This application is a continuation-in-part of Application Serial No. 796,804, filed November 25, 1991.

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. (Cohen, Oligodeoxynucleotides, CRC Press, Boca Raton, Florida (1989)); Uhlmann, et al., Chem. Rev. , Vol. 90, pgε. 543-584 (1990)). 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, pgε 1374-1375 (1991)). Furthermore, 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.

Natural oligonucleotides, which are negatively charged, however, are poor candidates for therapeutic agents due to their poor penetrability into the cell and their susceptibility to degradation by nucleases. Therefore, it is expected that relatively high concentrations of natural oligonucleotides would be required in order to achieve a therapeutic effect.

To overcome the above shortcomings, various strategies have been devised. 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)). 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)).

Examples of 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

SUBSTITUTESHEET

acidic pH levels, and therefore the cationic group could be lost under certain conditions. Conjugates with the positively charged molecule polylysine have been described by Lemaitre, et al., Proc. Nat. Acad. Sci. , Vol. 84, pgs. 648-652 (1987), and have been shown to be more active in cell culture than unmodified oligonucleotides. Polylysine, however, is not a preferred molecule for conjugation due to its relatively high toxicLty.

Mononucleotides with aminomethyl phosphonate moieties have been synthesized in order to study their susceptibility to nucleotide degrading enzymes. Holy, et al. (Journal of Carbohydrates, Nucleosides and Nucleotides, Vol. 1, pgs. 85-96 (1974)) disclose the synthesis of uridine-2' (3'-aminomethyl) phosphonate and thymidine -3'-aminomethyl phosphonate by the reaction of the, corresponding 5'-0-trityl nucleoside with N-benzyloxycarbonyl-aminomethyl phosphonate. Gulyaev, et al., (FEBS Letters, Vol. 22, pgs. 294-296 (1972)) disclose the formation of ribonucleoside 5'-aminomethyl phosphonates.

In accordance with an aspect of the present invention, there is provided an oligonucleotide wherein at least one nucleotide unit includes a phosphonate moiety having the following structural formula: 0

/

O = P - 0 -

\

X, wherein X is :

SUBSTITUTE SHEET

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. Preferably, 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. Each of R_, R ₃ , and R. may be the same or different. Most preferably, each of R„, R , and R. is hydrogen.

The term "oligonucleotide", as used herein, 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.

In general, the oligonucleotide has at least two nucleotide units, preferably at least five, more preferably from five to about 30 nucleotide units.

As hereinabove stated, 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. In one embodiment, an aminohydrocarbon phosphonate moiety may be attached to alternating nucleotide units of the oligonucleotide. In another embodiment, 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. Such 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.

In a most preferred embodiment, 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):

Alternatively, 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.

Reaction of one of the protected derivatives (1), (2), or (3) with a partially protected nucleoside, such as one having the structural formula (4):

wherein B is a protected or unprotected purine or pyrimidine base, in the presence of a condensing agent such as dicyclohexylcarbodiimide or triisopropylbenzene-sulfonyl chloride would produce an ester having the following structural formula

o- wherein is the protected amino group.

SUBSTITUTE SHEET

Preferably, 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.

Alternatively, 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. At the conclusion of the synthesis, the protecting groups can be removed from the aminomethyl moieties by treatment with ammonia or with amines such as ethylenediamine.

It is also contemplated that 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

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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.

Alternatively, 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.

In another embodiment, some oligonucleotides in accordance with the present invention may be prepared such that the oligonucleotides may be isolated as pure stereoiεomers 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 phosphorothioates.

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. Such 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. For example, 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. Thus, 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. Therefore, 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.

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.

The following table is illustrative of some additional applications of the subject compositions.

Area of Application Specific Application Targets

Infectious Diseases: Antivirals, Human AIDS, Herpes, CMV Antivirals, Animal Chicken Infectious Bronchitis

Pig Transmissible

Gastroenteritis Virus

Antibacterial, Human Drug Resistance Plasmids,

E. coli

Antiparasitic Agents Malaria

Sleeping Sickness

(Trypanosomes)

Cancer

Direct Anti-Tumor Oncogenes and their products

Agents

Adjunctive Therapy Drug Resistant Tumors-Genes and Products

Auto Immune Diseases T-cell receptors Rheumatoid Arthritis Type I Diabetes Systemic Lupus

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Multiple sclerosis

Organ Transplants Kidney-OTK3 cells cause

GVHD

The 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.

The above techniques in which the oligonucleotides may be employed 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. Preferably, 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 μmole/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. The preparations, particularly those which can be administered orally and which can be used for

the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration parenterally or orally, and 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. For example, 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. If desired, 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. For this purpose, 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. In order to produce coatings resistant to gastric juices, solutions of 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. In soft capsules, the active compounds are preferably dissolved -or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, 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. In addition, it is also posible to use 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. In addition, 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 carbσxymethyl cellulose, sorbitol and/or dextran. Optionally, the suspension may also contain stabilizers.

Additionally, 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.

It is also contemplated that oligonucleotides having aminoalkyl phosphonate moieties may be used as diagnostic probes. Thus, in accordance with another aspect of the present invention, there is provided an oligonucleotide wherein at least one of the nucleotide units of the oligonucleotide includes a phosphonate moiety having the following structural formula:

SUBSTITUTESHEET

l o ✓

O = I? - o -

X, wherein X is:

wherein 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. Specific examples of detectable markers which may be employed 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.

The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.

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

SUBSTITUTE SHEET

filtered through a sintered glass funnel and the eluant was treated with H _? 0. The resulting mixture was concentrated under high vacuum and the residue remaining was dissolved in methylene chloride. Upon addition of ethyl acetate, the desired product was precipitated out. The precipitate was collected, washed with ethyl acetate, and dried over _? 0.- to yield 1.2g of pure material.

Example 2

Preparation of the Triethylaminonium Salt of Phthalimidomethyl Phosphonate

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 _Ï‚ to yield 2.1 g (65%) of pure pthalimidomethyl phosphonate, triethylammonium salt.

Example 3

Preparation of 5'-dimethoxytrityl-thvmidine-3' -phthalimidomethyl phosphonate

The triethylammonium salt of phthalimidomethyIphosphonate (1.7 g, 5 mmol) was dried by coevaporation with pyridine (3 x 10 ml), dissolved in dry pyridine (40 ml) and treated with triisopropylbenzenesulfonyl chloride (3.0 g, 9.9 mmol) followed by a solution of 5'-O-dimethoxytritylthymidine (2.0 g., 3.67 mmol) in dry pyridine (40 ml) which was previously dried by coevaporation with pyridine. The resulting mixture was stirred at room temperature overnight under a dry nitrogen atmosphere and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography using CH ₂ Cl ₂ /Me0H/Et_N (30:1:0.3, 2.8 L followed by 30:2:0.3, 1.1 L) as solvent. The appropriate fractions were collected and combined to yield 1.8 g (57%) of a pure compound having the following structure:

(wherein B. is thymine) as a white foam.

Example Synthesis of an aminomethyl dinucleotide A commercially available nucleoside attached to a controlled pore glass (CPG) support, and having the following structural formula:

.(wherein B~ is a protected or unprotected purine or pyrimidine base) was treated with 3% dichloroacetic acid to remove the dimethoxytrityl (DMT) protecting group, and then reacted with the phthalimidomethyl nucleoside phosphonate

(6) of Example 3 in the presence of triisopropyl-3-nitro-l,2,4-triazole as coupling agent and

1-methylimidazole as catalyst in dry acetonitrile for 15 minutes to give a phthalimidomethyl dinucleotide having the following structural formula:

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

Example 5 Synthesis of a 3 ' aminomethyl end capped oligonucleotide

SU _BSTITUT ESHEET

The-protected dinucleotide (7) is prepared as described in Example 4. The protected dinucleotide is then loaded into a lμmole 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.

Example 6 Preparation of 5-'O-dimethoxytrityl-thymidyl-3'- phthalimidomethyl-phospho ^~ nv ^" l-5'-thvmidine, mixed isomers

5*-o-dimethoxytritylthymidine-3'-phthalimidomethylphospho na- te (Example 3, 2.0 g, 2.3 mmol) was dried by coevaporation with pyridine (3 x 15 ml), redissolved in dry pyridine (80 ml) and treated with l-(2,4,6)-trimethylbenzenesulfonyl-nitrotriazolide (0.75 g, 2.5 mmol), for 15 min. at room temp. 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-

SUBSTITUTESHEET

thymidyl-3'-phthalimidomethyl-phosphonyl-5'-thymidine, 1.5 g (65%).

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.

Example 8 Separation of 5'0-dimethσxytritylthymidyl-3'-pthalimido- methylphosphonyl -5'-thymidine by silica column chromatography

The residue from a 1.72 g preparation of mixed isomers of

5'-0-dimethoxytritylthymidyl-3'phthalimidomethylphosphony l-- 5'-thymidine was purified by column chromatography on silica gel (lOOg) using CH ₂ Cl ₂ /CH ₃ 0H/Et ₃ N (30:1:0.3) as the solvent.

Fractions 120-126 contained the faster eluting isomer, fractions 127-157 contained a mixture of both isomers, and fractions 158-170 contained the slower eluting isomer. The appropriate fractions were collected, evaporated to dryness and dried in vacuo over P.O _Ï‚ to give 0.17g of the faster eluting isomer, 0.2 g of the slower eluting isomer, and 0.7 g of a mixture of isomers.

Example 9 Synthesis of isomers of 5'-0-dimethoxytritylthymidyl-3' phthalimidomethylphosphonyl-5'-thymidine -3'-cyanoethyl-N, N-diisopropylaminophosphoramidite.

A sample of the faster isomer of 5'-O-dimethoxytritylthymidyl

-3'phthalimidomethylphosphonyl-5'-thymidine (0..42 g, 0.42 mmole) was is dried by coevaporation with pyridine, dissolved in dry acetonitrile (10 ml) under nitrogen, and treated with stirring with cyanoethoxy-

(N,N,N' ,N'-tetra-isopropylamino)- phosphine (0.33 ml, 1.05 mmol), tetrazole (30 mg), and diisopropylamine (0.08 ml, 0.58 mmol). After 50 minutes at room temperature the mixture was partitioned between 5% aqueous sodium bicarbonate and acetonitrile (50 ml of each). The organic layer was washed with water (2 x 50 ml), and concentrated in vacuo to a gum. The crude product were purified by column chromatography on silica gel (40g) using CH ₂ Cl ₂ /Me0H/Et ₃ N (100:2:1). The appropriate fractions were combined and evaporated to yield 0.36 g (71%) of the faster isomer of 5'-O-dimethoxytritylthy- midyl-3'-phthalimidomethylphosphonyl-5'-thymidine-3'-cyanoe ¬ thyl-N,N-diiεopropylaminophosphoramidite.

An identical procedure was followed to produce a phosphoramidite from the slower isomer.

Example 10 Procedures for oligonucleotide synthesis and deprotection

a) 5'-End capped oligonucleotide

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. 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. b) Synthesis of a tridecanucleotide with an alternating single isomer aminomethyl phosphonate/phosphodiester backbone.

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. 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 thirteen base oligonucleotide with single isomer aminomethyl phosphonate moieties alternating with phosphodiesters throughout the sequence.

SUBSTITUTESHEET

c) 3',5'-Aminomethyl phosphonate end capped oligonucleotide

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. -After nine subsequent coupling cycles with the commercial available monomer 5'-dimethoxytritylthymidine-3'-N,N-diisopropylamino-cyanoet- hoxyphosphoramidite, the final cycle again employs a 0.1 M solution of either the faster or slower isomer of phthalimidomethyl dinucleotide phosphoramidite of Example 9. 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. d) General procedure for oligonucleotide purification by HPLC

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.

Example 11 Synthesis of a biotinylated 3' aminomethyl oligonucleotide

SUBSTITUTE SHEET

The 3'-aminomethyl end capped oligonucleotide of Example 5 is placed in aqueous sodium bicarbonate buffer, pH 8. This solution is then treated with a solution of biotin N-hydroxysuccinimide ester (50 equivalents) in dimethylsulfoxide for 18 hours at room temperature. The resulting solution is passed through a Sephadex G25 column to remove the biotin and other small molecules and the fractions containing the oligonucleotide are concentrated and purified by high performance liquid chromatography using a C18 reversed phase silica column. The appropriate fractions are collected and evaporated to dryness to give the biotinylated 3'-aminomethyl end capped 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. By virtue of their positively-charged regions, the oligonucleotides of the present invention are taken up by the cell more readily and are less readily degraded because of their modified backbones. In the case of oligonucleotides having aminomethyl phosphonate moieties, the cationic groups are smaller and therefore less likely to disrupt base pairing than previously synthesized cationic oligonucleotides. Also, 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.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.