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WO1993011148A1 - Oligonucleotides comprenant des fractions de phosphonate d'aminohydrocarbure - Google Patents

Oligonucleotides comprenant des fractions de phosphonate d'aminohydrocarbure Download PDF

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Publication number
WO1993011148A1
WO1993011148A1 PCT/US1992/010043 US9210043W WO9311148A1 WO 1993011148 A1 WO1993011148 A1 WO 1993011148A1 US 9210043 W US9210043 W US 9210043W WO 9311148 A1 WO9311148 A1 WO 9311148A1
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oligonucleotide
oligonucleotides
hydrogen
hydrocarbon
markers
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PCT/US1992/010043
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English (en)
Inventor
Alan F. Cook
Reza Fathi
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Pharmagenics, Inc.
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Priority to JP5510176A priority Critical patent/JPH07501542A/ja
Priority to EP92925362A priority patent/EP0641354A4/fr
Publication of WO1993011148A1 publication Critical patent/WO1993011148A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention relates to oligonucleotides which bind to RNA (such as mRNA), DNA, proteins, or peptides, including, for example, oligonucleotides which inhibit mRNA function. More particularly, this invention relates to oligonucleotides in which one or more of the nucleotides include an aminohydrocarbon phosphonate moiety.
  • Watson-Crick base pairing enables an oligonucleotide to act as an antisense complement to a target sequence of an mRNA in order to block processing or effect translation arrest and regulate selectively gene expression.
  • Oligonucleotides have also been utilized to interfere with gene expression directly at the DNA level by formation of triple-helical (triplex) structures in part through Hoogsteen bonding interactions (Moffat, Science, Vol. 252, pg ⁇ 1374-1375 (1991)).
  • oligonucleotides have been shown to bind specifically to proteins (Oliphant, et al., Molec. Cell. Biol. Vol 9, pgs. 2944-2949 (1989)) and could thus be used to block undesirable protein function.
  • U.S. Patent No. 4,469,863 issued to Miller, et al., discloses the manufacture of nonionic nucleic acid alkyl and aryl phosphonates, and in particular nonionic nucleic acid methyl phosphonates.
  • U.S. Patent No. 4,757,055, also issued to Miller, et al. discloses a method for selectively controlling unwanted expression of foreign nucleic acid in an animal or in mammalian cells by binding the nucleic acid with a nonionic oligonucleotide alkyl or aryl phosphonate analogue.
  • Oligonucleotides have also been synthesized in which one non-bridging oxygen in each phosphodiester moiety is replaced by sulfur. Such analogues sometimes are referred to as phosphorothioate (PS) analogues, or "all PS” analogues, (Stein, et al., Nucl. Acids Res., Vol. 16, pgs. 3209-3221 (1988)).
  • PS phosphorothioate
  • Another approach has been to attach a targeting moiety, such as cholesterol, which improves the uptake of the oligonucleotide by a receptor-mediated process. (Stein et al. , Biochemistry, Vol. 30, pgs. 2439-2444 (1991)).
  • oligonucleotides with positive charges have been reported. Letsinger, et al. (JACS, Vol. 110, pgs. 4470-4471 (1988)) describe cationic oligonucleotides in which the backbone is modified by the attachment of diamino compounds to give positively charged oligonucleotides with phosphoramidate linkages. Phosphoramidate linkages, however, are known to be somewhat labile, especially at
  • an oligonucleotide wherein at least one nucleotide unit includes a phosphonate moiety having the following structural formula: 0
  • R- is a hydrocarbon, preferably alkylene, phenylene, or naphthylene, more preferably an alkyl group having from 1 to 15 carbon atoms, and most preferably 1 to 3 carbon atoms, with methylene being preferred.
  • Each of R_, R_, and R. is hydrogen or a hydrocarbon.
  • the hydrocarbon is an alkyl group having from 1 to 15 carbon atoms, more preferably from 1 to 3 carbon atoms, and most preferably a methyl group.
  • R_, R 3 , and R. may be the same or different.
  • each of Renfin, R , and R. is hydrogen.
  • oligonucleotide means that the oligonucleotide may be a ribonucleotide or a deoxyribonucleotide; i.e., the oligonucleotide may include ribose or deoxyribose sugars. Alternatively, the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof.
  • the oligonucleotide has at least two nucleotide units, preferably at least five, more preferably from five to about 30 nucleotide units.
  • At least one nucleotide unit of the oligonucleotide includes a phosphonate moiety which is an aminohydrocarbon phosphonate moiety, as hereinabove described.
  • An aminohydrocarbon phosphonate moiety may be attached to one or more nucleotide units at the 3 ' end and/or at the 5 f end of the oligonucleotide.
  • an aminohydrocarbon phosphonate moiety may be attached to alternating nucleotide units of the oligonucleotide.
  • an aminohydrocarbon phosphonate moiety may be attached to each nucleotide unit of the oligonucleotide.
  • the oligonucleotides also include any natural or unnatural, substituted or unsubstituted, purine or pyrimidine base.
  • purine and pyrimidine bases include. but are not limited to, natural purines and pyrimidines such as adenine, cytosine, thymine, guanine, uracil, or other purines and pyrimidines, such as isocytosine, 6-methyluracil, 4,6-dihydroxypyrimidine, hypoxanthine, xanthine, 2, 6-diamino purine, azacytosine, 5-methyl cytosine, and the like.
  • X is an aminomethyl moiety.
  • the synthesis of an oligonucleotide having such aminomethyl phosphonate moieties may be accomplished through the synthesis of a monomer unit with a protected aminomethyl functional group, followed by incorporation of one or more such monomer units into an oligonucleotide; or by synthesis of an oligonucleotide followed by subsequent attachment of the aminomethyl groups.
  • Monomer units which may be incorporated into an oligonucleotide may, in one embodiment, be prepared as follows:
  • Aminomethyl phosphonic acid may be reacted with a suitable reagent, such as trifluoroacetic anhydride, fluorenyloxycarbonylchloride, or phthalyl chloride to protect the amino group, and to give one of the following protected derivatives, (1), (2), or (3):
  • a suitable reagent such as trifluoroacetic anhydride, fluorenyloxycarbonylchloride, or phthalyl chloride
  • the phthalimide derivat ve (1) may be prepared by reaction of chloromethyl phosphonic acid with phthalimide, or by de ethylation of commercially available dimethylphthalimidomethyl phosphonate using trimethylsilyl bromide.
  • Hydroxymethyl phosphonic acid can also be used as a starting material for the synthesis of aminomethyl phosphonate derivatives.
  • the reaction of hydroxymethyl phosphonic acid with trifluoroacetic anhydride produces an ester which can be converted into a pyridinium intermediate, the reaction of which with ammonia produces aminomethyl phosphonic acid.
  • B is a protected or unprotected purine or pyrimidine base
  • a condensing agent such as dicyclohexylcarbodiimide or triisopropylbenzene-sulfonyl chloride would produce an ester having the following structural formula
  • the protected amino group is selected from
  • the ester having the structural formula 5 can be used as a monomer unit for oligonucleotide synthesis by coupling to a protected mononucleotide or oligonucleotide attached to a solid support. After the solid support-attached oligonucleotide is synthesized, the material is treated with ammonia to cleave the protecting groups and generate an oligonucleotide having one or more aminomethyl phosphonate moieties. Alternatively, the phthalimide protecting group can be removed by treatment with hydrazine or a substituted hydrazine to generate the aminomethyl compound. By this route, the aminomethyl modified units can be introduced at any position in the oligonucleotide as desired.
  • a modified mononucleotide may be prepared by reacting a partially protected nucleoside such as hereinabove described with a protected aminomethyl phosphite derivative to form a nucleoside phosphonamidite.
  • the nucleoside phosphonamidite can then be used in place of a nucleoside phosphoramidite in a DNA synthesizer.
  • the protecting groups can be removed from the aminomethyl moieties by treatment with ammonia or with amines such as ethylenediamine.
  • aminomethyl phosphonate moieties may be introduced into preformed oligonucleotides.
  • One approach is to carry out a synthesis of an oligonucleotide on a solid support using a DNA synthesizer, except that the iodine oxidation step which is normally used
  • SUBSTITUTESHEET to oxidize the phosphite intermediate to a phosphate is eliminated, and instead the oligonucleotide phosphite attached to the solid support is reacted with phthalimidomethyl bromide. Subsequent treatment with ammonia removes the phthalimido protecting group to give the aminomethyl oligonucleotide.
  • a methyl phosphonate oligonucleotide can be prepared by using commercially available nucleoside methyl phosphonamidites, and the methyl phosphonate oligonucleotide is then treated with iodine in pyridine to give a methyl pyridinium intermediate which can be converted into an aminomethyl oligonucleotide by treatment with ammonia.
  • oligonucleotides in accordance with the present invention may be prepared such that the oligonucleotides may be isolated as pure stereoi ⁇ omers in either the-R- or S- form.
  • Such oligonucleotides include those with one aminohydrocarbon phosphonate moiety at, or adjacent to, either the 3'-terminus or the 5'-terminus; oligonucleotides having aminohydrocarbon phosphonate moieties at both the 3'- and 5'-termini; oligonucleotides having aminohydrocarbon phosphonate moieties at internal positions, provided that the aminohydrocarbon phosphonate moieties are not present on adjacent nucleotide units; oligonucleotides in which aminohydrocarbon phosphonate moieties alternate with natural phosphodiester linkages throughout the entire sequence; and oligonucleotides possessing a mixture of aminohydrocarbon phosphonate and other modified backbone substituents, such as phosphoroth
  • Such oligonucleotides may, in one embodiment, be prepared by synthesizing protected aminohydrocarbon phosphonate dinucleotides which are mixtures of R- and S- isomers, followed by separation of the R- and S- isomers by
  • SUBSTITUTESHEET conventional means, such as high pressure liquid chromatography or silica gel column chromatography.
  • the pure isomers may then be attached to oligonucleotides by conventional means to produce single isomer aminohydrocarbon phosphonate oligonucleotides.
  • the administration of the oligonucleotides as pure steroisomers in either the R- or S- form may further improve the binding capabilities of the oligonucleotide and/or increase the resistance of the oligonucleotide to degradation by nucleases.
  • the oligonucleotides may include conjugate groups attached to the 3' or 5' termini to improve further the uptake of the oligonucleotide into the cell, the stability of the oligonucleotide inside the cell, or both.
  • conjugates include, but are not limited to, polyethylene glycol, polylysine, acridine, dodecanol, and cholesterol.
  • the oligonucleotides of the present invention may be employed to bind to RNA sequences by Watson-Crick hybridization, and thereby block RNA processing or translation.
  • the oligonucleotides of the present invention may be employed as "antisense" complements to target sequences of mRNA in order to_effect translation arrest and regulate selectively gene expression.
  • the oligonucleotides of the present invention may be employed to bind double-stranded DNA to form triplexes, or triple helices. Such triplexes inhibit the replication or transcription of DNA, thereby disrupting DNA synthesis or gene transcription, respectively. Such triplexes may also protect DNA binding sites from the action of enzymes such as DNA methylases.
  • the RNA or DNA of interest, to which the oligonucleotide binds may be present in a prokaryotic or eukaryotic cell, a virus, a normal cell, or a neoplastic cell.
  • the sequences may be bacterial sequences, plasmid sequences, viral sequences, chromosomal sequences, mitochondrial sequences, or plastid sequences.
  • the sequences may include open reading frames for coding proteins, mRNA, ribosomal RNA, snRNA, hnRNA, introns, or untranslated 5'- and 3'-sequences flanking open reading frames.
  • the target sequence may therefore be involved in inhibiting production of a particular protein, enhancing the expression of a particular gene by inhibiting the expression of a repressor, or the sequences may be involved in reducing the proliferation of viruses or neoplastic cells.
  • the oligonucleotides may be used in vitro or in vivo for modifying the phenotype of cells, or for limiting the proliferation of pathogens such as viruses, bacteria, protists, Mycoplasma species, Chlamydia or the like, or for inducing morbidity in neoplastic cells or specific classes of normal cells.
  • pathogens such as viruses, bacteria, protists, Mycoplasma species, Chlamydia or the like
  • the oligonucleotides may be administered to a host subject to or in a diseased state, to inhibit the transcription and/or expression of the native genes of a target cell.
  • the oligonucleotides may be used for protection from a variety of pathogens in a host, such as, for example, enterotoxigenie bacteria, Pneumococci, Neisseria organisms, Giardia organisms, Entamoebas, neoplastic cells, such as carcinoma cells, sarcoma cells, and lymphoma cells; specific B-cells; specific T-ce ls, such as helper cells, suppressor cells, cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, etc.
  • enterotoxigenie bacteria Pneumococci, Neisseria organisms, Giardia organisms, Entamoebas
  • neoplastic cells such as carcinoma cells, sarcoma cells, and lymphoma cells
  • specific B-cells specific T-ce ls, such as helper cells, suppressor cells, cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, etc.
  • CTL cytotoxic T
  • the oligonucleotides may be selected so as to be capable of interfering with transcription product maturation or production of proteins by any of the mechanisms involved with the binding of the subject composition to its target sequence. These echansims may include interference with processing, inhibition of transport across the nuclear membrane, cleavage by endonucleases, or the like.
  • the oligonucleotides may be complementary to such sequences as sequences expressing growth factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNA polymerases, antibiotic resistance, multiple drug resistance (mdr), genes involved with metabolic processes, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flanking sequences associated with the open reading frames.
  • Infectious Diseases Antivirals, Human AIDS, Herpes, CMV Antivirals, Animal Chicken Infectious Bronchitis
  • oligonucleotides of the present invention may be employed for binding to target molecules, such as, for example, proteins including, but not limited to, ligands, receptors, and/or enzymes, whereby such oligonucleotides inhibit or stimulate the activity of the target molecules.
  • target molecules such as, for example, proteins including, but not limited to, ligands, receptors, and/or enzymes, whereby such oligonucleotides inhibit or stimulate the activity of the target molecules.
  • oligonucleotides are also applicable to the inhibition of viral replication, as well as to the interference with the expression of genes which may contribute to cancer development.
  • the oligonucleotides of the present invention are administered in an effective binding amount to an RNA, a DNA, a protein, or a peptide.
  • the oligonucleotides are administered to a host, such as a human or non-human animal host, so as to obtain a concentration of oligonucleotide in the blood of from about 0.1 to about 100 ⁇ mole/1. It is also contemplated, however, that the oligonucleotides may be administered in vitro or ex vivo as well as in vivo.
  • the oligonucleotides may be administered in conjunction with an acceptable pharmaceutical carrier as a pharmaceutical composition.
  • Such pharmaceutical compositions may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Such oligonucleotides may be administered by intramuscular, intraperitoneal, intraveneous or subdermal injection in a suitable solution.
  • compositions which can be administered bucally or sublingually, including inclusion compounds contain from about 0.1 to 99 percent by weight of active ingredients, together with the excipient. It is also contemplated that the oligonucleotides may be administered topically.
  • the pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art.
  • the pharmaceutical preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes.
  • the process to be used will depend ultimately on the physical properties of the active ingredient used.
  • Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethylcellulose, sodium carboxymethylcellulose, arid/or polyvinyl pyrrolidone.
  • fillers such as sugar, for example, lactose or sucrose, mannitol or sorbitol
  • cellulose preparations and/or calcium phosphates for example, tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch or paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin,
  • disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are flow-regulating agents and lubricants, such as, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used.
  • Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with fillers such as " lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved -or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • suspensions of the active compounds as appropriate oil injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or_triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carb ⁇ xymethyl cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active ingredient depending upon its solubility, may be present both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • the diameters of the liposomes generally range from about 15 nm to about 5 microns.
  • oligonucleotides having aminoalkyl phosphonate moieties may be used as diagnostic probes.
  • an oligonucleotide wherein at least one of the nucleotide units of the oligonucleotide includes a phosphonate moiety having the following structural formula:
  • R_, R_, and R- are as hereinabove described, and R-. is a detectable marker.
  • Detectable markers which may be employed include, but are not limited to, colorimetric markers, fluorescent markers, enzyme markers, luminescent markers, radioactive markers, or ligand recognition reporter groups.
  • detectable markers include, but are not limited to, biotin and derivatives thereof (such as, for example, e-aminocaproyl biotin, and biotin amidocaproyl hydrazide), fluorescein (including derivatives such as fluorescein amine), rhodamine, alkaline phosphatase, horseradish peroxidase, and 2, 4-dinitrophenyl markers.
  • Such oligonucleotides which include a detectable marker may be used as DNA or RNA probes. The probes may be used as diagnostics as known in the art.
  • Example 1 Production of the pyridinium salt of phthalimidomethyl phosphonic acid To 2.0g (7.42 mmole) of dimethylphthalimidomethyl phosphonate, dried by coevaporation of pyridine and dissolved in 40 ml of dry pyridine, was added dropwise 2.45 ml (2.5 equivalents) of trimethylsilyl bromide under nitrogen. After 2.5 hours, the reaction mixture was
  • Dimethyl phthalimidomethyl phosphonate (2.0_g, 7.4 mmole) was dissolved in chloroform (15 ml), and bromotrimethylsilane (2 ml, 15 mmol) was added dropwise to the solution. After 2 hrs. the reaction mixture was concentrated under reduced pressure, and the residue was dissolved in chloroform (8 ml) followed by dropwise addition of triethylamine (20 ml) with cooling in ice bath. After stirring at room temperature for 2 hrs. the mixture was filtered and concentrated to dryness. The residue was dissolved in methanol (10 ml) and then added dropwise to anhydrous diethyl ether (4 ml). The precipitate was filtered, washed with ether and dried over P ? 0 ⁇ to yield 2.1 g (65%) of pure pthalimidomethyl phosphonate, triethylammonium salt.
  • the protected dinucleotide was then treated with dichloroacetic acid to remove the dimethoxytrityl protecting group, and then treated with ammonium hydroxide at 55°C to remove the phthalimido group and cleave the dinucleotide from the solid CPG support to give an aminomethyl dimer having the following structural formula 8, wherein each of
  • The-protected dinucleotide (7) is prepared as described in Example 4.
  • the protected dinucleotide is then loaded into a l ⁇ mole size column, installed on an Applied Biosystems DNA synthesizer (Model #394), and synthesis of a modified oligonucleotide is performed using standard phosphoramidite chemistry.
  • Deprotection is carried out with 28% aqueous ammonium hydroxide at 55°C and then freeze dried in vacuo.
  • the crude oligonucleotide is converted into its sodium salt form by passage of an aqueous solution through a cation exchange resin (Na ) using water as an eluant, and is purified by Sephadex G-25 column chromatography using water as an eluant to give the aminomethyl 3' end-capped oligonucleotide.
  • a cation exchange resin Na
  • Thymidine (0.6 g, 2.3 mmol) was dried by pyridine coevaporation in the same way, dissolved in pyridine (15 ml) and added to the solution of 5'-0-dimethyoxytritylthymidine-3'-phthalimidomethylphosphon- ate.
  • the reaction mixture was stirred at room temperature under a dry nitrogen atmosphere for 2-3 hrs., then diluted with aqueous sodium bicarbonate (5%, 300 ml) and extracted with ethyl acetate (3 x 200 ml). The organic layers were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the mixed isomers of 5'-0-dimethoxytrityl-
  • Example 7 Separation of isomers of 5'0-dimethoxytritylthymidyl-3' - phthalimidomethylphosphonyl-5'-thymidine by HPLC
  • the mixture of isomers of 5'0-dimethoxytritylthymidyl-3'- phthalimidomethylphosphony1-5'-thymidine from a 200 mg scale reaction was dissolved in triethylammonium acetate (0.1 M, TEAA)/ acetonitrile (60/40, 1.5 ml) and injected into a reversed phase C. column, Radial Pak Cartridge (Waters RCM 25 x 100 mm) .
  • the column was eluted with a linear gradient of TEAA/acetonitrile in which the concentration of acetonitrile increased from 35-80%.
  • the individual isomers were eluted at 31-35 and 39-41 minutes respectively.
  • This separation procedure was repeated six times and the appropriate fractions were pooled, extracted with ethyl acetate (3 x 50 ml), evaporated and dried in vacuo over P 2 0 5 .
  • This procedure yielded 80 mg of a faster isomer and 110 mg of a slower isomer, total yield 83%.
  • Analysis of the composites by analytical HPLC using a reversed phase C4 column (Radial Pak cartridge, 8x 100 mm, 15 urn, 300 A) indicated that pure isomers were obtained in each case.
  • a 12 base, thymine-containing oligonucleotide is prepared on a 1 umole scale using an Applied Biosystems Model 394 DNA synthesizer, with phosphoramidites and other reagents as supplied by the manufacturer. After nine coupling cycles with the commercially available monomer 5'dimethoxytritylthymidine-3 ' -N,N- diisopropylamino-cyanoethoxyphosphoramidite, the final cycle employs a 0.1 M solution of either the faster or slower isomer of the phthalimidomethyl dinucleotide phosphoramidite of Example 9.
  • the modified oligomer Upon completion of the synthesis, the modified oligomer is treated with concentrated ammonia for 20 min, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described below. This procedure produces a twelve base oligonucleotide with a single isomer aminomethyl phosphonate moiety at the 5'terminus.
  • a thymine-containing tridecanucleotide with an alternating, single isomer aminomethyl phosphonate/phosphodiester backbone is prepared on a 1 umole scale using an Applied Biosystem Model 394 DNA synthesizer, using a standard phosphoramidite cycle with either the faster or slower isomer of Example 9 as the phosphoramidite. Coupling times of 2 min. per cycle are used.
  • the modified oligomer is treated with concentrated ammonia for 20 min. , partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described below.
  • This procedure produces a thirteen base oligonucleotide with single isomer aminomethyl phosphonate moieties alternating with phosphodiesters throughout the sequence.
  • a 12 base thymine-containing oligonucleotide is prepared on a 1 umole scale using an Applied Biosystems Model 394 DNA synthesizer.
  • the initial cycle employs a 0.1 M solution of either the faster or slower isomer of phthalimidomethyl phosphonate dinucleotide phosphoramidite (Example 9) which is coupled to the solid support to which a thymidine residue is attached.
  • the oligonucleotide possessing a 5'-O-dimethoxytrityl group was purified by reverse phase HPLC (C4 Radial Pak Cartridge, 100 x 25 mm, 15u, 300A). After detritylation with 0.1 M acetic acid the product was again purified by reverse phase HPLC (C4 column) using a linear gradient of 0.1 M TEAA/acetonitrile, with the concentration of acetonitrile being varied from 5 to 70%. Deprotection was carried out using ethanol/ethylenediamine (1:1) at room temperature for 45 minutes to give the desired aminomethyl backbone modified oligonucleotide.
  • Advantages of the present invention include improved solubility of the positively charged oligonucleotides in aqueous solutions as compared with nonionic oligonucleotides, improved uptake into the cell as compared with natural oligonucleotides which are negatively charged and are poorly taken up by the cell, and resistance to degradation by nucleases as compared with natural oligonucleotides which are readily degraded by cellular enzymes.
  • the oligonucleotides of the present invention are taken up by the cell more readily and are less readily degraded because of their modified backbones.
  • the cationic groups are smaller and therefore less likely to disrupt base pairing than previously synthesized cationic oligonucleotides.
  • the carbon-phosphorus bonds are more stable than nitrogen-phosphorus bonds of other cationic oligonucleotides, and thus the oligonucleotides of the present invention are less likely to lose the cationic group by chemical or enzymatic hydrolysis.
  • Aminomethyl oligonucleotides bearing detectable markers such as reporter groups have the advantage that the reporter groups are on the outside of the duplex produced by hybridization to its target DNA or RNA and are therefore more accessible towards detection, and also do not interfere with the hybridization sites on the bases.

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Abstract

Oligonucléotide dans lequel au moins une unité de nucléotide comprend une fraction de phosphonate de formule (I), dans lequel X représente (a), R1 représente un hydrocarbure, de préférence du méthylène; R2, R3 et R4 représente indépendamment hydrogène ou un hydrocarbure; et R2, R3 et R4 peuvent être identiques ou différents. X représente préférablement une fraction d'aminométhyle. Ces oligonucléotides possèdent de meilleurs pouvoirs de liaison et résistent mieux aux nucléases. Dans une autre formulation, X peut représenter (b) ou (c), R1, R2 et R3 représentent alors les mêmes éléments que dans la formule précédente et R5 représente un marqueur repérable; par conséquent ces oligonucléotides sont utiles comme sondes de diagnostic.
PCT/US1992/010043 1991-11-25 1992-11-20 Oligonucleotides comprenant des fractions de phosphonate d'aminohydrocarbure WO1993011148A1 (fr)

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JP5510176A JPH07501542A (ja) 1991-11-25 1992-11-20 アミノ炭化水素ホスホン酸塩成分を有するオリゴヌクレオチド
EP92925362A EP0641354A4 (fr) 1991-11-25 1992-11-20 Oligonucleotides comprenant des fractions de phosphonate d'aminohydrocarbure.

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WO1997006183A1 (fr) * 1995-08-04 1997-02-20 Chiron Corporation Oligonucleotides cationiques, et procedes de synthese et d'utilisation de ceux-ci

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US4471113A (en) * 1982-02-03 1984-09-11 The United States Of America As Represented By The Department Of Energy Prodrugs based on phospholipid-nucleoside conjugates

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Biochemistry, Vol. 12(2), issued 1973 (Easton, PA), HARVEY et al., "Use of Phosphate-Blocking Groups in Ligase Joining of Oligodeoxyribonucleotides", pp. 208-214, entire document. *
Can. J. Biochem., Vol. 55(8), issued 1977, (Canada), ROBINSON et al., "Phosphonolipids. XXVII. Synthesis of Phosphonic Acid Analogs of Saturated and Unsaturated L- -lecithins", pp. 907-910; CHEM. ABSTR., Vol. 88(7), p. 559, Abstr. No. 50965f (1978); only Abstract supplied, entire document. *
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Sib. Khim. Zh., Vol. 1991(3), issued 1991(USSR), ZARYTOVA et al., "Modifications of Nucleic Acids in Stabilized Complementary Complexes. V. Thermodynamic Parameters of Complementary Complexes Formed with N-(2-hydroxyethyl)phenazenium Oligonucleotide Derivatives", pp. 24-29; CHEM. ABSTR., Vol. 115(25), p. 1058, Abstr. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006183A1 (fr) * 1995-08-04 1997-02-20 Chiron Corporation Oligonucleotides cationiques, et procedes de synthese et d'utilisation de ceux-ci
US6017700A (en) * 1995-08-04 2000-01-25 Bayer Corporation Cationic oligonucleotides, and related methods of synthesis and use

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EP0641354A1 (fr) 1995-03-08
JPH07501542A (ja) 1995-02-16
EP0641354A4 (fr) 1996-01-10
CA2122470A1 (fr) 1993-06-10
AU3144593A (en) 1993-06-28

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