_F is selected from the group consisting of H, t-butyl, dimethoxytrityl or tetrahydropyranyl.
[35" id="US-20010008936-A1-CLM-00035] 35. The composition of matter of
claim 30 wherein B_X is a pyrimidinyl-1 or purinyl-9 moiety.
[36" id="US-20010008936-A1-CLM-00036] 36. The composition of
claim 30 wherein B_X is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.
[37" id="US-20010008936-A1-CLM-00037] 37. The composition of
claim 30 wherein Q is O.
[38" id="US-20010008936-A1-CLM-00038] 38. The composition of
claim 30 having Sp stereochemistry.
[39" id="US-20010008936-A1-CLM-00039] 39. The composition of
claim 30 having Rp stereochemistry.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US6500945B2|2002-12-31|Nucleotides having chiral phosphorus linkages

---

US6699979B2|2004-03-02|Oligonucleotides having chiral phosphorus linkages

---

US5521302A|1996-05-28|Process for preparing oligonucleotides having chiral phosphorus linkages

---

US5434257A|1995-07-18|Binding compentent oligomers containing unsaturated 3&#39;,5&#39; and 2&#39;,5&#39; linkages

---

US5264564A|1993-11-23|Oligonucleotide analogs with novel linkages

---

CA2071483C|2001-04-17|Oligonucleotide analogs with novel linkages

---

US20150368643A1|2015-12-24|Oligonucleotides comprising alternating segments and uses thereof

---

US6271369B1|2001-08-07|Chimeric molecules targeted to viral RNAs

---

CA2184375C|2006-05-02|Oligonucleotide n3&#39;-p5&#39; phosphoramidates: synthesis and compounds; hybridization and nuclease resistance properties

---

US5635488A|1997-06-03|Compounds having phosphorodithioate linkages of high chiral purity

---

US6410702B1|2002-06-25|Modified internucleoside linkages |

---

KR100858465B1|2008-09-16|Oligonucleotide n3&#39;?p5&#39; thiophosphoramidates: their synthesis and use

---

EP1100809B1|2004-09-22|Rna targeted 2&#39;-modified oligonucleotides that are conformationally preorganized

---

US5587361A|1996-12-24|Oligonucleotides having phosphorothioate linkages of high chiral purity

---

CA2202280C|2000-08-15|Sugar modified nucleosides and their use for synthesis of oligonucleotides

---

JPH10510433A|1998-10-13|Oligonucleotides with high chiral purity phosphorothioate linkages

---

ES2244957T3|2005-12-16|MODIFIED OLIGONUCLEOTIDES, ITS PREPARATION, AS WELL AS ITS JOB.

---

JP2004500330A|2004-01-08|Guanidinium-functionalized oligomers and their preparation

---

Flamme et al.2019|Chemical methods for the modification of RNA

---

IL185569A|2012-02-29|Cyclic nucleoside analogues

---

EP1102787A1|2001-05-30|2&#39;-o-aminoethyloxyethyl-modified oligonucleotides

---

WO2001040515A1|2001-06-07|Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages

---

CN111684073A|2020-09-18|Method for producing nucleic acid molecule

---

WO2008025160A1|2008-03-06|Oxepane nucleosides and oligonucleotides, uses thereof and methods of making the same

---

WO1995031470A2|1995-11-23|Antisense inhibitors of gene expression

同族专利:

---

公开号 | 公开日

---

US5852188A|1998-12-22|

---

US6500945B2|2002-12-31|

---

US6239265B1|2001-05-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

US5635488A|1991-10-15|1997-06-03|Isis Pharmaceuticals, Inc.|Compounds having phosphorodithioate linkages of high chiral purity|

---

US5620963A|1991-10-15|1997-04-15|Isis Pharmaceuticals, Inc.|Oligonucleotides for modulating protein kinase C having phosphorothioate linkages of high chiral purity|

---

US5607923A|1991-10-15|1997-03-04|Isis Pharmaceuticals, Inc.|Oligonucleotides for modulating cytomegalovirus having phosphorothioate linkages of high chiral purity|

---

US5576302A|1991-10-15|1996-11-19|Isis Pharmaceuticals, Inc.|Oligonucleotides for modulating hepatitis C virus having phosphorothioate linkages of high chiral purity|

---

US5599797A|1991-10-15|1997-02-04|Isis Pharmaceuticals, Inc.|Oligonucleotides having phosphorothioate linkages of high chiral purity|

---

US5212295A|1990-01-11|1993-05-18|Isis Pharmaceuticals|Monomers for preparation of oligonucleotides having chiral phosphorus linkages|

---

US5852188A|1990-01-11|1998-12-22|Isis Pharmaceuticals, Inc.|Oligonucleotides having chiral phosphorus linkages|

---

US5506212A|1990-01-11|1996-04-09|Isis Pharmaceuticals, Inc.|Oligonucleotides with substantially chirally pure phosphorothioate linkages|

---

US5359052A|1991-08-05|1994-10-25|Polish Academy Of Sciences|Chalcophospholanes useful in the synthesis of oligonucleoside phosphorothioates, phosphorodithioates and related selenates|

---

US5512668A|1991-03-06|1996-04-30|Polish Academy Of Sciences|Solid phase oligonucleotide synthesis using phospholane intermediates|

---

US5646267A|1991-08-05|1997-07-08|Polish Academy Of Sciences|Method of making oligonucleotides and oligonucleotide analogs using phospholanes and enantiomerically resolved phospholane analogues|

---

ES2202447T3|1995-05-26|2004-04-01|Genta Incorporated|METHODS OF SYNTHESIS OF ORGAN-PHOSPHORATED DERIVATIVES.|

---

CA2233460A1|1995-09-01|1997-03-13|Polska Akademia Nauk Centrum Badan Molekularnych I Makomolekularnych|Compositions and methods for the synthesis of organophosphorus derivatives|

---

US5856465A|1996-05-24|1999-01-05|Polska Akademia Nauk Centrum Badan Molekularnych I Makromolekularnych|Compositions and methods for the synthesis of chirally pure organophosphorus nucleoside derivatives|AU2874892A|1991-10-15|1993-05-21|Isis Pharmaceuticals, Inc.|Oligonucleotides having chiral phosphorus linkages|

---

US5852188A|1990-01-11|1998-12-22|Isis Pharmaceuticals, Inc.|Oligonucleotides having chiral phosphorus linkages|

---

US6111094A|1990-08-14|2000-08-29|Isis Pharmaceuticals Inc.|Enhanced antisense modulation of ICAM-1|

---

US9096636B2|1996-06-06|2015-08-04|Isis Pharmaceuticals, Inc.|Chimeric oligomeric compounds and their use in gene modulation|

---

US7812149B2|1996-06-06|2010-10-12|Isis Pharmaceuticals, Inc.|2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations|

---

US5898031A|1996-06-06|1999-04-27|Isis Pharmaceuticals, Inc.|Oligoribonucleotides for cleaving RNA|

---

EP1765416A4|2004-06-03|2010-03-24|Isis Pharmaceuticals Inc|Double strand compositions comprising differentially modified strands for use in gene modulation|

---

US20040171028A1|1996-06-06|2004-09-02|Baker Brenda F.|Phosphorous-linked oligomeric compounds and their use in gene modulation|

---

US20050042647A1|1996-06-06|2005-02-24|Baker Brenda F.|Phosphorous-linked oligomeric compounds and their use in gene modulation|

---

PL184612B1|1997-04-25|2002-11-29|Pan|Method of obtaining p-chiral analoques of nucleotides|

---

DE19736591A1|1997-08-22|1999-02-25|Peter Prof Dr Hegemann|Preparing long nucleic acid polymers from linkable oligonucleotides|

---

US6211354B1|1998-05-06|2001-04-03|Tosch Corporation|Optically active DNA probe having phosphonic diester linkage|

---

US6867294B1|1998-07-14|2005-03-15|Isis Pharmaceuticals, Inc.|Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages|

---

AU1656601A|1999-11-12|2001-06-12|Isis Pharmaceuticals, Inc.|Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages|

---

US6677153B2|1999-11-29|2004-01-13|Avi Biopharma, Inc.|Antisense antibacterial method and composition|

---

US7220832B2|2000-03-31|2007-05-22|Merck & Co., Inc|Dermacentor variabilis GABA-gated chloride channels|

---

US6784291B2|2000-05-04|2004-08-31|Avi Biopharma, Inc.|Splice-region antisense composition and method|

---

CA2651881A1|2006-05-10|2008-03-27|Avi Biopharma, Inc.|Oligonucleotide analogs having cationic intersubunit linkages|

---

US6548251B1|2000-09-05|2003-04-15|Fidelity Systems, Inc.|Inhibition of molecular and biological processes using modified oligonucleotides|

---

EP1588139A4|2001-05-31|2006-10-04|Merck & Co Inc|Rhesus monkey, dog and ferret melanin-concentrating hormone type 2 receptor|

---

US7125982B1|2001-12-05|2006-10-24|Frayne Consultants|Microbial production of nuclease resistant DNA, RNA, and oligo mixtures|

---

EP2957568B1|2002-11-05|2016-12-21|Ionis Pharmaceuticals, Inc.|Compositions comprising alternating 2'-modified nucleosides for use in gene modulation|

---

JP2006515988A|2002-11-12|2006-06-15|ザリサーチファンデーションオブステイトユニバーシティオブニューヨーク|Translational regulation by non-translated small RNA |

---

US20070167384A1|2003-04-02|2007-07-19|Dharmacon, Inc.|Modified polynucleotides for use in rna interference|

---

US20040198640A1|2003-04-02|2004-10-07|Dharmacon, Inc.|Stabilized polynucleotides for use in RNA interference|

---

US20080311556A1|2003-08-07|2008-12-18|Iversen Patrick L|Sense Antiviral Compound and Method for Treating Ssrna Viral Infection|

---

US7480382B2|2003-09-30|2009-01-20|Microsoft Corporation|Image file container|

---

US20050222068A1|2003-10-23|2005-10-06|Mourich Dan V|Method and antisense composition for selective inhibition of HIV infection in hematopoietic cells|

---

US20050176125A1|2003-12-23|2005-08-11|Rosetta Inpharmatics Llc|Alternatively spliced isoform of CDC25A phosphatase and methods of use|

---

WO2005072527A2|2004-01-23|2005-08-11|Avi Biopharma, Inc.|Antisense oligomers and methods for inducing immune tolerance and immunosuppression|

---

EP1718747B1|2004-02-06|2009-10-28|Dharmacon, Inc.|Stabilized rnas as transfection controls and silencing reagents|

---

US20090280567A1|2004-02-06|2009-11-12|Dharmacon, Inc.|Stabilized sirnas as transfection controls and silencing reagents|

---

US8569474B2|2004-03-09|2013-10-29|Isis Pharmaceuticals, Inc.|Double stranded constructs comprising one or more short strands hybridized to a longer strand|

---

US7402574B2|2004-03-12|2008-07-22|Avi Biopharma, Inc.|Antisense composition and method for treating cancer|

---

US20050266469A1|2004-05-25|2005-12-01|Raymond Christopher K|Alternatively spliced isoforms of checkpoint kinase 1 |

---

US8394947B2|2004-06-03|2013-03-12|Isis Pharmaceuticals, Inc.|Positionally modified siRNA constructs|

---

US7150969B2|2004-06-04|2006-12-19|Rosetta Inpharmatics Llc|Alternatively spliced isoform of acetyl-CoA carboxylase 2 |

---

WO2006085973A2|2004-07-02|2006-08-17|Avi Biopharma, Inc.|Antisense antibacterial method and compound|

---

WO2006023880A2|2004-08-23|2006-03-02|Isis Pharmaceuticals, Inc.|Compounds and methods for the characterization of oligonucleotides|

---

US7884086B2|2004-09-08|2011-02-08|Isis Pharmaceuticals, Inc.|Conjugates for use in hepatocyte free uptake assays|

---

US8129352B2|2004-09-16|2012-03-06|Avi Biopharma, Inc.|Antisense antiviral compound and method for treating ssRNA viral infection|

---

US8357664B2|2004-10-26|2013-01-22|Avi Biopharma, Inc.|Antisense antiviral compound and method for treating influenza viral infection|

---

US7524829B2|2004-11-01|2009-04-28|Avi Biopharma, Inc.|Antisense antiviral compounds and methods for treating a filovirus infection|

---

US7935811B2|2004-11-22|2011-05-03|Dharmacon, Inc.|Apparatus and system having dry gene silencing compositions|

---

US7923207B2|2004-11-22|2011-04-12|Dharmacon, Inc.|Apparatus and system having dry gene silencing pools|

---

US7923206B2|2004-11-22|2011-04-12|Dharmacon, Inc.|Method of determining a cellular response to a biological agent|

---

WO2006066260A2|2004-12-17|2006-06-22|Thiosense, Inc.|Compositions of and methods for producing phosphorus-chiral monomers and oligomers|

---

AU2006213686A1|2005-02-09|2006-08-17|Avi Bio Pharma, Inc.|Antisense composition and method for treating muscle atrophy|

---

US20060223777A1|2005-03-29|2006-10-05|Dharmacon, Inc.|Highly functional short hairpin RNA|

---

US20060240032A1|2005-03-31|2006-10-26|Hinrichs David J|Immunomodulating compositions and methods for use in the treatment of human autoimmune diseases|

---

US20060293268A1|2005-05-05|2006-12-28|Rieder Aida E|Antisense antiviral compounds and methods for treating foot and mouth disease|

---

US7790694B2|2005-07-13|2010-09-07|Avi Biopharma Inc.|Antisense antibacterial method and compound|

---

US8067571B2|2005-07-13|2011-11-29|Avi Biopharma, Inc.|Antibacterial antisense oligonucleotide and method|

---

US8524676B2|2005-09-08|2013-09-03|Sarepta Therapeutics, Inc.|Method for treating enterovirus or rhinovirus infection using antisense antiviral compounds|

---

AU2006287530A1|2005-09-08|2007-03-15|Sarepta Therapeutics, Inc.|Antisense antiviral compound and method for treating picornavirus infection|

---

US8501704B2|2005-11-08|2013-08-06|Sarepta Therapeutics, Inc.|Immunosuppression compound and treatment method|

---

JP5713377B2|2005-12-28|2015-05-07|ザ スクリプス リサーチ インスティテュート|Natural antisense and non-coding RNA transcripts as drug targets|

---

US8785407B2|2006-05-10|2014-07-22|Sarepta Therapeutics, Inc.|Antisense antiviral agent and method for treating ssRNA viral infection|

---

WO2007139723A1|2006-06-01|2007-12-06|Trilink Biotechnologies|Chemicallymodified oligonucleotide primers for nucleic acid amplification|

---

US8252755B2|2006-09-22|2012-08-28|Dharmacon, Inc.|Duplex oligonucleotide complexes and methods for gene silencing by RNA interference|

---

US8188060B2|2008-02-11|2012-05-29|Dharmacon, Inc.|Duplex oligonucleotides with enhanced functionality in gene regulation|

---

ES2727549T3|2008-10-03|2019-10-17|Curna Inc|Treatment of diseases related to apolipoprotein a1 by inhibition of the natural antisense transcript to apolipoprotein a1|

---

CN102282155B|2008-12-02|2017-06-09|日本波涛生命科学公司|The synthetic method of the nucleic acid of phosphorus atoms modification|

---

CA2745811C|2008-12-04|2021-07-13|Joseph Collard|Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene|

---

MX2011005912A|2008-12-04|2011-06-17|Opko Curna Llc|Treatment of vascular endothelial growth factorrelated diseases by inhibition of natural antisense transcript to vegf.|

---

CN102712925B|2009-07-24|2017-10-27|库尔纳公司|It is diseases related that SIRTUINis treated by suppressing SIRTUINnatural antisense transcript|

---

JP6091752B2|2008-12-04|2017-03-08|クルナ・インコーポレーテッド|Treatment of erythropoietin-related diseases by suppression of natural antisense transcripts against EPO|

---

US8592386B2|2008-12-17|2013-11-26|Sarepta Therapeutics, Inc.|Antisense compositions and methods for modulating contact hypersensitivity or contact dermatitis|

---

DK2396038T3|2009-02-12|2016-02-01|Curna Inc|TREATMENT OF BRAIN-DERIVATED NEUROTROPHIC FACTOR-RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPTION TO BDNF|

---

ES2656290T3|2009-03-16|2018-02-26|Curna, Inc.|Treatment of diseases related to nuclear factorsimilar to 2by inhibition of natural antisense transcript to NRF2|

---

WO2010107740A2|2009-03-17|2010-09-23|Curna, Inc.|Treatment of delta-like 1 homologrelated diseases by inhibition of natural antisense transcript to dlk1|

---

JP6250930B2|2009-05-06|2017-12-20|クルナ・インコーポレーテッド|Treatment of TTP-related diseases by suppression of natural antisense transcripts against tristetraproline |

---

EP2427553A4|2009-05-06|2012-11-07|Opko Curna Llc|Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene|

---

KR101742334B1|2009-05-08|2017-06-01|큐알엔에이, 인크.|Treatment of dystrophin family related diseases by inhibition of natural antisense transcript to dmd family|

---

ES2664590T3|2009-05-18|2018-04-20|Curna, Inc.|Treatment of diseases related to reprogramming factors by inhibition of the natural antisense transcript to a reprogramming factor|

---

CN102549158B|2009-05-22|2017-09-26|库尔纳公司|By suppressing to treat the disease that TFE3 is related to IRS albumen 2for transcription factor E3natural antisense transcript|

---

WO2010138806A2|2009-05-28|2010-12-02|Curna, Inc.|Treatment of antiviral gene related diseases by inhibition of natural antisense transcript to an antiviral gene|

---

CN102695797B|2009-06-16|2018-05-25|库尔纳公司|By inhibiting to treat the relevant disease of glue protogene for the natural antisense transcript of glue protogene|

---

CA2765509C|2009-06-16|2021-08-17|Joseph Collard|Treatment of paraoxonase 1related diseases by inhibition of natural antisense transcript to pon1|

---

US8859515B2|2009-06-24|2014-10-14|Curna, Inc.|Treatment of tumor necrosis factor receptor 2related diseases by inhibition of natural antisense transcript to TNFR2|

---

EP2446037B1|2009-06-26|2016-04-20|CuRNA, Inc.|Treatment of down syndrome gene related diseases by inhibition of natural antisense transcript to a down syndrome gene|

---

SG177564A1|2009-07-06|2012-02-28|Ontorii Inc|Novel nucleic acid prodrugs and methods of use thereof|

---

AU2015255202B2|2009-07-06|2017-07-27|Wave Life Sciences Ltd.|Novel nucleic acid prodrugs and methods of use thereof|

---

ES2585360T3|2009-08-05|2016-10-05|Curna, Inc.|Treatment of diseases related to an insulin geneby inhibition of natural antisense transcription in an insulin gene |

---

CN104313027B|2009-08-11|2018-11-20|库尔纳公司|By inhibiting the natural antisense transcript of adiponectinto treat adiponectinrelated disease|

---

CN102482670B|2009-08-21|2018-06-15|库尔纳公司|CHIP relevant diseases are treated by inhibiting the natural antisense transcript of ' C-terminal of HSP70- interaction proteins ' |

---

KR101892760B1|2009-08-25|2018-08-28|큐알엔에이, 인크.|Treatment of 'iq motif containing gtpase activating protein'related diseases by inhibition of natural antisense transcript to iqgap|

---

WO2011038210A2|2009-09-25|2011-03-31|Curna, Inc.|Treatment of filaggrinrelated diseases by modulation of flg expression and activity|

---

EP2499248B1|2009-11-13|2017-01-04|Sarepta Therapeutics, Inc.|Antisense antiviral compound and method for treating influenza viral infection|

---

JP6025567B2|2009-12-16|2016-11-16|カッパーアールエヌエー，インコーポレイテッド|Treatment of MBTPS1-related diseases by inhibition of the natural antisense transcript against the membrane-bound transcription factor peptidase, site 1 |

---

KR101793753B1|2009-12-23|2017-11-03|큐알엔에이, 인크.|Treatment of uncoupling protein 2related diseases by inhibition of natural antisense transcript to ucp2|

---

JP5934106B2|2009-12-23|2016-06-15|カッパーアールエヌエー，インコーポレイテッド|Treatment of HGF-related diseases by inhibition of natural antisense transcripts against hepatocyte growth factor |

---

JP5982288B2|2009-12-29|2016-08-31|カッパーアールエヌエー，インコーポレイテッド|Treatment of tumor protein 63-related diseases by inhibition of natural antisense transcripts against tumor protein 63 |

---

CA2785173A1|2009-12-29|2011-07-28|Curna, Inc.|Treatment of nuclear respiratory factor 1related diseases by inhibition of natural antisense transcript to nrf1|

---

ES2677044T3|2009-12-31|2018-07-27|Curna, Inc.|Treatment of diseases related to insulin receptor substrate 2by inhibition of natural antisense transcript for IRS2 and transcription factor E3 |

---

CN102906264B|2010-01-04|2017-08-04|库尔纳公司|IRF8 relevant diseases are treated by suppressing the natural antisense transcript of interferon regulatory factor 8 |

---

WO2011085066A2|2010-01-06|2011-07-14|Curna, Inc.|Treatment of pancreatic developmental gene related diseases by inhibition of natural antisense transcript to a pancreatic developmental gene|

---

CA2786535C|2010-01-11|2019-03-26|Curna, Inc.|Treatment of sex hormone binding globulinrelated diseases by inhibition of natural antisense transcript to shbg|

---

DK2529015T3|2010-01-25|2018-02-26|Curna Inc|TREATMENT OF RNASE H1-RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO RNASE H1|

---

CA2790506A1|2010-02-22|2011-08-25|Curna, Inc.|Treatment of pyrroline-5-carboxylate reductase 1related diseases by inhibition of natural antisense transcript to pycr1|

---

WO2011123745A2|2010-04-02|2011-10-06|Opko Curna Llc|Treatment of colony-stimulating factor 3related diseases by inhibition of natural antisense transcript to csf3|

---

JP5978203B2|2010-04-09|2016-08-24|カッパーアールエヌエー，インコーポレイテッド|Treatment of fibroblast growth factorfibroblast growth factor FGF21) related diseases by inhibition of natural antisense transcripts against FGF21|

---

KR20130101442A|2010-05-03|2013-09-13|큐알엔에이, 인크.|Treatment of sirtuinrelated diseases by inhibition of natural antisense transcript to a sirtuin |

---

TWI531370B|2010-05-14|2016-05-01|可娜公司|Treatment of par4 related diseases by inhibition of natural antisense transcript to par4|

---

CN102971423B|2010-05-26|2018-01-26|库尔纳公司|MSRA relevant diseases are treated by suppressing Methionine Sulfoxide Reductase Anatural antisense transcript|

---

EP3299464B1|2010-05-26|2019-10-02|CuRNA, Inc.|Treatment of atonal homolog 1related diseases by inhibition of natural antisense transcript to atoh1|

---

CN103003288B|2010-05-28|2019-06-21|萨勒普塔医疗公司|The oligonucleotide analogs of subunit linkage and/or end group with modification|

---

CA3102008A1|2010-06-02|2011-12-08|Alnylam Pharmaceuticals, Inc.|Compositions and methods directed to treating liver fibrosis|

---

KR101914309B1|2010-06-23|2018-11-02|큐알엔에이, 인크.|Treatment of sodium channel voltage-gated, alpha subunitrelated diseases by inhibition of natural abtisense transcript to scna|

---

KR101886452B1|2010-07-14|2018-09-07|큐알엔에이, 인크.|Treatment of discs large homolog related diseases by inhibition of natural antisense transcript to dlg|

---

US8198429B2|2010-08-09|2012-06-12|Avi Biopharma, Inc.|Antisense antiviral compounds and methods for treating a filovirus infection|

---

US10017763B2|2010-09-03|2018-07-10|Sarepta Therapeutics, Inc.|dsRNA molecules comprising oligonucleotide analogs having modified intersubunit linkages and/or terminal groups|

---

EP2620428B1|2010-09-24|2019-05-22|Wave Life Sciences Ltd.|Asymmetric auxiliary group|

---

KR101886457B1|2010-10-06|2018-08-07|큐알엔에이, 인크.|Treatment of sialidase 4related diseases by inhibition of natural antisense transcript to neu4|

---

WO2012054723A2|2010-10-22|2012-04-26|Opko Curna Llc|Treatment of alpha-l-iduronidaserelated diseases by inhibition of natural antisense transcript to idua|

---

US10000752B2|2010-11-18|2018-06-19|Curna, Inc.|Antagonat compositions and methods of use|

---

RU2608493C2|2010-11-23|2017-01-18|Курна, Инк.|Treating diseases, associated with nanog, by inhibition of natural antisense nanog transcript|

---

WO2012078967A2|2010-12-10|2012-06-14|Alnylam Pharmaceuticals, Inc.|Compositions and methods for increasing erythropoietinproduction|

---

US9127275B2|2010-12-10|2015-09-08|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting expression of klf-1 and bcl11a genes|

---

CN103813810B|2011-03-29|2021-08-03|阿尔尼拉姆医药品有限公司|Compositions and methods for inhibiting expression of TMPRSS6 gene|

---

US10086043B2|2011-04-03|2018-10-02|The General Hospital Corporation|Efficient protein expression in vivo using modified RNA |

---

CN103620036B|2011-06-09|2016-12-21|库尔纳公司|FXN relevant disease is treated by the natural antisense transcript of suppression Frataxin |

---

JP6110372B2|2011-06-21|2017-04-05|アルナイラム ファーマシューティカルズ， インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ．|Angiopoietin-like 3iRNA composition and method of use thereof|

---

EP2723390B1|2011-06-23|2017-12-27|Alnylam Pharmaceuticals, Inc.|Serpina1 sirnas: compositions of matter and methods of treatment|

---

MX347361B|2011-07-19|2017-04-12|Wave Life Sciences Ltd|Methods for the synthesis of functionalized nucleic acids.|

---

JP2014526887A|2011-08-01|2014-10-09|アルナイラムファーマシューティカルズ，インコーポレイテッド|How to improve the success rate of hematopoietic stem cell transplantation|

---

CA2847811C|2011-09-06|2019-10-22|Curna, Inc.|Treatment of diseases related to alpha subunits of sodium channels, voltage-gatedwith small molecules|

---

US20130085139A1|2011-10-04|2013-04-04|Royal Holloway And Bedford New College|Oligomers|

---

CN108864192A|2011-11-18|2018-11-23|萨勒普塔医疗公司|Function modified oligonucleotides and its subunit|

---

BR112014014740B1|2011-12-22|2021-08-24|Alios Biopharma, Inc|NUCLEOSIDE COMPOUNDS, NUCLEOTIDES AND THEIR ANALOGUES, THEIR USE AND PHARMACEUTICAL COMPOSITION|

---

US9441007B2|2012-03-21|2016-09-13|Alios Biopharma, Inc.|Substituted nucleosides, nucleotides and analogs thereof|

---

USRE48171E1|2012-03-21|2020-08-25|Janssen Biopharma, Inc.|Substituted nucleosides, nucleotides and analogs thereof|

---

CN110438125A|2012-03-15|2019-11-12|科纳公司|By inhibiting the natural antisense transcript of brain derived neurotrophic factorto treat BDNF related disease|

---

US9133461B2|2012-04-10|2015-09-15|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting expression of the ALAS1 gene|

---

JP6268157B2|2012-07-13|2018-01-24|株式会社Ｗａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｊａｐａｎ|Asymmetric auxiliary group|

---

RU2677639C2|2012-07-13|2019-01-18|Шин Ниппон Биомедикал Лэбораториз, Лтд.|Chiral nucleic acid adjuvant|

---

WO2014012081A2|2012-07-13|2014-01-16|Ontorii, Inc.|Chiral control|

---

WO2014071358A2|2012-11-05|2014-05-08|Foundation Medicine, Inc.|Novel ntrk1 fusion molecules and uses thereof|

---

WO2014130922A1|2013-02-25|2014-08-28|Trustees Of Boston University|Compositions and methods for treating fungal infections|

---

BR112015022876A2|2013-03-14|2017-11-07|Alnylam Pharmaceuticals Inc|agent, pharmaceutical composition and method for inhibiting expression of complement component c5, use of said agent, vector and cell.|

---

CA2912834A1|2013-05-22|2014-11-27|Alnylam Pharmaceuticals, Inc.|Tmprss6 irna compositions and methods of use thereof|

---

CA2912826A1|2013-05-22|2014-11-27|Alnylam Pharmaceuticals, Inc.|Serpina1 irna compositions and methods of use thereof|

---

WO2014197835A2|2013-06-06|2014-12-11|The General Hospital Corporation|Methods and compositions for the treatment of cancer|

---

CN103319555A|2013-07-01|2013-09-25|淮海工学院|Preparation method of 2'-methoxy adenosine|

---

MX2016004230A|2013-10-02|2016-10-21|Alnylam Pharmaceuticals Inc|Compositions and methods for inhibiting expression of the lect2 gene.|

---

SG11201602631XA|2013-10-04|2016-05-30|Alnylam Pharmaceuticals Inc|Compositions and methods for inhibiting expression of the alas1 gene|

---

EP3060680B1|2013-10-21|2019-02-27|The General Hospital Corporation|Methods relating to circulating tumor cell clusters and the treatment of cancer|

---

SG11201604692UA|2013-12-12|2016-07-28|Alnylam Pharmaceuticals Inc|Complement component irna compositions and methods of use thereof|

---

WO2015095527A1|2013-12-20|2015-06-25|The General Hosptial Corporation|Methods and assays relating to circulating tumor cells|

---

JPWO2015108047A1|2014-01-15|2017-03-23|株式会社新日本科学|Chiral nucleic acid adjuvant having immunity induction activity and immunity induction activator|

---

US10322173B2|2014-01-15|2019-06-18|Shin Nippon Biomedical Laboratories, Ltd.|Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent|

---

JPWO2015108048A1|2014-01-15|2017-03-23|株式会社新日本科学|Chiral nucleic acid adjuvant and antitumor agent having antitumor activity|

---

CN113278617A|2014-01-16|2021-08-20|波涛生命科学有限公司|Chiral design|

---

JP6594902B2|2014-02-11|2019-10-23|アルナイラムファーマシューティカルズ，インコーポレイテッド|Keto hexokinaseiRNA compositions and methods of use thereof|

---

TW201607559A|2014-05-12|2016-03-01|阿尼拉製藥公司|Methods and compositions for treating a SERPINC1-associated disorder|

---

CA2948381A1|2014-05-22|2015-11-26|Alnylam Pharmaceuticals, Inc.|Angiotensinogenirna compositions and methods of use thereof|

---

EP3148564B1|2014-06-02|2020-01-08|Children's Medical Center Corporation|Methods and compositions for immunomodulation|

---

EP3191591A1|2014-09-12|2017-07-19|Alnylam Pharmaceuticals, Inc.|Polynucleotide agents targeting complement component c5 and methods of use thereof|

---

JOP20200115A1|2014-10-10|2017-06-16|Alnylam Pharmaceuticals Inc|Compositions And Methods For Inhibition Of HAO1 ) Gene Expression|

---

WO2016061487A1|2014-10-17|2016-04-21|Alnylam Pharmaceuticals, Inc.|Polynucleotide agents targeting aminolevulinic acid synthase-1and uses thereof|

---

WO2016069694A2|2014-10-30|2016-05-06|Alnylam Pharmaceuticals, Inc.|Polynucleotide agents targeting serpinc1and methods of use thereof|

---

JOP20200092A1|2014-11-10|2017-06-16|Alnylam Pharmaceuticals Inc|HEPATITIS B VIRUSiRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2016081444A1|2014-11-17|2016-05-26|Alnylam Pharmaceuticals, Inc.|Apolipoprotein c3irna compositions and methods of use thereof|

---

AU2016219263A1|2015-02-13|2017-08-17|Alnylam Pharmaceuticals, Inc.|Patatin-like phospholipase domain containing 3iRNA compositions and methods of use thereof|

---

MA41795A|2015-03-18|2018-01-23|Sarepta Therapeutics Inc|EXCLUSION OF AN EXON INDUCED BY ANTISENSE COMPOUNDS IN MYOSTATIN|

---

WO2016164746A1|2015-04-08|2016-10-13|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting expression of the lect2 gene|

---

US11020417B2|2015-06-04|2021-06-01|Sarepta Therapeutics, Inc|Methods and compounds for treatment of lymphocyte-related diseases and conditions|

---

EP3307316A1|2015-06-12|2018-04-18|Alnylam Pharmaceuticals, Inc.|Complement component c5 irna compositions and methods of use thereof|

---

EP3310918B1|2015-06-18|2020-08-05|Alnylam Pharmaceuticals, Inc.|Polynucleotide agents targeting hydroxyacid oxidaseand methods of use thereof|

---

WO2016209862A1|2015-06-23|2016-12-29|Alnylam Pharmaceuticals, Inc.|Glucokinaseirna compositions and methods of use thereof|

---

WO2017011286A1|2015-07-10|2017-01-19|Alnylam Pharmaceuticals, Inc.|Insulin-like growth factor binding protein, acid labile subunitand insulin-like growth factor 1irna compositions and methods of use thereof|

---

CA2996873A1|2015-09-02|2017-03-09|Alnylam Pharmaceuticals, Inc.|Programmed cell death 1 ligand 1irna compositions and methods of use thereof|

---

MA46427A|2015-10-09|2018-08-15|Wave Life Sciences Ltd|COMPOSITIONS OF OLIGONUCLEOTIDES AND RELATED PROCESSES|

---

MA45295A|2016-04-19|2019-02-27|Alnylam Pharmaceuticals Inc|HIGH DENSITY LIPOPROTEIN BINDING PROTEINRNA COMPOSITION AND METHODS FOR USING THEM|

---

US20190256845A1|2016-06-10|2019-08-22|Alnylam Pharmaceuticals, Inc.|COMPLEMENT COMPONENT C5 iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING PAROXYSMAL NOCTURNAL HEMOGLOBINURIA |

---

JP2020502142A|2016-12-16|2020-01-23|アルナイラム ファーマシューティカルズ， インコーポレイテッドＡｌｎｙｌａｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ．|Method for treating or preventing a TTR-related disease using a transthyretiniRNA composition|

---

AU2018252193A1|2017-04-14|2019-10-17|Tollnine, Inc.|Immunomodulating polynucleotides, antibody conjugates thereof, and methods of their use|

---

EP3652317A1|2017-07-13|2020-05-20|Alnylam Pharmaceuticals, Inc.|Lactate dehydrogenase airna compositions and methods of use thereof|

---

US20210261959A1|2017-11-01|2021-08-26|Alnylam Pharmaceuticals, Inc.|Complement component c3 irna compositions and methods of use thereof|

---

US20200385719A1|2017-11-16|2020-12-10|Alnylam Pharmaceuticals, Inc.|Kisspeptin 1irna compositions and methods of use thereof|

---

WO2019100039A1|2017-11-20|2019-05-23|Alnylam Pharmaceuticals, Inc.|Serum amyloid p componentirna compositions and methods of use thereof|

---

WO2019126097A1|2017-12-18|2019-06-27|Alnylam Pharmaceuticals, Inc.|High mobility group box-1irna compositions and methods of use thereof|

---

TW202016304A|2018-05-14|2020-05-01|美商阿尼拉製藥公司|Angiotensinogenirna compositions and methods of use thereof|

---

TW202023574A|2018-08-13|2020-07-01|美商阿尼拉製藥公司|Hepatitis b virusdsrna agent compositions and methods of use thereof|

---

EP3837367A1|2018-08-16|2021-06-23|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting expression of the lect2 gene|

---

AU2019344776A1|2018-09-18|2021-01-21|Alnylam Pharmaceuticals, Inc.|KetohexokinaseiRNA compositions and methods of use thereof|

---

US10913951B2|2018-10-31|2021-02-09|University of Pittsburgh—of the Commonwealth System of Higher Education|Silencing of HNF4A-P2 isoforms with siRNA to improve hepatocyte function in liver failure|

---

AU2019405961A1|2018-12-20|2021-07-22|Humabs Biomed Sa|Combination HBV therapy|

---

AU2019406186A1|2018-12-20|2021-07-15|Praxis Precision Medicines, Inc.|Compositions and methods for the treatment of KCNT1 related disorders|

---

AU2020209186A1|2019-01-16|2021-09-09|Genzyme Corporation|Serpinc1 iRNA compositions and methods of use thereof|

---

CA3139325A1|2019-05-13|2020-11-19|Vir Biotechnology, Inc.|Compositions and methods for treating hepatitis b virusinfection|

---

WO2021022109A1|2019-08-01|2021-02-04|Alnylam Pharmaceuticals, Inc.|SERPIN FAMILY F MEMBER 2iRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021022108A2|2019-08-01|2021-02-04|Alnylam Pharmaceuticals, Inc.|CARBOXYPEPTIDASE B2iRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021030522A1|2019-08-13|2021-02-18|Alnylam Pharmaceuticals, Inc.|SMALL RIBOSOMAL PROTEIN SUBUNIT 25iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021046122A1|2019-09-03|2021-03-11|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting expression of the lect2 gene|

---

WO2021067747A1|2019-10-04|2021-04-08|Alnylam Pharmaceuticals, Inc.|Compositions and methods for silencing ugt1a1 gene expression|

---

WO2021076828A1|2019-10-18|2021-04-22|Alnylam Pharmaceuticals, Inc.|Solute carrier family member irna compositions and methods of use thereof|

---

TW202134435A|2019-10-22|2021-09-16|美商阿尼拉製藥公司|Complement component c3 irna compositions and methods of use thereof|

---

WO2021087036A1|2019-11-01|2021-05-06|Alnylam Pharmaceuticals, Inc.|HUNTINGTINiRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021087325A1|2019-11-01|2021-05-06|Alnylam Pharmaceuticals, Inc.|Compositions and methods for silencing dnajb1-prkaca fusion gene expression|

---

TW202132568A|2019-11-13|2021-09-01|美商阿尼拉製藥公司|Methods and compositions for treating an angiotensinogen-associated disorder|

---

WO2021102373A1|2019-11-22|2021-05-27|Alnylam Pharmaceuticals, Inc.|Ataxin3rnai agent compositions and methods of use thereof|

---

WO2021119226A1|2019-12-13|2021-06-17|Alnylam Pharmaceuticals, Inc.|Human chromosome 9 open reading frame 72irna agent compositions and methods of use thereof|

---

WO2021126734A1|2019-12-16|2021-06-24|Alnylam Pharmaceuticals, Inc.|Patatin-like phospholipase domain containing 3irna compositions and methods of use thereof|

---

WO2021154941A1|2020-01-31|2021-08-05|Alnylam Pharmaceuticals, Inc.|Complement component c5 irna compositions for use in the treatment of amyotrophic lateral sclerosis |

---

WO2021163066A1|2020-02-10|2021-08-19|Alnylam Pharmaceuticals, Inc.|Compositions and methods for silencing vegf-a expression|

---

WO2021167841A1|2020-02-18|2021-08-26|Alnylam Pharmaceuticals, Inc.|Apolipoprotein c3irna compositions and methods of use thereof|

---

WO2021178607A1|2020-03-05|2021-09-10|Alnylam Pharmaceuticals, Inc.|Complement component c3 irna compositions and methods of use thereof for treating or preventing complement component c3-associated diseases|

---

WO2021178736A1|2020-03-06|2021-09-10|Alnylam Pharmaceuticals, Inc.|KETOHEXOKINASEiRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021188611A1|2020-03-18|2021-09-23|Alnylam Pharmaceuticals, Inc.|Compositions and methods for treating subjects having a heterozygous alanine-glyoxylate aminotransferase genevariant|

---

WO2021195307A1|2020-03-26|2021-09-30|Alnylam Pharmaceuticals, Inc.|Coronavirus irna compositions and methods of use thereof|

---

WO2021202443A2|2020-03-30|2021-10-07|Alnylam Pharmaceucticals, Inc.|Compositions and methods for silencing dnajc15 gene expression|

---

WO2021207167A1|2020-04-06|2021-10-14|Alnylam Pharmaceuticals, Inc.|Compositions and methods for silencing myoc expression|

---

WO2021207189A1|2020-04-07|2021-10-14|Alnylam Pharmaceuticals, Inc.|Compositions and methods for silencing scn9a expression|

---

WO2021206922A1|2020-04-07|2021-10-14|Alnylam Pharmaceuticals, Inc.|Transmembrane serine protease 2irna compositions and methods of use thereof|

---

WO2021206917A1|2020-04-07|2021-10-14|Alnylam Pharmaceuticals, Inc.|ANGIOTENSIN-CONVERTING ENZYME 2iRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021222065A1|2020-04-27|2021-11-04|Alnylam Pharmaceuticals, Inc.|Apolipoprotein eirna agent compositions and methods of use thereof|

---

WO2021222549A1|2020-04-30|2021-11-04|Alnylam Pharmaceuticals, Inc.|Complement factor birna compositions and methods of use thereof|

---

WO2021231675A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of argininosuccinate synthetase |

---

WO2021231680A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of methyl-cpg binding protein 2 |

---

WO2021231692A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of otoferlin |

---

WO2021231679A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of gap junction protein beta 2 |

---

WO2021231691A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of retinoschisin 1 |

---

WO2021231698A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of argininosuccinate lyase |

---

WO2021231673A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of leucine rich repeat kinase 2 |

---

WO2021231685A1|2020-05-15|2021-11-18|Korro Bio, Inc.|Methods and compositions for the adar-mediated editing of transmembrane channel-like protein 1 |

---

WO2021237097A1|2020-05-21|2021-11-25|Alnylam Pharmaceuticals, Inc.|Compositions and methods for inhibiting marc1 gene expression|

---

WO2021234459A2|2020-05-22|2021-11-25|Wave Life Sciences Ltd.|Double stranded oligonucleotide compositions and methods relating thereto|

---

WO2021252557A1|2020-06-09|2021-12-16|Alnylam Pharmaceuticals, Inc.|Rnai compositions and methods of use thereof for delivery by inhalation|

---

WO2021257782A1|2020-06-18|2021-12-23|Alnylam Pharmaceuticals, Inc.|XANTHINE DEHYDROGENASEiRNA COMPOSITIONS AND METHODS OF USE THEREOF|

---

WO2021262840A1|2020-06-24|2021-12-30|Vir Biotechnology, Inc.|Engineered hepatitis b virus neutralizing antibodies and uses thereof|

法律状态:
2002-03-11| AS| Assignment|Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:COOK, PHILLIP DAN;REEL/FRAME:012706/0510 Effective date: 19980722 |

---

2004-06-15| CC| Certificate of correction|

---

2006-05-24| FPAY| Fee payment|Year of fee payment: 4 |

---

2010-08-09| REMI| Maintenance fee reminder mailed|

---

2010-12-31| LAPS| Lapse for failure to pay maintenance fees|

---

2011-01-31| STCH| Information on status: patent discontinuation|Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |

---

2011-02-22| FP| Lapsed due to failure to pay maintenance fee|Effective date: 20101231 |

优先权:

---

申请号 | 申请日 | 专利标题

---

US46335890A| true| 1990-01-11|1990-01-11||

---

US56697790A| true| 1990-08-13|1990-08-13||

---

PCT/US1991/000243|WO1991010671A1|1990-01-11|1991-01-11|Compositions and methods for detecting and modulating rna activity and gene expression|

---

US07/777,670|US5212295A|1990-01-11|1991-10-15|Monomers for preparation of oligonucleotides having chiral phosphorus linkages|

---

US08/058,023|US5521302A|1990-01-11|1993-05-05|Process for preparing oligonucleotides having chiral phosphorus linkages|

---

US08/635,009|US5852188A|1990-01-11|1996-04-19|Oligonucleotides having chiral phosphorus linkages|

---

US09/208,533|US6239265B1|1990-01-11|1998-12-09|Oligonucleotides having chiral phosphorus linkages|

---

US09/784,917|US6500945B2|1990-01-11|2001-02-16|Nucleotides having chiral phosphorus linkages|US09/784,917| US6500945B2|1990-01-11|2001-02-16|Nucleotides having chiral phosphorus linkages|

---

US10/150,696| US6699979B2|1991-10-15|2002-05-17|Oligonucleotides having chiral phosphorus linkages|