CA2122470A1 - Oligonucleotides having aminohydrocarbon phosphonate moieties - Google Patents
Oligonucleotides having aminohydrocarbon phosphonate moietiesInfo
- Publication number
- CA2122470A1 CA2122470A1 CA 2122470 CA2122470A CA2122470A1 CA 2122470 A1 CA2122470 A1 CA 2122470A1 CA 2122470 CA2122470 CA 2122470 CA 2122470 A CA2122470 A CA 2122470A CA 2122470 A1 CA2122470 A1 CA 2122470A1
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- CA
- Canada
- Prior art keywords
- oligonucleotide
- oligonucleotides
- hydrogen
- hydrocarbon
- markers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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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
Abstract
An oligonucleotide wherein at least one nucleotide unit includes a phosphonate moiety having the structural formula (I) , wherein X is (a), R1 is a hydrocarbon, preferably methylene, and each of R2, R3, and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different. Preferably, X
is an aminomethyl moiety. Such oligonucleotides having improved binding capabilities and improved resistance to nucleases.
Alternatively, X may be (b) or (c), wherein R1, R2, and R3 are as hereinabove described, and R5 is a detectable marker, thus making such oligonucleotides useful as diagnostic probes.
is an aminomethyl moiety. Such oligonucleotides having improved binding capabilities and improved resistance to nucleases.
Alternatively, X may be (b) or (c), wherein R1, R2, and R3 are as hereinabove described, and R5 is a detectable marker, thus making such oligonucleotides useful as diagnostic probes.
Description
W(~ 93/1 1 14X 2 1 2 2 4 7 0 Pcr/US92/10043 OLIGONUCI.EOTIDES ~AVING ~INO~YDRQCARBON
PllOSP~0~113 MOIETIES
Thi~ application is a continuation-in-part of Application Serial No. 79~,804, filed November 25, l991.
This invention relates to oligonucleotides which bind to RNA (such as mRNA), DNA, protein~, 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 enakles an oligonucleoti~e 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 expre~sion. ~Cohen, OliaodeoxYnucleotides, CRC Presæ, Boca Raton, Florida (lg89)); Uhlmann, et al., Chem. Rev., Vol. 90, pgs. 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 bondi~g interactions (Moffat, Science, Vol. 252, pgs 1374-137~ (l991)). ~urthermore, oligonucleotides have been shown to bind specifically to protein~ (Oliphant, et al., Molec. Cell. Biol. Vol g, pgs.
2944-2949 (19B9)) and could thus be used to block undesirable protein function.
SuasT~
WO93/1114X 2 122 ~7 0 -2- PCT/US92/10043 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. Thereore, it is expected that relatively high concentrations of natural oligonucleotides would be reguired in order to achieve a therapeutic effect.
To overcome the above shoxtcomings, various strategies have been devised. U.S. Patent No. 4,469,863, issued to Miller, et al., discloses the manufac'ure 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 phosphodie ter moiety is replaced by sulfur. Such analogues sometimes are referred to as phosphorothioate (PS) analogues, or "all PS"
analogue~, (Stein, et al., Nucl. Acids Res., Vol. 16, pgs.
3209-3221 (1988)). Another approach has been to attach a targeting mo~ety, such as cholesterol, which improves the uptake of the oligonucleotide by a receptor-mediated process. (Stein et al., Biochemistry, Vol. 30, pgs.
243g-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 compoundæ to give positively charged oligonucleotides with phosphoramidate linkages. Phosphoramidate linkages, however, are known to be somewhat labile, especially at SUBSTITUTE SHEEr W093/1114X 2 i 2 2 1 7 0 PCT/US92/10043 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, howe~er, is not a preferred molecule for conjugation due to its relatively high toxicity.
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. l, pgs.
85-96 (1974)) disclose the synthesis of uridine-2' (3'-aminomethyl) phosphonate and thymidine -3'-aminomethyl pho~phonate by the reaction of th~ corresponding 5'-0-trityl nucleo~ide 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:
O = P - O -X, wherein X is:
Rl-N-R3 R4.
SUBSTITUTE SHEEl-2 122 4~ 0 _4_ Rl is a hydrocarbon, preferably alkylene, phenylene, or naphthylene, more preferably an alkyl group having from l to 15 carbon atoms, and most preferably l to 3 carbon atoms, with methylene being preferred. Each of R2 R3, and R4 is hydrogen or a hydrocarbon. Preferably, the hydrocarbon is an alkyl group having from l to 15 carbon atoms, more preferably from l to 3 carbon atoms, and most preferably a methyl group. Each of R2, R3, and R4 may be the same or different. Most preferably, each of R2, R3, and R4 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 S' 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, SU~STITUTE SHEF~
wo 93/11148 2 I 2 2 ~ 7 0 ` P~/US92/10043 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 pho~phonate moieties may be accomplished through the synthesis of a monomer unit with a protected aminomethyl group, followed by incorporation of one or more such monomer units into an oli~onucleotide; 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, ~uch 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):
o ~ ~ CI~Iz~ CO~
OH OA~
-- ~o~ Gg =~--C h,'~ o - O ~
o~ 3 Alternatively, the phthalimide derivative (1) may be prepared by reaction of chloromethyl phosphonic acid with pht~alimide, or by demethylation of commercially available TITI IT~ FFT
212~ 4~ -6- : ~
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):
~c~3 C~o ~ C--~¢~
(~) wherein B is a protected or unprotected purine or pyrimidine base, in the presence of a condensing aqent such as dicyclohexylcarbodiimide or triisopropylbenzene-sulfonyl chloride would produce an ester having the following structural formula (5):
~C~3 ,~
C~C~C- 0~
~ )/
S~--C
o wherein Q is the protected amino group.
SUBSTITUTE SHEET
WOg3/11148 21~2470 PCT/US92/10~3 Preferably, the protected amino group is selected from the group consisting of:
~a~
Cb~ c~
cc) ~ C ~7 - o--C ~
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 ~olid support. After the solid support~attached oligonucleo~ide 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 rémoved by treatment with hydrazine or a substituted hydrazine to ~enerate 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 æuch as hereinabove described with a protected aminomethyl phosphite derlvative to form a nucleoside phosphonamidite.
The nucleo8ide phosphonamidite can then be used in place of a nucleoside pho~phoramidite in a DNA synthesizer. At the conclusion of the synthesis, the protecting groups can be r~moved from the aminomethyl moieties by treatment with ammonia or with amines such as ethylenediamine.
It is also contemplated that aminomethyl phoiphonate moieties may be introduced into preformed oligonucleotides.
One appro~ch 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 ,~
.. :.
, ~
W093/1114X 2 1 2 2 4 7 0 PCT/US92/1~43 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. Subseguent 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 stereo~omers in either the--R- or S- form. Such oligonucleotide~ include those with one aminohydrocarbon phoæphonate 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 am~nohydrocarbon 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 phosphonat~ 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 SUE~STITUTE SHEET
W093/l1148 - 2 1 2 2 ~ 7 0 PCTtUSg2~10043 conventional means, such as high pressure liquid chromatography or sîlica 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 fùrther improve the binding capabilities of the oligonucleotide and/or increase the resi~tance of the oligonucleotide to deqradation by nucleases.
The oligonucleotides may include conjugate groups attached to the 3' or 5' termini to improve further the uptake of the oligonucieotide 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 oligonucleotideæ of the present invention may be employed to bind to RNA seguences by Wat~on-Crick hybridization, and thereby block RNA proce~sing or translation. For example, the oligonucLeotides of the present invention may be employed as "antisense" complements to target sequences of mRNA in order tQ_e~fect 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 Sl~3ST~TUTE SHEET
WO 93/1 1 14X 2 1 2 2 ~ ~ ~ PCT/US92/10043~
seguences, 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 readin~
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 uced in vitro or in vivo for m~diEying the phenotype of cells, or for limiting the proliferation of pathogens such as viruses, bacteria, protists, MYcoplasma species, Chlam~dia 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 andjor expression of the native genes of a target cell. Therefore, the oliqonucleotides may be used for protection from a variety of pathogens in a host, such as, for example, enterotoxigenic bacteria, Pneumococci, Nei~seria organisms, Giardia organisms, Entamoebas, neopla~tic cells, such as carcinoma cells, sarcoma cells, and lymphoma cells; specific B-cells;
8pecific T-cells, 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 mechansims may include interference with proces~ing, inhibition of transport acro~s the nuclear membrane, cleavage by endonucleases, or the like.
SUE~STITUTE SHEET
W093/1114X 2 1 2 ~, ~ 7 0 PCT/US92/10043 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 resi~tance (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 s~quences a~sociated with the open reading frames.
The following table is illustrative of some additional applications of the subject compositions.
Area of ADDlication Specific AvPlicati-on Taraets Inectious 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 A~ents Ad~unctive Therapy Drug Resistant Tumors-Genes and Products Auto Immune Diseases T-cell receptors Rheumatoid ~rthritis Type I Diabetes Systemic Lupus SUBSTITUTE SHEET
W093/1114X 2 1 2 2 ~ ~ O PCT/US92/10043 Multiple sclerosis '' Organ Transplants Kidney-OTK3 cells cause GVHD
The oligonucleotides of the present invention may be employed for binding to target molecules, ~uch 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 repl-ication, as well as to the interference with the expression of genes which may contribute to cancer development.
The oli~onucleotides 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 ad'ministered 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 O.l to about 100 ~mole/l. 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 w~th 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 WO93/1114X 2 ~ 2 ,~ ~17 a PCT/US92/10043 -l3-the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be admini~tered rectally, such as suppositories, as well as suitable solutions f~r administration parenterally or orally, and compositions which can be administered bucally or sublingually, including inclusion compounds, contain from about O.l 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 itQelf well known in the art. For example, the pharmaceutical preparations may be made by means of conventional mixing, granulating, draqee-making, dissolving or lyophilizing processes. The proce~s to be used will depend ultimately on the physical propertie~ of the active ingredient used.
Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucrose, mannitol or ~orbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen pho~phate, as well as binders such as starch or paste, u~ing, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum traqacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethylcellulose, sodium carboxymethylcellulo~e, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl pyrrol$done, agar, or alginic acid or a saIt thereof, such as ~odium 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 SU~STITUTE SHER
WO93~11148 212 2 4 7 0 - PCT/US92/10043 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 ~olutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, ~olutions 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, 8ealed 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 ~actose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissoIved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liguid 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.
SUBSTlTUT~ ~:~FFT
WO93/1114X 2 1 2 . '1 ~ O PCT/US92/10043 -l5-Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspen~ions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lipophilic ~olvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. A~ueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
Optionally, the suspenQion may also contain stabilizers.
Additionally, the compounds of the present invention may al~o be administered encapsulated in liposomes,-wherein the active ingredient is contained either di6persed 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 i8 generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids ~uch as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, andjor 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:
SUBSTITUTE SHEEl~
WO93~11148 2 1 2 2 ~ 7 ~ PCT/US92/1~043 O = ~- o -X, wherein X is:
,R2 ~R2 -Rl-N -R3, or -Rl-N-R5, wherein Rl, R2, and R3 are as hereinabove described, and R5 i 8 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 em~loye~
include, but are not limited to, biotin and derivatives thereof (such as, for example, e-aminocaproyl biotin, and biotin amidocaproyl hydrazlde), fluorescein (including derivatives such as fluorescein amine), rhodamine, alkaline phosphatase, horseradish peroxidase, and 2, 4-dinitrophenyl markers. Such oligonucleotides which include a de~ectable marker may be used as DNA or RNA probes. The probes may be used a~ diagnostics as known in the art.
The invention will now be described with respect to the fo~lowing example~; however, the scope of the preRent invention i8 not intended to be limited thereby.
Exam~le 1 Production of the pyridinium salt of Dhthalimidomethvl Dhos~ gnic ~
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 :
2 1 ~1 ~47 0 ``
filtered through a sintered glass funnel and the eluant was treated with H20. The resulting mixture was concentrated under high vacuum and the residue remaining was dissolved in methylene chloride. Upon addition of ethyl a~etate, the desired product was precipitated out. The precipitate was collected, washed with ethyl acetate, and dried over P205 to yield 1.2g of pure material.
ExamPle 2 PreDaration of the Triethylaminonium Salt of Phthalimidomethyl PhosDhonate .- Dimethyl phthalimidomethyl phosphonate (2.0 ~, 1.4 mmole) was di~solved in chloroform (15 ml), and bromotrimethyl~ilane (2 ml, 15 mmol) wa~ added dropwise to `
the solution. After 2 hrs. the reaction mixture was concentrated under reduced pre~sure, and the residue was dissolved in chloroform (8 ml) followed by dropwisé addition of triethylamine (20 ml) with cooling in ice bath. After stirring at room temperature for 2 hrs. the mixtuxe 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 P205 to yield 2.1 g (65X) of pure pthalimidomethyl pho~phonate, triethylammonium salt.
ExamDle 3 PreDaration of 5'-dimethoxvtritYl-thvmidine-3' -DhthalimidomethYl PhosPhonate SVBSTtTUTE SHEET
The triethylammonium salt of phthalimidomethylphosphonate (1.7 g, 5 mmol) was dried by coevaporation with pyridine (3 x l0 ml), dissolved in dry pyridine (40 ml) and treated with triisopropylbenzenesulfonyl chloride (3.0 g, 9.9 mmol) followed by a solution of 5'-0-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 pre~ure. The re~idue was purified by silica gel column chromatography u~ing CH2Cl2/MeOH~Et3N (30:l:0.3, 2.8 L followed by 30:2:0.3, l.l L) as solvent. The appropriate fractions were colIected and combined to yield 1.8 ~ (57%) of a pure compoOu~d having the following structure:
o~ . 6 o~
- o (wherein Bl is thymine) as a white foam.
ExamDle 4 SYnthesis of an aminomethYl d nucleotide A commercially available nucleoside attached to a controlled pore gla~s (CPG) support, and having the following structural formula:
D~r- O ~o~l 2 ~_ O
~h/f~- CP~
SuE3sTlTuT~
W093/11148 2 1 2 2 ~ 7 0 PCT/US92~10043 (wherein B2 is a protected or unprotected purine or pyrimidine base) was treated with 3% dichloroacetic acid to remove the dimethoxytrityl (DMT) protectinq group, and then reacted with the phthalimidomethyl nucleoside phosphonate (6) of Example 3 in the presence of trii~opropyl-3-nitro-1,2,4-triazole as coupling agent and 1-methylimidazole as catalyst in dry acetonitrile for 15 minutes to give a phthalimidomethyl dinucleotide having the following structural ~ormula: ~
O
p_ o ~!?
O
C~
The protected dinucleotide was then treated with dichloroacetic acid to remove the dimethoxytrityl protecting group, and then treated with ammonium hydroxide at 55C to remove the phthalimido group and cleave the dinucleotide from the solid CPG ~upport to gi~e an aminomethyl dimer having the following ~tructural formula 8, wherein each of Bl and B2 i~ an unprotected purine or pyrimidine base.
t~o 0'~
0~
Example 5 SYnthesis of a 3' aminomethYl end SUBSTITUTE. SHEEl' ~ -20-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 55C and then freeze dried in vacuo. The crude oligonucleotide is con~erted 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 a~ an eluant to give the aminomethyl 3' end-capped oligonucleotide.
Example 6 Preparation of 5-'0-dimethoxYtritvl-thYmidYl-3'-Dhthalimidomethvl-DhosPhonvl-5'-thvmidine, mixed isomers 5'-0-dimethoxytritylthymidine-3'-phthalimidomethylpho phona-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 1-(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, di~ olved in pyridine (15 ml) and added to the solution of 5'-0-dimethyoxytritylthymidine-3'-phthalimidomethylphosphon-~te. The reaction mixture was stirred at room temperature under a dry nltrogen atmosphere for 2-3 hrs., then diluted with aqueous ~odium 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-SUBSTITUTE SHEEl' .
W093/11148 ~1 2 2 ~ 7 ~ PCT/US92/10043 thymidyl-3'-phthalimidomethyl-phosphonyl-5'-thymidine, 1.5 g (65%).
Example 7 SeParation of i~omers of S'0-dimethox~tritvlthvmidvl-3'-Dhthalimidomethvlpho~phonyl-5'-thvmidine by HPLC
~ The mixture of isomers of 5'0-dimethoxytritylthymidyl-3'-phthalimidomethylphosphonyl-S'-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 C4 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 P205. This procedure yielded 80 mg of a faster isomer and 110 mg of a slower isomer, total yield 83%. Analysis of the composites by analyti~al HPLC using a rever~ed phase C*
column (Radial Pak cartridge, 8x 100 mmh 15 um, 300 A) indicated that pure isomers were obtained in each case.
ExamPle 8 SeDaration of 5'0-dimethoxvtritvlthvmidvl-3'-Dthalimido-methviDhocDbonvl -5'-thymi~ne~ bv ~ilica column chromato~raphY
The residue from a 1.72 g preparation of mixed isomers of 5'-0-dimethoxytritylthymidyl-3'phthalimidomethylphosphonyl--5'-thymidine was purified by column chromatography on silica gel (lOOg) using CH2Cl~/CH30H/Et3N (30:1:0.3) as the solvent.
SUE3~;TIT(JT~ FT
W093/1l148 212 2 4 7 0 PCT/US92/10043..
^ -22-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 P205 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.
ExamDle 9 S~nthe~is of isomers of S'-0-dimethoxYtritylth~midYl-3' DhthalimidomethvlDhosphonyl-5'-thymidine -3'-cYanoethYl-N, N-diisoproDYlaminoDhosPhoramidite.
A sample of the faster iso~er of 5'-0-dimethoxytritylthymidyl -3'phthalimidomethylphosphonyl-5'-thymidine (O.g2 g,~0.42 mmole) was i8 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 ~odium bicarbonate and acetonitrile (S0 ml of each). The organic layer was washed with water (2 x S0 ml) and concentrated in vacuo to a gum. The crude product were purified by column chromatography on silica gel (40g) using CH2C12/MeOH/Et3N
(100:2:1). The appropriate fractions were combined and evaporated to yield 0.36 g (71%) of the faster isomer of 5'-0-dimethoxytritylthy-midyl-3'-phthalimidomethylphosphonyl-S'-thymidine-3'-cyanoe-thyl-N,N-diisopropylaminophosphoramidite.
An~identical procedure was followed to produce a phosphoramidite.from the slower isomer.
Exam~le 10 Procedures for oliqonucleotide s~nthesis and deProtection WO93/1114X 212 ~ ~ 7 0 PCT/USg2/10043 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 ynthesizer, 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 i8 treated with concentrated ammonia for 20 min, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described ~elow.
This procedure produces a twelve base oligonucleotide with a Yingle isomer aminomethyl phosphonate moiety at the 5'terminus.
b) Synthesis of a tridecanucleotide with an alternating single i80~er uoinomethyl phosphonate/phosphodie~ter backbone.
A thymine-containing tridecanucleotide with an alternating, single isomer aminomethyl phosphonate/phosphodiester backbone i~ prepared on a 1 umole scale u~ing an Applied Biosystem Model 394 DNA synthesizer, using a standard phosphoramidite cycle with either the fa~ter or slower i~omer of Example 9 as the phosphoramidite.
Coupling time~ 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, lyaphilized to dryness and purified as described below.
This procedure produces a thirteen base oligonucleotide with single i~omer aminomethyl phosphonate moieties alternating with pbosphodiesters throughout the sequence.
SUE~STITUTE SHEET
W093/1l148 212 2 4 7 0 PCT/US92/10043 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
~olution of either the fa~ter 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 dryne~s and purified as described below.
d) General procedure for oligonucleotide purification by E~PLC ' The oligonucleotide possessing a 5'-0-dimethoxytrityl ~roup was purified by reverse phase HPLC (C4 Radial Pak Cartridge, 100 x 25 mm, l5u, 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 O.1 M TEAA/aceton~trile, 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 oliaonucleotide S(IBSTITUTE SHE~T
W043/l1148 21 2 2 ~. 7 0 PCT/US92/10043 .
The 3'-aminomethyl end capped oligonucleotide of Example 5 i~ placed in aqueous sodium bicarbonate buffer, pH
8. This solution i8 then treated with a solution of bi~tin N-hydroxysuccinimide ester (50 equivalents) in dimethylsulfoxide for 18 hours at room temperature. The resulting solution i~ passed through a Sephadex G25 column to remove the bio~in and other ~mall molecules and the fractions containing the olîgonucleotide are concentrated and purified by high performance liquid chromatography using a C18 rever~ed pha~e silica column. The appropriate fractions are collected and evaporated to drynes~ to give the biotinylated 3'-aminomethyl end capped oligonucleotide.
Advantages of the present invention include improved olubility o the positively charged oligonucleotides in agueou~ ~olution~ as compared with nonionic oligonucleotides, improved uptake into the cell a~ compared with natural oligonucleotides which are negatively charged and are poorly taken up by the cell, and re~istance to degradation by nucleases a~ compared with natural oligonucleotides which are readily degraded by cellular enzymes. By virtue of their positively-charged regions, the oligonucleotide~ of the present invention are ta~en up by the cell more readily and are less readily degraded because of their modified backbone~. In the ca~e of oligonucleotides having aminomethyl phosphonate moieties, the cationic groups are ~maller and therefore les~ likely to disrupt base pairing than previously synthesized cationic oligonucleotides. Also, the carbon-phosphorus bonds are more ~table than nitrogen-phosphorus bonds of other cationic oligonucleotides, and thus the oligonucleotides of the present invention are less likely to lo~e the cationic group by chemical or enzymatic hydrolysis.
~ minomethyl oligonucleotides bearing detectable markers such as reporter groups have the advantage that the reporter C~ I~'rtT~ ITr ~J~_ .
WO93~11148 - PCT/US92/10043 2122~70 -26-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 preqent invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and ~till be within the scope of the accompanying claims.
! :UBSTITUTE~ S~EET
PllOSP~0~113 MOIETIES
Thi~ application is a continuation-in-part of Application Serial No. 79~,804, filed November 25, l991.
This invention relates to oligonucleotides which bind to RNA (such as mRNA), DNA, protein~, 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 enakles an oligonucleoti~e 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 expre~sion. ~Cohen, OliaodeoxYnucleotides, CRC Presæ, Boca Raton, Florida (lg89)); Uhlmann, et al., Chem. Rev., Vol. 90, pgs. 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 bondi~g interactions (Moffat, Science, Vol. 252, pgs 1374-137~ (l991)). ~urthermore, oligonucleotides have been shown to bind specifically to protein~ (Oliphant, et al., Molec. Cell. Biol. Vol g, pgs.
2944-2949 (19B9)) and could thus be used to block undesirable protein function.
SuasT~
WO93/1114X 2 122 ~7 0 -2- PCT/US92/10043 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. Thereore, it is expected that relatively high concentrations of natural oligonucleotides would be reguired in order to achieve a therapeutic effect.
To overcome the above shoxtcomings, various strategies have been devised. U.S. Patent No. 4,469,863, issued to Miller, et al., discloses the manufac'ure 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 phosphodie ter moiety is replaced by sulfur. Such analogues sometimes are referred to as phosphorothioate (PS) analogues, or "all PS"
analogue~, (Stein, et al., Nucl. Acids Res., Vol. 16, pgs.
3209-3221 (1988)). Another approach has been to attach a targeting mo~ety, such as cholesterol, which improves the uptake of the oligonucleotide by a receptor-mediated process. (Stein et al., Biochemistry, Vol. 30, pgs.
243g-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 compoundæ to give positively charged oligonucleotides with phosphoramidate linkages. Phosphoramidate linkages, however, are known to be somewhat labile, especially at SUBSTITUTE SHEEr W093/1114X 2 i 2 2 1 7 0 PCT/US92/10043 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, howe~er, is not a preferred molecule for conjugation due to its relatively high toxicity.
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. l, pgs.
85-96 (1974)) disclose the synthesis of uridine-2' (3'-aminomethyl) phosphonate and thymidine -3'-aminomethyl pho~phonate by the reaction of th~ corresponding 5'-0-trityl nucleo~ide 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:
O = P - O -X, wherein X is:
Rl-N-R3 R4.
SUBSTITUTE SHEEl-2 122 4~ 0 _4_ Rl is a hydrocarbon, preferably alkylene, phenylene, or naphthylene, more preferably an alkyl group having from l to 15 carbon atoms, and most preferably l to 3 carbon atoms, with methylene being preferred. Each of R2 R3, and R4 is hydrogen or a hydrocarbon. Preferably, the hydrocarbon is an alkyl group having from l to 15 carbon atoms, more preferably from l to 3 carbon atoms, and most preferably a methyl group. Each of R2, R3, and R4 may be the same or different. Most preferably, each of R2, R3, and R4 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 S' 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, SU~STITUTE SHEF~
wo 93/11148 2 I 2 2 ~ 7 0 ` P~/US92/10043 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 pho~phonate moieties may be accomplished through the synthesis of a monomer unit with a protected aminomethyl group, followed by incorporation of one or more such monomer units into an oli~onucleotide; 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, ~uch 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):
o ~ ~ CI~Iz~ CO~
OH OA~
-- ~o~ Gg =~--C h,'~ o - O ~
o~ 3 Alternatively, the phthalimide derivative (1) may be prepared by reaction of chloromethyl phosphonic acid with pht~alimide, or by demethylation of commercially available TITI IT~ FFT
212~ 4~ -6- : ~
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):
~c~3 C~o ~ C--~¢~
(~) wherein B is a protected or unprotected purine or pyrimidine base, in the presence of a condensing aqent such as dicyclohexylcarbodiimide or triisopropylbenzene-sulfonyl chloride would produce an ester having the following structural formula (5):
~C~3 ,~
C~C~C- 0~
~ )/
S~--C
o wherein Q is the protected amino group.
SUBSTITUTE SHEET
WOg3/11148 21~2470 PCT/US92/10~3 Preferably, the protected amino group is selected from the group consisting of:
~a~
Cb~ c~
cc) ~ C ~7 - o--C ~
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 ~olid support. After the solid support~attached oligonucleo~ide 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 rémoved by treatment with hydrazine or a substituted hydrazine to ~enerate 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 æuch as hereinabove described with a protected aminomethyl phosphite derlvative to form a nucleoside phosphonamidite.
The nucleo8ide phosphonamidite can then be used in place of a nucleoside pho~phoramidite in a DNA synthesizer. At the conclusion of the synthesis, the protecting groups can be r~moved from the aminomethyl moieties by treatment with ammonia or with amines such as ethylenediamine.
It is also contemplated that aminomethyl phoiphonate moieties may be introduced into preformed oligonucleotides.
One appro~ch 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 ,~
.. :.
, ~
W093/1114X 2 1 2 2 4 7 0 PCT/US92/1~43 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. Subseguent 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 stereo~omers in either the--R- or S- form. Such oligonucleotide~ include those with one aminohydrocarbon phoæphonate 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 am~nohydrocarbon 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 phosphonat~ 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 SUE~STITUTE SHEET
W093/l1148 - 2 1 2 2 ~ 7 0 PCTtUSg2~10043 conventional means, such as high pressure liquid chromatography or sîlica 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 fùrther improve the binding capabilities of the oligonucleotide and/or increase the resi~tance of the oligonucleotide to deqradation by nucleases.
The oligonucleotides may include conjugate groups attached to the 3' or 5' termini to improve further the uptake of the oligonucieotide 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 oligonucleotideæ of the present invention may be employed to bind to RNA seguences by Wat~on-Crick hybridization, and thereby block RNA proce~sing or translation. For example, the oligonucLeotides of the present invention may be employed as "antisense" complements to target sequences of mRNA in order tQ_e~fect 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 Sl~3ST~TUTE SHEET
WO 93/1 1 14X 2 1 2 2 ~ ~ ~ PCT/US92/10043~
seguences, 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 readin~
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 uced in vitro or in vivo for m~diEying the phenotype of cells, or for limiting the proliferation of pathogens such as viruses, bacteria, protists, MYcoplasma species, Chlam~dia 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 andjor expression of the native genes of a target cell. Therefore, the oliqonucleotides may be used for protection from a variety of pathogens in a host, such as, for example, enterotoxigenic bacteria, Pneumococci, Nei~seria organisms, Giardia organisms, Entamoebas, neopla~tic cells, such as carcinoma cells, sarcoma cells, and lymphoma cells; specific B-cells;
8pecific T-cells, 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 mechansims may include interference with proces~ing, inhibition of transport acro~s the nuclear membrane, cleavage by endonucleases, or the like.
SUE~STITUTE SHEET
W093/1114X 2 1 2 ~, ~ 7 0 PCT/US92/10043 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 resi~tance (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 s~quences a~sociated with the open reading frames.
The following table is illustrative of some additional applications of the subject compositions.
Area of ADDlication Specific AvPlicati-on Taraets Inectious 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 A~ents Ad~unctive Therapy Drug Resistant Tumors-Genes and Products Auto Immune Diseases T-cell receptors Rheumatoid ~rthritis Type I Diabetes Systemic Lupus SUBSTITUTE SHEET
W093/1114X 2 1 2 2 ~ ~ O PCT/US92/10043 Multiple sclerosis '' Organ Transplants Kidney-OTK3 cells cause GVHD
The oligonucleotides of the present invention may be employed for binding to target molecules, ~uch 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 repl-ication, as well as to the interference with the expression of genes which may contribute to cancer development.
The oli~onucleotides 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 ad'ministered 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 O.l to about 100 ~mole/l. 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 w~th 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 WO93/1114X 2 ~ 2 ,~ ~17 a PCT/US92/10043 -l3-the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be admini~tered rectally, such as suppositories, as well as suitable solutions f~r administration parenterally or orally, and compositions which can be administered bucally or sublingually, including inclusion compounds, contain from about O.l 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 itQelf well known in the art. For example, the pharmaceutical preparations may be made by means of conventional mixing, granulating, draqee-making, dissolving or lyophilizing processes. The proce~s to be used will depend ultimately on the physical propertie~ of the active ingredient used.
Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucrose, mannitol or ~orbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen pho~phate, as well as binders such as starch or paste, u~ing, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum traqacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethylcellulose, sodium carboxymethylcellulo~e, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl pyrrol$done, agar, or alginic acid or a saIt thereof, such as ~odium 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 SU~STITUTE SHER
WO93~11148 212 2 4 7 0 - PCT/US92/10043 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 ~olutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, ~olutions 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, 8ealed 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 ~actose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissoIved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liguid 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.
SUBSTlTUT~ ~:~FFT
WO93/1114X 2 1 2 . '1 ~ O PCT/US92/10043 -l5-Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspen~ions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lipophilic ~olvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. A~ueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
Optionally, the suspenQion may also contain stabilizers.
Additionally, the compounds of the present invention may al~o be administered encapsulated in liposomes,-wherein the active ingredient is contained either di6persed 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 i8 generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids ~uch as lecithin and sphingomycelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, andjor 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:
SUBSTITUTE SHEEl~
WO93~11148 2 1 2 2 ~ 7 ~ PCT/US92/1~043 O = ~- o -X, wherein X is:
,R2 ~R2 -Rl-N -R3, or -Rl-N-R5, wherein Rl, R2, and R3 are as hereinabove described, and R5 i 8 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 em~loye~
include, but are not limited to, biotin and derivatives thereof (such as, for example, e-aminocaproyl biotin, and biotin amidocaproyl hydrazlde), fluorescein (including derivatives such as fluorescein amine), rhodamine, alkaline phosphatase, horseradish peroxidase, and 2, 4-dinitrophenyl markers. Such oligonucleotides which include a de~ectable marker may be used as DNA or RNA probes. The probes may be used a~ diagnostics as known in the art.
The invention will now be described with respect to the fo~lowing example~; however, the scope of the preRent invention i8 not intended to be limited thereby.
Exam~le 1 Production of the pyridinium salt of Dhthalimidomethvl Dhos~ gnic ~
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 :
2 1 ~1 ~47 0 ``
filtered through a sintered glass funnel and the eluant was treated with H20. The resulting mixture was concentrated under high vacuum and the residue remaining was dissolved in methylene chloride. Upon addition of ethyl a~etate, the desired product was precipitated out. The precipitate was collected, washed with ethyl acetate, and dried over P205 to yield 1.2g of pure material.
ExamPle 2 PreDaration of the Triethylaminonium Salt of Phthalimidomethyl PhosDhonate .- Dimethyl phthalimidomethyl phosphonate (2.0 ~, 1.4 mmole) was di~solved in chloroform (15 ml), and bromotrimethyl~ilane (2 ml, 15 mmol) wa~ added dropwise to `
the solution. After 2 hrs. the reaction mixture was concentrated under reduced pre~sure, and the residue was dissolved in chloroform (8 ml) followed by dropwisé addition of triethylamine (20 ml) with cooling in ice bath. After stirring at room temperature for 2 hrs. the mixtuxe 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 P205 to yield 2.1 g (65X) of pure pthalimidomethyl pho~phonate, triethylammonium salt.
ExamDle 3 PreDaration of 5'-dimethoxvtritYl-thvmidine-3' -DhthalimidomethYl PhosPhonate SVBSTtTUTE SHEET
The triethylammonium salt of phthalimidomethylphosphonate (1.7 g, 5 mmol) was dried by coevaporation with pyridine (3 x l0 ml), dissolved in dry pyridine (40 ml) and treated with triisopropylbenzenesulfonyl chloride (3.0 g, 9.9 mmol) followed by a solution of 5'-0-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 pre~ure. The re~idue was purified by silica gel column chromatography u~ing CH2Cl2/MeOH~Et3N (30:l:0.3, 2.8 L followed by 30:2:0.3, l.l L) as solvent. The appropriate fractions were colIected and combined to yield 1.8 ~ (57%) of a pure compoOu~d having the following structure:
o~ . 6 o~
- o (wherein Bl is thymine) as a white foam.
ExamDle 4 SYnthesis of an aminomethYl d nucleotide A commercially available nucleoside attached to a controlled pore gla~s (CPG) support, and having the following structural formula:
D~r- O ~o~l 2 ~_ O
~h/f~- CP~
SuE3sTlTuT~
W093/11148 2 1 2 2 ~ 7 0 PCT/US92~10043 (wherein B2 is a protected or unprotected purine or pyrimidine base) was treated with 3% dichloroacetic acid to remove the dimethoxytrityl (DMT) protectinq group, and then reacted with the phthalimidomethyl nucleoside phosphonate (6) of Example 3 in the presence of trii~opropyl-3-nitro-1,2,4-triazole as coupling agent and 1-methylimidazole as catalyst in dry acetonitrile for 15 minutes to give a phthalimidomethyl dinucleotide having the following structural ~ormula: ~
O
p_ o ~!?
O
C~
The protected dinucleotide was then treated with dichloroacetic acid to remove the dimethoxytrityl protecting group, and then treated with ammonium hydroxide at 55C to remove the phthalimido group and cleave the dinucleotide from the solid CPG ~upport to gi~e an aminomethyl dimer having the following ~tructural formula 8, wherein each of Bl and B2 i~ an unprotected purine or pyrimidine base.
t~o 0'~
0~
Example 5 SYnthesis of a 3' aminomethYl end SUBSTITUTE. SHEEl' ~ -20-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 55C and then freeze dried in vacuo. The crude oligonucleotide is con~erted 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 a~ an eluant to give the aminomethyl 3' end-capped oligonucleotide.
Example 6 Preparation of 5-'0-dimethoxYtritvl-thYmidYl-3'-Dhthalimidomethvl-DhosPhonvl-5'-thvmidine, mixed isomers 5'-0-dimethoxytritylthymidine-3'-phthalimidomethylpho phona-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 1-(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, di~ olved in pyridine (15 ml) and added to the solution of 5'-0-dimethyoxytritylthymidine-3'-phthalimidomethylphosphon-~te. The reaction mixture was stirred at room temperature under a dry nltrogen atmosphere for 2-3 hrs., then diluted with aqueous ~odium 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-SUBSTITUTE SHEEl' .
W093/11148 ~1 2 2 ~ 7 ~ PCT/US92/10043 thymidyl-3'-phthalimidomethyl-phosphonyl-5'-thymidine, 1.5 g (65%).
Example 7 SeParation of i~omers of S'0-dimethox~tritvlthvmidvl-3'-Dhthalimidomethvlpho~phonyl-5'-thvmidine by HPLC
~ The mixture of isomers of 5'0-dimethoxytritylthymidyl-3'-phthalimidomethylphosphonyl-S'-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 C4 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 P205. This procedure yielded 80 mg of a faster isomer and 110 mg of a slower isomer, total yield 83%. Analysis of the composites by analyti~al HPLC using a rever~ed phase C*
column (Radial Pak cartridge, 8x 100 mmh 15 um, 300 A) indicated that pure isomers were obtained in each case.
ExamPle 8 SeDaration of 5'0-dimethoxvtritvlthvmidvl-3'-Dthalimido-methviDhocDbonvl -5'-thymi~ne~ bv ~ilica column chromato~raphY
The residue from a 1.72 g preparation of mixed isomers of 5'-0-dimethoxytritylthymidyl-3'phthalimidomethylphosphonyl--5'-thymidine was purified by column chromatography on silica gel (lOOg) using CH2Cl~/CH30H/Et3N (30:1:0.3) as the solvent.
SUE3~;TIT(JT~ FT
W093/1l148 212 2 4 7 0 PCT/US92/10043..
^ -22-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 P205 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.
ExamDle 9 S~nthe~is of isomers of S'-0-dimethoxYtritylth~midYl-3' DhthalimidomethvlDhosphonyl-5'-thymidine -3'-cYanoethYl-N, N-diisoproDYlaminoDhosPhoramidite.
A sample of the faster iso~er of 5'-0-dimethoxytritylthymidyl -3'phthalimidomethylphosphonyl-5'-thymidine (O.g2 g,~0.42 mmole) was i8 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 ~odium bicarbonate and acetonitrile (S0 ml of each). The organic layer was washed with water (2 x S0 ml) and concentrated in vacuo to a gum. The crude product were purified by column chromatography on silica gel (40g) using CH2C12/MeOH/Et3N
(100:2:1). The appropriate fractions were combined and evaporated to yield 0.36 g (71%) of the faster isomer of 5'-0-dimethoxytritylthy-midyl-3'-phthalimidomethylphosphonyl-S'-thymidine-3'-cyanoe-thyl-N,N-diisopropylaminophosphoramidite.
An~identical procedure was followed to produce a phosphoramidite.from the slower isomer.
Exam~le 10 Procedures for oliqonucleotide s~nthesis and deProtection WO93/1114X 212 ~ ~ 7 0 PCT/USg2/10043 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 ynthesizer, 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 i8 treated with concentrated ammonia for 20 min, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described ~elow.
This procedure produces a twelve base oligonucleotide with a Yingle isomer aminomethyl phosphonate moiety at the 5'terminus.
b) Synthesis of a tridecanucleotide with an alternating single i80~er uoinomethyl phosphonate/phosphodie~ter backbone.
A thymine-containing tridecanucleotide with an alternating, single isomer aminomethyl phosphonate/phosphodiester backbone i~ prepared on a 1 umole scale u~ing an Applied Biosystem Model 394 DNA synthesizer, using a standard phosphoramidite cycle with either the fa~ter or slower i~omer of Example 9 as the phosphoramidite.
Coupling time~ 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, lyaphilized to dryness and purified as described below.
This procedure produces a thirteen base oligonucleotide with single i~omer aminomethyl phosphonate moieties alternating with pbosphodiesters throughout the sequence.
SUE~STITUTE SHEET
W093/1l148 212 2 4 7 0 PCT/US92/10043 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
~olution of either the fa~ter 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 dryne~s and purified as described below.
d) General procedure for oligonucleotide purification by E~PLC ' The oligonucleotide possessing a 5'-0-dimethoxytrityl ~roup was purified by reverse phase HPLC (C4 Radial Pak Cartridge, 100 x 25 mm, l5u, 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 O.1 M TEAA/aceton~trile, 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 oliaonucleotide S(IBSTITUTE SHE~T
W043/l1148 21 2 2 ~. 7 0 PCT/US92/10043 .
The 3'-aminomethyl end capped oligonucleotide of Example 5 i~ placed in aqueous sodium bicarbonate buffer, pH
8. This solution i8 then treated with a solution of bi~tin N-hydroxysuccinimide ester (50 equivalents) in dimethylsulfoxide for 18 hours at room temperature. The resulting solution i~ passed through a Sephadex G25 column to remove the bio~in and other ~mall molecules and the fractions containing the olîgonucleotide are concentrated and purified by high performance liquid chromatography using a C18 rever~ed pha~e silica column. The appropriate fractions are collected and evaporated to drynes~ to give the biotinylated 3'-aminomethyl end capped oligonucleotide.
Advantages of the present invention include improved olubility o the positively charged oligonucleotides in agueou~ ~olution~ as compared with nonionic oligonucleotides, improved uptake into the cell a~ compared with natural oligonucleotides which are negatively charged and are poorly taken up by the cell, and re~istance to degradation by nucleases a~ compared with natural oligonucleotides which are readily degraded by cellular enzymes. By virtue of their positively-charged regions, the oligonucleotide~ of the present invention are ta~en up by the cell more readily and are less readily degraded because of their modified backbone~. In the ca~e of oligonucleotides having aminomethyl phosphonate moieties, the cationic groups are ~maller and therefore les~ likely to disrupt base pairing than previously synthesized cationic oligonucleotides. Also, the carbon-phosphorus bonds are more ~table than nitrogen-phosphorus bonds of other cationic oligonucleotides, and thus the oligonucleotides of the present invention are less likely to lo~e the cationic group by chemical or enzymatic hydrolysis.
~ minomethyl oligonucleotides bearing detectable markers such as reporter groups have the advantage that the reporter C~ I~'rtT~ ITr ~J~_ .
WO93~11148 - PCT/US92/10043 2122~70 -26-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 preqent invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and ~till be within the scope of the accompanying claims.
! :UBSTITUTE~ S~EET
Claims (36)
1. An oligonucleotide wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the following structural formula:
wherein X is:
, and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different.
wherein X is:
, and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different.
2. The oligonucleotide of Claim 1 wherein R1 is alkylene, phenylene, or naphthylene.
3. The oligonucleotide of Claim 2 wherein R1 is an alkylene group having from 1 to about 15 carbon atoms.
4. The oligonucleotide of Claim 3 wherein R1 is methylene.
5. The oligonucleotide of Claim 4 wherein each of R2, R3, and R4 is hydrogen.
6. The oligonucleotide of Claim 1 wherein the oligonucleotide is a deoxyribonucleotide.
7. The oligonucleotide of Claim 1 wherein the oligonucleotide is a ribonucleotide.
8. A composition for binding to an RNA, or DNA, a protein, or a peptide, comprising:
(a) an oligonucleotide, wherein the oligonucleotide is a ribonucleotide or deoxyribonucleotide, and wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the following structural formula:
wherein X is:
and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different; and (b) an acceptable pharmaceutical carrier, wherein said oligonucleotide is present in an effective binding amount to an RNA, a DNA, a protein, or peptide.
(a) an oligonucleotide, wherein the oligonucleotide is a ribonucleotide or deoxyribonucleotide, and wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the following structural formula:
wherein X is:
and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different; and (b) an acceptable pharmaceutical carrier, wherein said oligonucleotide is present in an effective binding amount to an RNA, a DNA, a protein, or peptide.
9. The composition of Claim 8 wherein R1 is alkylene, phenylene or naphthylene.
10. The composition of Claim 9 wherein R1 is an alkylene group having from 1 to about 15 carbon atoms.
11. The composition of Claim 10 wherein R1 is methylene.
12. The composition of Claim 11 wherein each of R2, R3, and R4 is hydrogen.
13. The composition of Claim 8 wherein the oligonucleotide is a deoxyribonucleotide.
14. The composition of Claim 8 wherein the oligonucleotide is a ribonucleotide.
15. In a process wherein an oligonucleotide is administered for binding to an RNA, a DNA, a protein, or a peptide, the improvement comprising:
administering to a host an effective binding amount of an oligonucleotide, wherein the oligonucleotide is a ribonucleotide or deoxyribonucleotide, and wherein at least one nucleotide unit of the oligonucleotide incldues a phosphonate moiety having the following structural formula:
wherein X is:
, and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different.
administering to a host an effective binding amount of an oligonucleotide, wherein the oligonucleotide is a ribonucleotide or deoxyribonucleotide, and wherein at least one nucleotide unit of the oligonucleotide incldues a phosphonate moiety having the following structural formula:
wherein X is:
, and wherein R1 is a hydrocarbon, and each of R2, R3 and R4 is hydrogen or a hydrocarbon, and each of R2, R3, and R4 may be the same or different.
16. The process of Claim 15 wherein R1 is alkylene, phenylene or naphthylene.
17. The process of Claim 16 wherein R1 is an alkylene group having from 1 to about 15 carbon atoms.
18. The process of Claim 17 wherein R1 is methylene.
19. The process of Claim 18 wherein each of R2, R3, and R4 is hydrogen.
20. The process of Claim 15 wherein the oligonucleotide is a deoxyribonucleotide.
21. The process of Claim 15 wherein the oligonucleotide is a ribonucleotide.
22. An oligonucleotide, wherein the oligonucleotide is a ribonucleotide or deoxyribonucleotide, and wherein at least one of the nucleotide units of the oligonucleotide includes a phosphonate moiety having the following structural formula:
wherein X is:
or wherein R1 is a hydrocarbon, R2 is hydrogen or a hydrocarbon, R3 is hydrogen or a hydrocarbon, and each of R2 and R3 may be the same or different, and R5 is a detectable marker.
wherein X is:
or wherein R1 is a hydrocarbon, R2 is hydrogen or a hydrocarbon, R3 is hydrogen or a hydrocarbon, and each of R2 and R3 may be the same or different, and R5 is a detectable marker.
23. The oligonucleotide of Claim 22 wherein X is:
.
.
24. The oligonucleotide of Claim 23 wherein R1 is alkylene, phenylene, or naphthylene.
25. The oligonucleotide of Claim 24 wherein R1 is an alkylene group having from 1 to about 15 carbon atoms.
26. The oligonucleotide of Claim 25 wherein R1 is methylene.
27. The oligonucleotide of Claim 26 wherein each of R2 and R3 is hydrogen.
28. The oligonucleotide of Claim 23 wherein R5 is selected from the group consisting of colorimetric markers, fluorescent markers, luminescent markers, radioactive markers, enzyme markers, and ligand recognition reporter groups.
29. The oligonucleotide of Claim 22 wherein X is:
.
.
30. The oligonucleotide of Claim 29 wherein R1 is alkylene, phenylene, or naphthylene.
31. The oligonucleotide of Claim 30 wherein R1 is an alkylene group having from 1 to about 15 carbon atoms.
32. The oligonucleotide of Claim 31 wherein R1 is methylene.
33. The oligonucleotide of Claim 32 wherein R2 is hydrogen.
34. The oligonucleotide of Claim 29 wherein R5 is selected from the group consisting of colorimetric markers, fluorescent markers, luminescent markers, radioactive markers, enzyme markers, and ligand recognition reporter groups.
35. The oligonucleotide of Claim 22 wherein the oligonucleotide is a deoxyribonucleotide.
36. The oligonucleotide of Claim 22 wherein the oligonucleotide is a ribonucleotide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79680491A | 1991-11-25 | 1991-11-25 | |
| US796,804 | 1991-11-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2122470A1 true CA2122470A1 (en) | 1993-06-10 |
Family
ID=25169100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2122470 Abandoned CA2122470A1 (en) | 1991-11-25 | 1992-11-20 | Oligonucleotides having aminohydrocarbon phosphonate moieties |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0641354A4 (en) |
| JP (1) | JPH07501542A (en) |
| AU (1) | AU3144593A (en) |
| CA (1) | CA2122470A1 (en) |
| WO (1) | WO1993011148A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6017700A (en) * | 1995-08-04 | 2000-01-25 | Bayer Corporation | Cationic oligonucleotides, and related methods of synthesis and use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
1992
- 1992-11-20 EP EP92925362A patent/EP0641354A4/en not_active Withdrawn
- 1992-11-20 CA CA 2122470 patent/CA2122470A1/en not_active Abandoned
- 1992-11-20 JP JP5510176A patent/JPH07501542A/en active Pending
- 1992-11-20 WO PCT/US1992/010043 patent/WO1993011148A1/en not_active Application Discontinuation
- 1992-11-20 AU AU31445/93A patent/AU3144593A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0641354A4 (en) | 1996-01-10 |
| JPH07501542A (en) | 1995-02-16 |
| AU3144593A (en) | 1993-06-28 |
| WO1993011148A1 (en) | 1993-06-10 |
| EP0641354A1 (en) | 1995-03-08 |
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