WO1995031572A1 - Aminohydrocarbon phosphonate oligonucleotides and uses therefor - Google Patents

Abstract

A method for modulating the activity of a protein or nucleic acid in vivo by binding it with an oligonucleotide wherein at least one nucleotide unit includes an aminohydrocarbon phosphonate moiety having formula (I), wherein X is (II), and wherein R1 is a hydrocarbon, preferably alkylene, and R2, R3 and R4 are each independently hydrogen or a hydrocarbon. Preferably, R1 is methyl, ethyl or propyl moiety. It is also preferred to use the pure stereoisomers of such compounds rather than their racemic mixtures. Such pure stereoisomers having improved binding capabilities and improved resistance to nucleases. Alternatively, X may be (III), wherein R1, R2, and R3 are as hereinabove described, and R5 is a detectable marker, thus making such oligonucleotides useful as diagnostic probes or for screening libraries of cells.

Description

AMINOHYDROCARBON PHOSPHONATE OLIGONUCLEOTIDES

AND USES THEREFOR

This invention relates to oligonucleotides which bind to RNA (such as mRNA), DNA, proteins, or peptides, including, for example, oligonucleotides which inhibit mRNA function. More particularly, this invention relates to oligonucleotides in which one or more of the

nucleotides include an aminohydrocarbon phosphonate moiety.

Watson-Crick base pairing enables an oligonucleotide to act as an antisense complement to a target sequence of an mRNA in order to block processing or effect

translation arrest and regulate selectively gene

expression. (Cohen, Oligodeoxynucleotides, CRC Press, Boca Raton, Florida (1989)); Uhlmann et al . , Chem. Rev. 90:543-584 (1990)). Oligonucleotides have also been utilized to interfere with gene expression directly at the DNA level by formation of triple-helical (triplex) structures in part through Hoogsteen bonding interactions (Moffat, Science, 252:1374-1375 (1991)). Furthermore, oligonucleotides have been shown to bind specifically to proteins (Oliphant et al . , Molec. Cell . Biol . 9:2944-2949 (1989)) and could thus be used to block undesirable protein function.

Natural oligonucleotides, which are negatively charged, however, are poor candidates for therapeutic agents due to their poor penetrability into the cell and their susceptibility to degradation by nucleases.

Therefore, it is expected that relatively high

concentrations of natural oligonucleotides would be required in order to achieve a therapeutic effect.

To overcome the above shortcomings, various

strategies have been devised. U.S. Patent No. 4,469,863, issued to Miller et al ., discloses the manufacture of nonionic nucleic acid alkyl and aryl phosphonates, and in particular nonionic nucleic acid methyl phosphonates. U.S. Patent No. 4,757,055, also issued to Miller et al . , discloses a method for selectively controlling unwanted expression of foreign nucleic acid in an animal or in mammalian cells by binding the nucleic acid with a nonionic oligonucleotide alkyl or aryl phosphonate analogue.

Oligonucleotides have also been synthesized in which one non-bridging oxygen in each phosphodiester moiety is replaced by sulfur. Such analogues sometimes are

referred to as phosphorothioate (PS) analogues, or "all PS" analogues, (Stein et al . , Nucl . Acids Res . 16:3209-3221 (1988)). Another approach has been to attach a targeting moiety, such as cholesterol, which improves the uptake of the oligonucleotide by a receptor-mediated process. (Stein et al . , Biochemistry, 30:2439-2444

(1991)). Examples of oligonucleotides with positive charges have been reported. Letsinger et al . JACS, 110:4470-4471 (1988) describe cationic oligonucleotides in which the backbone is modified by the attachment of diamino

compounds to give positively charged oligonucleotides with phosphoramidate linkages. Phosphoramidate linkages, however, are known to be somewhat labile, especially at 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 . , 84:648-652 (1987), and have been shown to be more active in cell culture than unmodified oligonucleotides.

Polylysine, however, is not a preferred molecule for conjugation due to its relatively high 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, 1:85-96 (1974) disclose the synthesis of uridine-2' (3')-aminomethyl phosphonate and thymidine -3'-aminomethyl phosphonate by the reaction of the

corresponding 5'-0-trityl nucleoside with N-benzyloxycarbonyl-aminomethyl phosphonate. Gulyaev et al . , FEBS Letters, 22:294-296 (1972) disclose the

formation of ribonucleoside 5'-aminomethyl phosphonates.

An alternate approach to the synthesis of backbone-modified cationic substituents is to prepare

(aminoalkyl)phosphonates in which the anionic oxygen is replaced by a cationic group via a P-C bond. The

synthesis of (aminomethyl)phosphonates has been reported. See Fathi et al . (1994) Bioconjugate Chem., 5:47-57.

During the course of this prior work, it became evident that although (aminomethyl)phosphonates possessed interesting properties, they were shown to be somewhat unstable in aqueous solution.

Japanese Patent Applications Kokai 61-44,353(1986) and 61-57,596(1986) disclose polynucleotides which are labeled with a fluorescent marker or enzyme label, respectively, bound through an aminoalkyl bridge to a phosphonate, and which are used for identification and extraction of target genes. Each describes the synthesis of a decamer having aminoethylphosphonate groups.

In order to identify more stable derivatives for genetic manipulation and therapeutic applications, other representatives and preparations of this class of compounds were therefore evaluated and the present invention relates to certain of such compounds and their advantageous properties for certain uses.

During the course of arriving at the present invention aminoalkylphosphonate oligonucleotides have been prepared and their physicochemical, enzymatic and biological properties have been studied. The individual R and S stereoisomers of the protected dimers have been isolated, characterized and incorporated into

oligonucleotides such as those in which the backbone consists of alternating aminoalkylphosphonate and

phosphodiester nucleotide units. The R isomer of the 2-aminoethyl phosphonate oligonucleotide forms a hybrid with its complementary sequence which is more stable than either its corresponding aminomethyl derivative, the aminoethylphosphonate racemic mixture or the

corresponding natural counterpart. The specificity of hybridization was examined by hybridization of an

oligonucleotide containing one (2-aminoethyl)phosphonate to partially complementary oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing

(aminomethyl)phosphonates, oligonucleotide (2-aminoethyl)-phosphonates are completely stable in aqueous solution. These oligonucleotides are stable towards enzymatic degradation but do not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in serum-containing media resulted in minimal degradation over 24 hours.

Advantages of the present invention include improved solubility of the positively charged oligonucleotides in aqueous solutions as compared with nonionic

oligonucleotides, improved uptake into the cell as compared with natural oligonucleotides which are

negatively charged and are poorly taken up by the cell, and resistance to degradation by nucleases as compared with natural oligonucleotides which are readily degraded by cellular enzymes. By virtue of their positively-charged regions, the oligonucleotides of the present invention are taken up by the cell more readily and are less readily degraded because of their modified

backbones. In the case of oligonucleotides having aminomethyl phosphonate moieties, the cationic groups are smaller and therefore less likely to disrupt base pairing than previously synthesized cationic oligonucleotides. Also, the carbon-phosphorus bonds are more stable than nitrogen-phosphorus bonds of other cationic

oligonucleotides, and thus the oligonucleotides of the present invention are less likely to lose the cationic group by chemical or enzymatic hydrolysis.

Figure 1. Synthesis of mononucleotide (2-aminoethyl)phosphonates.

Figure 2. Dinucleotide (2-aminoethyl)phosphonates.

Figure 3. Oligonucleotide (2-aminoethyl)phosphonates. Figure 4. Dinucleotide (3-aminopropyl)phosphonates. Figure 5. Synthesis of monomer phthalimidoethyl

phosphonamidites.

In one aspect, the invention provides a method for modulating the activity of a target molecule, such as a protein, carbohydrate or nucleic acid, which comprises contacting a cell in which the target molecule activity is to be modulated with an oligonucleotide wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the formula:

wherein X is:

and wherein R₁ is a hydrocarbon, and each of R₂, R₃ and R₄ is independently hydrogen or a

hydrocarbon. Examples of protein target molecules include cytokine or can be selected from the group consisting of basic fibroblast growth factor, gamma interferon and gp30 glycoprotein.

In another aspect, the invention provides a method for selectively regulating gene expression which comprises contacting a cell in which the expression of a selected gene is to be regulated with a complementary oligonucleotide having at least one nucleotide unit that includes a phosphonate moiety with the formula: wherein X is

and wherein R₁ is a hydrocarbon, and each of R₂, R₃ and R₄ is independently hydrogen or a hydrocarbon.

In accordance with another aspect of the present invention, there is provided an oligonucleotide

composition wherein at least one nucleotide unit includes a single isolated isomeric form of a phosphonate moiety having the formula:

wherein X is:

R₁ is a hydrocarbon, preferably alkylene, phenylene, or naphthylene, more preferably an alkylene group having from 1 to 15 carbon atoms, and most preferably 1 to about 5 carbon atoms, with methylene, ethylene and propylene being preferred. R₁ can also be selected from the groups consisting of vinylene, substituted vinylene, ethynylene, allyl and substituted allyl. The substituted vinylene and substituted allyl are preferably substituted with methyl or ethyl moieties. Each of R₂, R₃, and R₄ is independently hydrogen or a hydrocarbon. Preferably, the hydrocarbon is an alkyl group having from 1 to 15 carbon atoms, more preferably from 1 to 3 carbon atoms, and most preferably a methyl group. Most preferably, each of R₂, R₃, and R₄ is hydrogen.

The oligonucleotide can also be conjugated to at least one moiety that enhances its uptake into cells. Such moieties can, for example, be polyethylene glycol, polylysine, peptides, acridine, dodecanol, cholesterol, biotin, folate, glutathione and mannose-6-phosphate.

The term "oligonucleotide", as used herein, means that the oligonucleotide may be a ribonucleotide or a deoxyribonucleotide; i.e., the oligonucleotide may include ribose or deoxyribose sugars. Alternatively, the oligonucleotide may include other 5-carbon or 6-carbon sugars, such as, for example, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof.

In general, the oligonucleotide has at least two nucleotide units, preferably at least five, more

preferably from five to about 30 nucleotide units.

As hereinabove stated, at least one nucleotide unit of the oligonucleotide includes a phosphonate moiety which is an aminohydrocarbon phosphonate moiety, as hereinabove described. An aminohydrocarbon phosphonate moiety may be attached to one or more nucleotide units at the 3' end and/or at the 5' end of the oligonucleotide. In one embodiment, an aminohydrocarbon phosphonate moiety may be attached to alternating nucleotide units of the oligonucleotide. In another embodiment, an

aminohydrocarbon phosphonate moiety may be attached to each nucleotide unit of the oligonucleotide.

The oligonucleotides also include any natural or unnatural, substituted or unsubstituted, purine or pyrimidine base. Such purine and pyrimidine bases include, but are not limited to, natural purines and pyrimidines such as adenine, cytosine, thymine, guanine, uracil, or other purines and pyrimidines, such as

isocytosine, 6-methyluracil, 4,6-dihydroxypyrimidine, hypoxanthine, xanthine, 2, 6-diamino purine, azacytosine, 5-methyl cytosine, and the like.

In a most preferred embodiment, X is an aminomethyl, aminoethyl or aminopropyl moiety, particularly the stereoisomeric isolates thereof. The synthesis of an oligonucleotide having such aminoethyl or aminopropyl phosphonate moieties may be accomplished through the synthesis of a monomer unit with a protected aminomethyl, aminoethyl or aminopropyl functional group, followed by incorporation of one or more such monomer units into an oligonucleotide; or by synthesis of an oligonucleotide followed by subsequent attachment of the aminomethyl, aminoethyl or aminopropyl groups.

Monomer units which may be incorporated into an oligonucleotide, may, in one embodiment, be prepared as follows.

Aminomethyl phosphonic acid may be reacted with a suitable reagent, such as trifluoroacetic anhydride, fluorenyloxycarbonylchloride, or phthalyl chloride to protect the amino group, and to give one of the following protected derivatives 1, 2 or 3:

Alternatively, the phthalimide derivative 1 may be prepared by reaction of chloromethyl phosphonic acid with phthalimide, or by demethylation of commercially available dimethylphthalimidomethyl phosphonate using trimethylsilyl bromide.

Hydroxymethyl phosphonic acid can also be used as a starting material for the synthesis of aminomethyl phosphonate derivatives. The reaction of hydroxymethyl phosphonic acid with trifluoroacetic anhydride produces an ester which can be converted into a pyridinium intermediate, the reaction of which with ammonia produces aminomethyl phosphonic acid.

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

wherein B is a protected or unprotected purine or

pyrimidine base, in the presence of a condensing agent such as dicyclohexylcarbodiimide or triisopropylbenzenesulfonyl chloride would produce an ester having the formula 5:

wherein Q is the protected amino group.

Preferably, the protected amino group is selected from the group consisting of:

The ester having formula 5 can be used as a monomer unit for oligonucleotide synthesis by coupling to a protected mononucleotide or oligonucleotide attached to a solid support. After the solid support-attached

oligonucleotide is synthesized, the material is treated with ethylenediamine to cleave the protecting groups and generate an oligonucleotide having one or more

aminomethyl phosphonate moieties. Alternatively, the phthalimide protecting group can be removed by treatment with hydrazine or a substituted hydrazine to generate the aminomethyl compound. By this rewve, the aminomethyl modified units can be introduced at any position in the oligonucleotide as desired.

Alternatively, a modified mononucleotide may be prepared by reacting a partially protected nucleoside such as hereinabove described with a protected aminomethyl phosphite derivative to form a nucleoside phosphonamidite. The nucleoside phosphonamidite can then be used in place of a nucleoside phosphoramidite in a DNA synthesizer. At the conclusion of the synthesis, the protecting groups can be removed from the aminomethyl moieties by treatment with ammonia or with amines such as ethylenediamine.

Aminomethyl phosphonate moieties can also be introduced into preformed oligonucleotides. One approach is to carry out a synthesis of an oligonucleotide on a solid support using a DNA synthesizer, except that the iodine oxidation step which is normally used to oxidize the phosphite intermediate to a phosphate is eliminated, and instead the oligonucleotide phosphite attached to the solid support is reacted with phthalimidomethyl bromide. Subsequent treatment with ethylenediamine removes the phthalimido protecting group to give the aminomethyl oligonucleotide.

In another embodiment, some oligonucleotides in accordance with the present invention may be prepared such that the oligonucleotides may be isolated as pure stereoisomers in either the R- or S- form. Such

oligonucleotides include those with one aminohydrocarbon phosphonate moiety at, or adjacent to, either the 3'-terminus or the 5'-terminus; oligonucleotides having aminohydrocarbon phosphonate moieties at both the 3'- and 5'-termini; oligonucleotides having aminohydrocarbon phosphonate moieties at internal positions,

oligonucleotides in which aminohydrocarbon phosphonate moieties alternate with natural phosphodiester linkages throughout the entire sequence; and oligonucleotides possessing a mixture of aminohydrocarbon phosphonate and other modified backbone substituents, such as

phosphorothioates. Surprisingly, oligonucleotides with alternating aminomethylphosphonate and natural phosphate moieties are internalized into cells more readily than their ethyl counterparts. Oligonucleotides derivatized with

alternating sequences of either the aminomethyl or the aminoethyl phosphonate and natural phosphate moieties hybridize better to DNA than does natural DNA, and further, they selectively bind better to DNA than to RNA, thereby having the potential for enhanced specificity for direct gene regulation at the chromosomal level.

Although the aminomethyl derivatives are internalized into the cell more readily, the aminoethyl derivatives hybridize somewhat better than the aminomethyl

derivatives.

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 conventional means, such as high pressure liquid chromatography or silica gel column chromatography. The pure isomers may then be incorporated into

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 further improves the binding capabilities of the oligonucleotide and/or increases the resistance of the oligonucleotide to degradation by nucleases.

For example, the aminoalkylphosphonates cannot be degraded by certain DNA-cleaving enzymes, such as SI nuclease and mung bean nuclease, which readily cleave natural DNA sequences. The oligonucleotides may include conjugate groups attached to the 3' or 5' termini to improve further the uptake of the oligonucleotide into the cell, the

stability of the oligonucleotide inside the cell, or both. Such conjugates include, but are not limited to, polyethylene glycol, polylysine, acridine, dodecanol, and cholesterol.

The oligonucleotides of the present invention may be employed to bind to RNA sequences by Watson-Crick hybridization, and thereby block RNA processing or translation. For example, the oligonucleotides of the present invention may be employed as "antisense"

complements to target sequences of mRNA in order to effect translation arrest and regulate selectively gene expression.

The oligonucleotides of the present invention may be employed to bind double-stranded DNA to form triplexes, or triple helices. Such triplexes inhibit the

replication or transcription of DNA, thereby disrupting DNA synthesis or gene transcription, respectively. Such triplexes may also protect DNA binding sites from the action of enzymes such as DNA methylases.

The RNA or DNA of interest, to which the

oligonucleotide binds, may be present in a prokaryotic cell, eukaryotic cell or a virus. The sequences may be bacterial sequences, plasmid sequences, viral sequences, chromosomal sequences, mitochondrial sequences, or plastid sequences. The sequences may include open reading frames for coding proteins, mRNA, ribosomal RNA, snRNA, hnRNA, introns, or untranslated 5'- and 3'-sequences flanking open reading frames. The target sequence may therefore be involved in inhibiting

production of a particular protein, enhancing the expression of a particular gene by inhibiting the expression of a repressor, or the sequences may be involved in reducing the proliferation cf viruses or unwanted cells.

The oligonucleotides may be used in vitro or in vivo for modifying the phenotype of cells, or for limiting the proliferation of pathogens such as viruses, bacteria, protists, Mycoplasma species, Chlamydia or the like, or for inducing morbidity in specific classes of cells.

Thus, the oligonucleotides may be administered to a host subject to or in a diseased state, to inhibit the transcription and/or expression of the native genes of a target cell. Therefore, the oligonucleotides may be used for protection from a variety of pathogens in a host, such as, for example, enterotoxigenic bacteria,

Pneumococci , Neisseria organisms, Giardia organisms, Entamoebas; specific B-cells; specific 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 processing, inhibition of transport across the nuclear membrane, cleavage by endonucleases, or the like.

The oligonucleotides may be complementary to such sequences as sequences expressing growth factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNA polymerases, antibiotic resistance, multiple drug resistance (mdr), genes involved with metabolic processes, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flanking sequences associated with the open reading frames.

The oligonucleotides of the present invention may be employed for binding to target molecules, such as, for example, proteins including, but not limited to, ligands, receptors, and/or enzymes, whereby such oligonucleotides inhibit or stimulate the activity of the target

molecules. The above techniques in which the

oligonucleotides may be employed are also applicable to the inhibition of viral replication.

The oligonucleotides of the present invention are administered in an effective binding amount to an RNA, a DNA, a protein or a peptide. Preferably, the

oligonucleotides are administered to a host, such as a human or non-human animal host, so as to produce a concentration of oligonucleotide in the blood of from about 0.1 to about 100 μmole/l. It is also contemplated, however, that the oligonucleotides may be administered in vi tro or ex vivo as well as in vivo.

The oligonucleotides may be administered in

conjunction with an acceptable pharmaceutical carrier as a pharmaceutical composition. Such pharmaceutical compositions may contain suitable excipients and

auxiliaries which facilitate processing of the active compounds into preparations which can be used

pharmaceutically. Such oligonucleotides may be

administered by intramuscular, intraperitoneal,

intraveneous or subdermal injection in a suitable

solution. The preparations, particularly those which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees and capsules, and preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration parenterally or orally, and compositions which can be administered bucally or sublingually, including inclusion compounds, contain from about 0.1 to 99 percent by weight of active ingredients, together with the excipient. It is also contemplated that the oligonucleotides may be

administered topically.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example, the pharmaceutical preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. The process to be used will depend ultimately on the physical properties of the active ingredient used.

Suitable excipients are, in particular, fillers such as sugar, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium

phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch or paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxypropylmethylcellulose, sodium

carboxymethylcellulose, 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 pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulating agents and lubricants, such as, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as

acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses.

Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a

plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the oligonucleotide in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, it is also posible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oil injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous

injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. Optionally, the suspension may also contain stabilizers.

Additionally, the compounds of the present invention may also be administered encapsulated in liposomes, wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active ingredient, depending upon its solubility, may be present both in the aqueous layer, in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns.

Also contemplated as an aspect of the invention is a method for detecting a target protein or carbohydrate in a sample which comprises (i) contacting the sample with an oligonucleotide that selectively binds with the traget wherein at least one nucleotide unit of said

oligonucleotide includes a phosphonate moiety have the formula

wherein X is:

** and wherein R₁ is a hydrocarbon, and R₂ and R₃ are each independently hydrogen or a hydrocarbon and R₅ is

detectable marker; and

(ii) observing any detectable response.

It is also contemplated that the stereoisomerically pure 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:

wherein X is:

wherein R₁, R₂, and R₃ are as hereinabove described, and R₅ is a detectable marker. Detectable markers which may be employed include, but are not limited to, colorimetric markers, fluorescent markers, enzyme markers luminescent markers, radioactive markers, or ligand recognition reporter groups. Specific examples of detectable markers which may be employed include, but are not limited to, biotin and derivatives thereof (such as, for example, e-aminocaproyl biotin, and biotin amidocaproyl hydrazide), fluorescein (including derivatives such as fluorescein amine), rhodamine, alkaline phosphatase, horseradish peroxidase, and 2, 4-dinitrophenyl markers. Such oligonucleotides which include a detectable marker may be used as DNA or RNA probes. The probes may be used as diagnostics as known in the art.

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

Oligonucleotides were synthesized using an Applied Biosystems Model 394 DNA synthesizer. 5'-O-dimethoxytritylthymidine was purchased from Peninsula Laboratories, Inc. (Belmont, CA) and unless otherwise stated all other chemicals were obtained from Aldrich Company. Ultraviolet spectra were obtained with a

Shimadzu UV 160U spectrophotometer using 1 mL quartz cuvettes. Extinction coefficients of oligonucleotides were obtained using a published procedure. See Rychlick and Rhoads (1989) Nucleic Acids Res., 17:8543-8551.

Unless otherwise stated, mono- and dinucleotides were purified using a Dynamax 60A silica column (41.4 mm × 25 cm, Rainin, Emeryville, CA). Oligonucleotide HPLC

purifications were performed using a Waters 600E system controller equipped with a multi-solvent delivery system and a model 991 photodiode array detector. Trityl-on separations were performed using a Waters RCM (8 mm × 10 cm) C₄ column for analytical purposes and a C₄ RCM (25 mm × 10 cm) column for preparative use with a gradient of 0.1 M triethylammonium acetate buffer pH 7.0

(TEAA) /acetonitrile. Oligonucleotides were converted into the sodium form using a Bio-Rad 50W-X8 resin, sodium form. NMR spectra were recorded using a Varian Unity Plus 500 MHz spectrometer. Unless otherwise stated, NMR spectra were run in DMSO-d6. ³¹P NMR were broad band decoupled, referenced to H₃PO₄as an external standard, and ¹H NMR spectra were referenced to external

tetramethylsilane as standard.

Example 1

Production of the pyridinium salt of phthalimidomethyl phosphonic acid To 2.0g (7.42 mmole) of dimethylphthalimidomethyl phosphonate, dried by coevaporation of pyridine and dissolved in 40 ml of dry pyridine, was added dropwise 2.45 ml (2.5 equivalents) of trimethylsilyl bromide under nitrogen. After 2.5 hours, the reaction mixture was filtered through a sintered glass funnel and the eluant was treated with H₂O. The resulting mixture was

concentrated under high vacuum and the residue remaining was dissolved in methylene chloride. Upon addition of ethyl acetate, the desired product was precipitated out. The precipitate was collected, washed with ethyl acetate, and dried over P₂O₅ to yield 1.2g of pure material.

Example 2

Preparation of the Triethylammonium Salt of Phthalimidomethyl Phosphonate

Dimethyl phthalimidomethyl phosphonate (2.0 g, 7.4 mmole) was dissolved in chloroform (15 ml), and

bromotrimethylsilane (2 ml, 15 mmol) was added dropwise to the solution. After 2 hrs. the reaction mixture was concentrated under reduced pressure, and the residue was dissolved in chloroform (8 ml) followed by dropwise addition of triethylamine (20 ml) with cooling in ice bath. After stirring at room temperature for 2 hrs. the mixture was filtered and concentrated to dryness. The residue was dissolved in methanol (10 ml) and then added dropwise to anhydrous diethyl ether (4 ml). The

precipitate was filtered, washed with ether and dried over P₂O₅ to yield 2.1 g (65%) of pure pthalimidomethyl phosphonate, triethylammonium salt.

Example 3

Preparation of 5'-dimethoxytrityl-thymidine-3'

-phthalimidomethyl phosphonate

The triethylammonium salt of

phthalimidomethylphosphonate (1.7 g, 5 mmol) was dried by coevaporation with pyridine (3 × 10 ml), dissolved in dry pyridine (40 ml) and treated with

triisopropylbenzenesulfonyl chloride (3.0 g, 9.9 mmol) followed by a solution of 5'-O-dimethoxytritylthymidine (2.0 g., 3.67 mmol) in dry pyridine (40 ml) which was previously dried by coevaporation with pyridine. The resulting mixture was stirred at room temperature overnight under a dry nitrogen atmosphere and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography using

CH₂Cl₂/MeOH/Et₃N (30:1:0.3, 2.8 L followed by 30:2:0.3, l.l L) as solvent. The appropriate fractions were collected and combined to yield 1.8 g (57%) of a pure compound having the following structure 6:

(wherein B₁ is thymine) as a white foam.

Example 4

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

(wherein B₂ is a protected or unprotected purine or pyrimidine base) was treated with 3% dichloroacetic acid to remove the dimethoxytrityl (DMT) protecting group, and then reacted with the phthalimidomethyl nucleoside phosphonate (6) of Example 3 in the presence of

triisopropyl-3-nitro-1,2,4-triazole as coupling agent and 1-methylimidazole as catalyst in dry acetonitrile for 15 minutes to give a phthalimidomethyl dinucleotide having structure 7: The protected dinucleotide was then treated with dichloroacetic acid to remove the dimethoxytrityl protecting group, and then treated with ethylenediamine at 55°C to remove the phthalimido group and cleave the dinucleotide from the solid CPG support to give an aminomethyl dimer having the following structure 8,

wherein each of B₁ and B₂ is an unprotected purine or pyrimidine base.

Example 5

Synthesis of a 3' aminomethyl end capped oligonucleotide

The protected phthalimido dinucleotide 7 is prepared as described in Example 4. The protected dinucleotide is then loaded into a lμmole size column, installed on an Applied Biosystems DNA synthesizer (Model #394), and synthesis of a modified oligonucleotide is performed using standard phosphoramidite chemistry. Deprotection is carried out with ethylenediamine at 55°C and then freeze dried in vacuo. The crude oligonucleotide is converted into its sodium salt form by passage of an aqueous solution through a cation exchange resin (Na⁺) using water as an eluant, and is purified by Sephadex G-25 column chromatography using water as an eluant to give the aminomethyl 3' end-capped oligonucleotide.

Example 6

Preparation of 5-'O-dimethoxytrityl-thymidyl-3'- phthalimidomethyl-phosphonyl-5'-thymidine, mixed isomers

5'-O-dimethoxytritylthymidine-3'-phthalimidomethyl-phosphonate (Example 3, 2.0 g, 2.3 mmol) was dried by coevaporation with pyridine (3 × 15 ml), redissolved in dry pyridine (80 ml) and treated with 1-(2,4,6)trimethyl- benzenesulfonyl-nitrotriazolide (0.75 g, 2.5 mmol), for 15 min. at room temp. Thymidine (0.6 g, 2.3 mmol) was dried by pyridine coevaporation in the same way, dissolved in pyridine (15 ml) and added to the solution of 5'-0-dimethyoxytritylthymidine-3'-phthalimidomethylphosphonate. The reaction mixture was stirred at room temperature under a dry nitrogen

atmosphere for 2-3 hrs., then diluted with aqueous sodium bicarbonate (5%, 300 ml) and extracted with ethyl acetate

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

Example 7

Separation of isomers of 5'O-dimethoxytritylthymidyl-3'- phthalimidomethylphosphonyl-5'-thymidine by HPLC

The mixture of isomers of 5'O-dimethoxytritylthymidyl-3'- phthalimidomethylphosphonyl-5'-thymidine from a 200 mg scale reaction was dissolved in triethylammonium acetate (0.1 M, TEAA)/ acetonitrile

(60/40, 1.5 ml) and injected into a reversed phase C₄ column, Radial Pak Cartridge (Waters RCM 25 × 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 × 50 ml), evaporated and dried in vacuo over P₂O₅. This procedure yielded 80 mg of a faster (Sp) isomer and 110 mg of a slower (Rp) isomer, total yield 83%. Analysis of the composites by

analytical HPLC using a reversed phase C4 column (Radial Pak cartridge, 8× 100 mm, 15 μm, 300 A) indicated that pure isomers were obtained in each case.

Example 8

Separation of 5'O-dimethoxytritylthymidyl-3'-phthalimido methylphosphonyl -5'-thymidine by silica column chromatography

The residue from a 1.72 g preparation of mixed isomers of 5'-O-dimethoxytritylthymidyl-3'phthalimidomethylphosphonyl-5'-thymidine was purified by column chromatography on silica gel (100g) using

CH₂Cl₂/CH₃OH/Et₃N (30:1:0.3) as the solvent. Fractions 120-126 contained the faster eluting (Sp) isomer, fractions 127-157 contained a mixture of both isomers, and fractions 158-170 contained the slower eluting (Rp) isomer. The appropriate fractions were collected, evaporated to dryness and dried in vacuo over P₂O₅ to give 0.17g of the faster eluting isomer, 0.2 g of the slower eluting isomer, and 0.7 g of a mixture of isomers.

Example 9

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

A sample of the Sp isomer of 5'-0-dimethoxytritylthymidyl -3'phthalimidomethylphosphonyl-5'-thymidine (0.42 g, 0.42 mmole) was dried by

coevaporation with pyridine, dissolved in dry

acetonitrile (10 ml) under nitrogen, and treated with stirring with cyanoethoxy- (N,N,N',N'-tetra-isopropylamino) phosphine (0.33 ml, 1.05 mmol), tetrazole (30 mg), and diisopropylamine (0.08 ml, 0.58 mmol). After 50 minutes at room temperature the mixture was partitioned between 5% aqueous sodium bicarbonate and acetonitrile (50 ml of each). The organic layer was washed with water (2 × 50 ml) and concentrated in vacuo to a gum. The crude

product were purified by column chromatography on silica gel (40g) using CH₂Cl₂/MeOH/Et₃N (100:2:1). The

appropriate fractions were combined and evaporated to yield 0.36 g (71%) of the Sp isomer of 5'-O-dimethoxytritylthymidyl-3'-phthalimidomethylphosphonyl-5'-thymidine-3'-cyanoethyl-N,N-diisopropylaminophosphoramidite.

An identical procedure was followed to produce a phosphoramidite from the slower (Rp) isomer. Example 10

Procedures for oligonucleotide synthesis and deprotection a) 5 ' -End capped oligonucleotide

A 12 base, thymine-containing oligonucleotide is prepared on a 1 umole scale using an Applied Biosystems Model 394 DNA synthesizer, with phosphoramidites and other reagents as supplied by the manufacturer. After nine coupling cycles with the commercially available monomer 5'dimethoxytritylthymidine-3'-N,N-diisopropylamino-cyanoethoxyphosphoramidite, the final cycle employed a 0.1 M solution of either the Rp or Sp isomer of the phthalimidomethyl dinucleotide

phosphoramidite of Example 9. Upon completion of the synthesis, the modified oligomer was treated with ethylenediamine at 55° for 1 hour, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described below. This procedure produced a twelve base oligonucleotide with a single isomer

aminomethyl phosphonate moiety at the 5'terminus. b) Synthesis of a tridecanucleotide with an alternating single isomer aminomethyl phosphonate/phosphodiester backbone.

A thymine-containing tridecanucleotide with an alternating, single isomer aminomethyl

phosphonate/phosphodiester backbone was prepared on a 1 umole scale using an Applied Biosystem Model 394 DNA synthesizer, using a standard phosphoramidite cycle with either the faster (Sp) or slower (Rp) isomer of Example 9 as the phosphoramidite. Coupling times of 2 min. per cycle was used. Upon completion of the synthesis, the modified oligomer is treated with ethylenediamine for 1 hour at 55°, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as

described below. This procedure produced a thirteen base

oligonucleotide with single isomer aminomethyl

phosphonate moieties alternating with phosphodiesters throughout the sequence. c) 3',5'-Aminomethyl phosphonate end-capped

oligonucleotide

A 12 base thymine-containing oligonucleotide was prepared on a 1 μmole scale using an Applied Biosystems Model 394 DNA synthesizer. The initial cycle employed a 0.1 M solution of either the faster (Sp) or slower (Rp) isomer of phthalimidomethyl phosphonate dinucleotide phosphoramidite (Example 9) which was coupled to the solid support to which a thymidine residue was attached. After nine subsequent coupling cycles with the commercial available monomer 5'-dimethoxytritylthymidine-3'-N,N-diisopropylamino-cyanoethoxyphosphoramidite, the final cycle again employed a 0.1 M solution of either the Rp or Sp isomer of phthalimidomethyl dinucleotide

phosphoramidite of Example 9. Upon completion of the synthesis, the modified oligomer was treated with

ethylenediamine for 1 hour at 55°, partially concentrated under a stream of nitrogen, lyophilized to dryness and purified as described below. d) General procedure for oligonucleotide purification by HPLC

The oligonucleotide possessing a 5'-O-dimethoxytrityl group was purified by reverse phase HPLC (C4 Radial Pak Cartridge, 100 × 25 mm, 15u, 300A). After detritylation with 0.1 M acetic acid the product was again purified by reverse phase HPLC (C4 column) using a linear gradient of 0.1 M TEAA/acetonitrile, with the concentration of acetonitrile being varied from 5 to 70%. Example 11

Synthesis of a biotinylated 3' aminomethyl oligonucleotide

The 3'-aminomethyl end capped oligonucleotide of Example 5 was placed in aqueous sodium bicarbonate buffer, pH 8. This solution was then treated with a solution of biotin N-hydroxysuccinimide ester (50 equivalents) in dimethylsulfoxide for 18 hours at room temperature. The resulting solution was passed through a Sephadex G25 column to remove the biotin and other small molecules and the fractions containing the

oligonucleotide were concentrated and purified by high performance liquid chromatography using a C18 reversed phase silica column. The appropriate fractions were collected and evaporated to dryness to give the

biotinylated 3'-aminomethyl end capped oligonucleotide.

Aminomethyl oligonucleotides bearing detectable markers such as reporter groups have the advantage that the reporter groups are on the outside of the duplex produced by hybridization to its target DNA or RNA and are therefore more accessible towards detection, and also do not interfere with the hybridization sites on the bases.

Example 12

Synthesis of Aminoethyl Phosphonates

Dimethyl (2-Phthalimidoethyl)phosphonate (10).

N-(2-Hydroxyethyl)phthalimide (11), (3.0 g, 15.7 mmol) was dissolved in CH₂Cl₂ (120 mL), cooled to -78°C in a dry ice bath and treated with dry pyridine (3 mL), followed by trifluoromethanesulfonic anhydride (3.4 mL, 20 mmol). The reaction mixture was stirred at approx. -65°C under dry nitrogen for 1.5 hours and then partitioned between hexane (300 mL)/ 2 X (100 mL) 0.5 N HCl. The organic layer was washed once with water (100 mL), followed by 5% aqueous NaHCO₃ (100 mL), water (100 mL), and brine (100 mL), and then dried over sodium sulfate. The solid was removed by filtration and the filtrate was evaporated to dryness to yield 4.14 g of the triflate as a white solid which was used directly without any further purification. ¹H NMR: δ (ppm) 7.87 (m, 4H, Phthalyl), 4.44 (t, 2H, CH₂, J = 4.96 Hz), 3.91 (t, 2H, CH₂, J = 4.96).

Dimethyl phosphite (1.76 mL, 19.2 mmol) was added dropwise with stirring under dry nitrogen to a suspension of sodium hydride (0.46 g,19.2 mmol) in CH₂Cl₂ (5 mL). This mixture was treated with a solution of

phthalimidoethyl triflate (4.14 g, 12.8 mmol) in CH₂Cl₂ (15 mL) dropwise with cooling in an ice bath. The flask was allowed to warm to room temperature and stored for 40 min. The solution was extracted with ice-cold 0.5 N HCl (2 × 50 mL), followed by water (50 mL), 5% aqueous NaHCO₃ (50 mL), water (50 mL), and brine (50 mL). The organic layer was dried over sodium sulfate, filtered, and the filtrate was evaporated to dryness. The residue was purified by silica column chromatography using a linear gradient of 0.5-1% CH₃OH in CH₂Cl₂ and the appropriate fractions were collected and concentrated to dryness to yield 3.33 g (75%) of pure 10 as a white solid. ¹H NMR: δ (ppm) 7.72 (m, 4H, phthalyl), 4.31 (m, 2H, CH₂), 3.47 (m, 2H, CH₂). ³¹P NMR: d (ppm) 19.04 (t, CH₂-P, J = 9.6 Hz).

2-(Phthalimidoethyl)phosphonate, triethylammonium salt (9).

An ice-cold solution of 10 (3.8 g, 13.4 mmol) in chloroform (20 mL) was treated dropwise with stirring with bromotrimethylsilane (4.5 g, 29.5 mmol) under dry nitrogen. After 2 h the reaction mixture was

concentrated under reduced pressure, and the residue was redissolved in chloroform (10 mL) followed by addition of triethylamine (40 mL) with cooling in an ice bath.

After 20 min the ice bath was removed and reaction was stirred for an additional 1.5 h. The mixture was filtered and the filtrate was concentrated to dryness. The residue was dissolved in chloroform (5 mL) and added dropwise to ether (800 mL) to give a precipitate which was filtered, washed with ether and dried over P₂O₅ to yield 9 4.0 g (66%) as the triethylammonium salt which was directly used for the next step without any

purification.

5'-O-(Dimethoxytrityl)thymidine-3'-[2-(phthalimido)ethyl] phosphonate (12).

5'-O-(Dimethoxytrityl)thymidine (1.0 g , 1.8 mmol) was dried by coevaporation with pyridine (3 × 5 mL), dissolved in dry pyridine (100 mL), and treated with triisopropylbenzenesulfonyl chloride (TPS-Cl, 3.3g, 10.8 mmol) followed by 9 (0.92 g, 2 mmol). The resulting suspension was stirred overnight under a dry nitrogen atmosphere, diluted with 5% NaHCO₃ (150 mL), and

extracted with hexane (100 ml) and ethyl acetate (3 × 100 mL). The ethyl acetate layers were combined, washed with water (100 mL) and dried over anhydrous magnesium

sulfate. The crude product was purified by column chromatography on silica gel (80 g), using 2-5% methanol in CH₂Cl₂ containing 1% triethylamine. The appropriate fractions were combined and concentrated to yield 0.7 g (45 %) of desired product as white solid. ¹H NMR: δ (ppm) 11.35 (bs, 1H, NH), 7.49 (s, 1H, H₆), 6.8-7.38 (m, 13H, trityl), 6.19 (t, 1H, H₁,), 4.77 (m, 1H, H₃,), 4.09 (s, 1H, H₄,), 3.69 (s, 6H, OMe), 3.62 (m, 2H, CH₂), 3.2-3.6 (m, 2H, H₅,_,5"), 2.32 (m, 2H, H₂,_,2"), 1.68 (m, 2H, CH₂), 1.4 (s, 3H, 5-CH₃). ³¹P NMR: δ (ppm) 17.35 (s). 5'-[[5'-O-(Dimethoxytrityl)thymid-3'-yl]-[2-(phthalimido)ethyl]phosphonyl]thymidine (13).

A mixture of 12 (0.4 g, 0.45 mmol) and thymidine (0.11 g, 0.45 mmol) was dried by coevaporation with pyridine (2 × 5 mL), redissolved in dry pyridine (20 mL), and treated with TPS-Cl (0.69 g, 2.3mmol) followed by tetrazole (0.48 g, 6.8 mmol). The reaction mixture was stirred under dry nitrogen for three hours and

partitioned between 5% NaHCO₃ (100 mL) and ethyl acetate (2 × 50 mL). The organic layer was washed with water (100 mL), dried over sodium sulfate and evaporated to a foam which was purified by column chromatography on silica gel (55 g), using 2-3% methanol in CH₂Cl₂

containing 1% triethylamine. The appropriate fractions were combined and concentrated to yield 0.23 g (50%) of mixture of 13 as a mixture of isomers. The mixture of isomers (0.25 g) was dissolved in

acetonitrile/triethylamine (1:1, 4 mL) and injected onto a reversed-phase C₄ Vydac column (5 × 25 cm) which was eluted with a linear gradient from 35 to 80% acetonitrile in 0.1 M TEAA. The Sp and Rp isomers were eluted at 39.4 and 45.07 minutes respectively and the appropriate fractions were pooled and concentrated to white solids. This procedure yielded 50 mg of the slower, Rp isomer 13a and 60 mg of the faster, Sp isomer 13b.

13a: ¹H NMR: δ (ppm) 11.38 (s, 1H, NH), 11.25 (s, 1H, NH) 7.58-7.8 (m, 4H, phthalimido), 7.41 (s, 1H, H₆), 6.8-7.38 (m, 14H, trityl, H₆), 6.2 (t, 1H, H₁,), 6.05 (t, 1H, H₁,), 5.28 (d, 1H, 3'-OH), 5.19 (m, 1H, H₃,), 3.9-4.2 (m, 3H, H₃,, H₅,_,5"), 3.89 (m, 4H, H₄,_), 3.8 (m, 1H, H₄,₎, 3.65 (s, 6H, OCH₃), 3.2-3.5 (m, 4H, CH₂, H₅,_,5"), 2.2-2.4 (m, 4H, H₂,_,2", CH₂), 1.9 (m, 2H, H₂,_,2"), 1.65 (s, 3H, CH₃), 1.4 (s, 3H, CH₃). 13b: ¹H NMR: δ (ppm) 11.39 (s, 1H, NH), 11.3 (s, 1H, NH) 7.6-7.8 (m, 4H, phthalimido), 7.4 (s, 2H, H₆), 6.8-7.38 (m, 13H, trityl), 6.16 (t, 1H, H₁,), 6.0 (t, 1H, H₁,), 5.4 (d, 1H, 3' -OH) , 5.17 (m, 1H, H₃,), 4.3 (m, 1H, H₃,), 3.85-4.2 (m, 4H, H₄,₅,₅,,), 3.8 (m, 2H, CH₂), 3.64 (s, 6H, OCH₃), 3.2 (m, 2H, H₅,_,5"), 2.3 (m, 4H, H₂,_,2", CH₂), 1.9 (m, 2H, H₂,_,2"), 1.65 (s, 3H, CH₃), 1.4 (s, 3H, CH₃).

Pyridinium [2-(3,4,5,6-tetrabromophthalimido)ethyl]phosphonate (14).

(2-Aminoethyl) phosphonic acid (2g, 16 mmol) was dissolved in water (20 mL), neutralized with 1N NaOH to pH 7.0 and passed through a column of Dowex 50X2-400 (40 mL, pyridinium form) ion exchange resin. The column was washed with water (3 × 50 mL) and the combined eluates were concentrated in vacuo to give 2.33 g (52%) of the pyridinium salt of (2-aminoethyl)phosphonic acid as a white solid. A portion of this material (1g, 3.53 mmol) was suspended in DMF (100 mL) and treated with 3,4,5,6-tetrabromophthalic anhydride (9.8 g, 21.2 mmol) under reflux overnight. The reaction mixture was cooled to room temperature to precipitate the product. The

precipitate was filtered, washed with DMF (3 × 50 mL) followed by hexane (2 × 50 mL) and dried in vacuo over P₂O₅ to give 14 (2.57 g, 70%) as an off-white solid. ¹H NMR: δ (ppm) 8.5, 7.7, 7.3 (m, 12H, pyridinium), 3.7 (m, 2H, CH₂), 1.8 (m, 2H, CH₂). ³¹P NMR: δ (ppm) 21.6 (s).

5'-O-(Dimethoxytrityl)thymidine 3'-[2-(3,4,5,6-tetrabromo- phthalimido)ethyl]phosphonate (15).

5'-O-(Dimethoxytrityl)thymidine (0.822 g , 1.5 mmol) was dried by coevaporation with pyridine (2 × 5 mL), dissolved in dry pyridine (40 mL), and treated with triisopropylbenzenesulfonyl chloride (TPS-Cl, 1.45g, 4.8 mmol) followed by 14 (1.0 g, 1.814 mmol). The resulting suspension was stirred overnight under a dry nitrogen atmosphere, diluted with water (150 mL), and extracted with ethyl acetate (2 × 75 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness, and the residue was dissolved in CH₂Cl₂/CH₃OH (99:1, 10 mL) and filtered through a one micron glass filter. The filtrate was purified on a

Dynamax normal phase silica column (8 m, 21.4 mm × 25 cm) with a gradient of 1-20% CH₃OH in CH₂Cl₂ containing 0.1% triethylamine (TEA). The fractions eluting at 26-34 minutes were collected and concentrated to yield 0.32 g (19%) of pure 15 as an off-white foam. ¹H NMR: δ

(ppm)11.33 (s, 1H, NH), 7.49 (s, 1H, H₆), 6.8-7.38 (m, 13H, trityl), 6.19 (t, 1H, H₁,), 4.77 (m, 1H, H₃,), 4.09 (s, 1H, H₄,), 3.69 (s, 6H, OMe), 3.62 (m, 2H, CH₂), 3.2-3.6 (m, 2H, H₅,_,5"), 2.32 (m, 2H, H₂,_,2"), 1.68 (m, 2H, CH₂), 1.4 (s, 3H, 5-CH₃). ³¹P NMR: δ (ppm) 17.35 (s).

5'-[[5'-O-(Dimethoxytrityl)thymid-3'-yl]-[2-(3,4,5,6-tetrabromo- phthalimido)ethyl]phosphonyl]thymidine (16).

Compound 15 (9 g, 7.6 mmol) and thymidine (1.85 g, 7.6 mmol) were dried by coevaporation with pyridine (2 × 10 mL), redissolved in dry pyridine (20 mL), and treated with TPS-Cl (8.06 g) followed by tetrazole (0.53 g). The reaction mixture was stirred under dry nitrogen overnight and partitioned between aqueous sodium bicarbonate (300 mL, 2.5%) and methylene chloride (2 × 150 mL). The organic layer was washed with water (100 mL), dried over sodium sulfate and evaporated to a foam. This material (10 g) was dissolved in CH₂Cl₂ (30 mL), filtered through a glass funnel to remove insoluble materials, and injected in three separate aliquots onto a normal phase silica column which was eluted at 40 mL/min with a linear gradient from 0-10% isopropanol in CH₂Cl₂ containing 0.1% pyridine. 16a and 16b were eluted at 32.2 and 31.0 minutes respectively. The appropriate fractions were pooled and evaporated to give white solids. This procedure yielded 470 mg of 16a, 500 mg of 16b, and 690 mg of a mixture of isomers.

16a: ¹H NMR: δ (ppm) 11.35 (s, 1H, NH), 11.27 (s, 1H, NH) 7.44 (s, 2H, H₆), 6.8-7.43 (m, 13H, trityl), 6.15 (t, 1H, H₁,), 6.09 (t, 1H, H₁,), 5.41 (d, 1H, 3'-OH) , 5.12 (m, 1H, H₃), 4.05-4.21 (m, 3H, H₃,_,5,_,5"), 3.88 (m, 1H, H₄,), 3.68

(m, 1H, H₄) 3.6 (s, 6H, OCH₃), 3.22 (m, 2H, CH₂), 3.15 (m, 2H, H₅,_,5"), 2.42 (m, 2H, H₂,_,2"), 2.2 (m, 4H, H₂,_,2", CH₂), 1.67

(s, 3H, CH₃), 1.35 (s, 3H, CH₃). ³¹P NMR: δ (ppm) 30.45

(s) .

16b: ¹H NMR: δ (ppm) 11.36 (s, 1H, NH), 11.26 (s, 1H, NH) 7.45 (s, 1H, H₆), 7.39 (s, 1H, H₆), 6.8-7.34 (m, 13H, trityl), 6.16 (t, 1H, H₁,), 6.08 (t, 1H, H₁,), 5.4 (d, 1H, 3'-OH), 5.15 (m, 1H, H₃,), 4.1 (m, 1H, H₃,), 3.66-4.0 (m, 3H, H₄,_,5,_,5,,), 3.6 (s, 6H, OCH₃), 3.4 (m, 2H, CH₂), 3.18 (m, 2H, H₅,_,5"), 2.45 (m, 2H, H₂,_,2"), 2.18-2.32 (m, 4H, H₂,_,2", CH₂), 1.68 (s, 3H, CH₃), 1.4 (s, 3H, CH₃). ³¹P NMR: δ (ppm) 31.03 (8).

Synthesis of 5'-O-(Dimethoxytrityl)thymidyl-3'-[2-(3,4,5,6-tetrabromophthalimido)ethyl]phosphonyl-5'-thymidine-3'-cyanoethyl-N,N-diisopropylamino-phosphoramidite (20).

A sample of 16 (500 mg, 0.38 mmol) was dried by coevaporation with pyridine, dissolved in dry

acetonitrile (5 mL) and methylene chloride (7 mL) under nitrogen, and treated with stirring with 2-cyanoethoxy- (N,N,N',N'-tetra-isopropylamino)-phosphine (1.2 mL, 3.8 mmol), diisopropylamine (0.3 mL, 2.1 mmol), and

tetrazole (106 mg, 1.52 mmol). After 2 h at room temp the mixture was partitioned between 5% aqueous sodium

bicarbonate and methylene chloride (50 mL of each). The aqueous layer was extracted with methylene chloride (2 × 70 mL) and the combined organic layers were washed with water (2 × 50 mL) and dried over magnesium sulfate. The solids were removed by filtration and the filtrate was concentrated to a gum which was purified by column chromatography on silica gel (55 g). The column was eluted with a linear gradient of 0-15% ethanol in methylene chloride containing Et₃N (0.1%) and the

appropriate fractions were combined and evaporated to yield the purified material as a foam. This material was dissolved in methylene chloride (10 mL) and added dropwise with stirring to pentane (450 mL). The

precipitate was collected and dried to yield 448 mg (78%) of pure 10. ³¹P nmr δ (ppm) 31.239 (s), 150.858 (s).

Pyridinium [2-(3,4,5,6-tetrachlorophthalimido)ethyl]phosphonate (17).

The procedure as described for 14 was followed using pyridinium (2-aminoethyl)phosphonate (1g, 3.53 mmol) suspended in IMF (100 mL) and 3,4,5,6-tetrachlorophthalic anhydride (6.05 g, 21.2 mmol). After isolation 1.94 g, (89%) of 17 was obtained. ¹H NMR: δ (ppm) 78.5, 7.7, 7.3 (m, 12H, pyridinium), 3.7 (m, 2H, CH2), 1.8 (m, 2H, CH₂). ³¹P NMR: δ (ppm) 21.4 (s).

5'-O-(Dimethoxytrityl)thymidine 3'-[2-(3,4,5,6-tetrachlorophthalimido)ethyl]phosphonate (18).

5'-O-(Dimethoxytrityl)thymidine (1.08 g , 1.99 mmol) was dried by coevaporation with pyridine (2 × 5 mL), dissolved in dry pyridine (40 mL), and treated with TPS-Cl (1.92 g, 6.33 mmol) followed by 17 (1.0 g, 1.814 mmol). The resulting suspension was stirred overnight under a dry nitrogen atmosphere, diluted with water (150 mL), and extracted with ethyl acetate (2 × 75 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness and purified by silica gel column chromatography (45 g, 130-270 mesh) using a gradient of 1-6% CH₃OH/CH₂Cl₂ containing 0.1% TEA as the solvent. The appropriate fractions were collected, combined and evaporated to yield 1.10 g (63%) of 18 as an off-white foam. ¹H NMR: δ (ppm) 11.32 (s, 1H, NH), 7.47 (s, 1H, H₆), 6.84-7.35 (m, 13 H, trityl), 6.12 (t, 1H, H₁,), 4.75 (m, 1H, H₃), 4.04 (m, 1H, H₄,), 3.68 (s, 6H, OMe), 3.64 (m, 2H, H₂), 3.16-3.23 (m, 2H, H₅,), 2.31 (m, 2H, H₂,), 1.70 (m, 2H, H₂), 1.38 (s, 3H, CH₃). ³¹P NMR: δ (ppm) 16.4 (s).

5'-[[5'-O-(Dimethoxytrityl)thymid-3'-yl]-[2-(3,4,5,6- tetrachlorophthalimido)ethyl]phosphonyl]thymidine (19).

Compound 18 (1.0 g, 1.124 mmol) and thymidine (0.272 g, 1.124 mmol) were dried by coevaporation with pyridine (2 × 5 mL), redissolved in dry pyridine (20 mL), and treated with TPS-Cl (1.19 g, 3.935 mmol) followed by tetrazole (0.086 g, 1.237 mmol). The reaction mixture was stirred under dry nitrogen overnight and isolated as described for 16. The fractions eluting at 20-26 min were evaporated to give 19 as a mixture of isomers, 0.58 g (50%).

The mixture of isomers from two experiments (1.1 g) were combined, dissolved in CH₂Cl₂ and injected onto a normal phase silica column (Dynamax-60A, 41.4 mm) which was eluted with a linear gradient from 0-6% isopropanol in CH₂Cl₂ containing 0.1% pyridine. The Sp and Rp isomers were eluted at 39-43 and 49-74 minutes respectively. The appropriate fractions were pooled and concentrated to white solids. This procedure yielded 400 mg of Rp isomer 19a, 90 mg of Sp isomer 19b, and 500 mg of a mixture of isomers. 19a: ¹H NMR: δ (ppm) 11.34 (s, 1H, NH) , 11.26 (s, 1H, NH) 7.45 (s, 2H, H₆) , 6.8-7.3 (m, 13H, trityl) , 6.16 (t, 1H, H₁,) , 6.10 (t, 1H, H₁,) , 5.1 (m, 1H, H₃,) , 4.1-4.26 (m, 3H, H₃,_,5,_,5") , 4.14 (m, 1H, H₄,) , 3.88 (m, 1H, H_4'), 3.7 (s, 6H, OCH₃) , 3.35 (m, 2H, CH₂) , 3.21-3.31 (m, 2H, H₅,_,5"), 2.44 (m, 2H, H₂,_,2") , 2.25 (m, 2H, CH₂) , 2.07 (m, 2H, H₂,_,2"), 1.69 (s, 3H, CH₃), 1.4 (s, 3H, CH₃) . ³¹P NMR: δ (ppm) 29.52 (s) .

19b: ¹H NMR: δ (ppm) 7.46 (s, 1H, H₆) , 7.40 (s, 1H, H₆) 6.8-7.3 (m, 13H, trityl) , 6.18 (t, 1H, H₁,), 6.09 (t, 1H, H₁,), 5.13 (m, 1H, H₃,) , 4.0-4.12 (m, 3H, H₃,_,5,_,5"), 3.9 (m, 1H, H₄,) , 3.77 (m, 3H, H₄,, CH₂) , 3.70 (s, 6H, OCH₃) , 3.01-3.3 (m, 2H, H₅,_,5") , 2.46 (m, 2H, H₂,_,2") , 2.24 (m, 2H, CH₂) , 2.0 (m, 2H, H₂,_,2"), 1.70 (s, 3H, CH₃), 1.43 (s, 3H, CH₃) . ³¹P NMR: δ (ppm) 29.26 (s) .

5'-O-(Dimethoxytrityl)thymidyl-3'-[2-(3,4,5,6-tetrachloro- phthalimido)ethyl]phosphonyl-5'-thymidine-3'-cyanoethyl-N, N-diisopropylaminophosphoramidite (21).

A sample of 19a (320 mg, 0.304 mmol) was dried by coevaporation with pyridine, dissolved in dry methylene chloride (10 mL) under nitrogen, and treated with stirring with 2-cyanoethoxy-(N,N,N',N'-tetraisopropylamino)-phosphine (724 mL, 2.28 mmol),

diisopropyl-amine (178 mL, 1.274 mmol), and tetrazole (64 mg, 0.913 mmol). After 1 h at room temp the mixture was partitioned between 5% aqueous sodium bicarbonate (100 ml) and methylene chloride (2 × 50 mL). The aqueous layer was extracted with methylene chloride (2 × 50 mL) and the combined organic layers were washed with water (2 × 50 mL) and dried over magnesium sulfate. The solids were removed by filtration and the filtrate was concentrated to a gum which was purified by column chromatography on silica gel (20 g). The column was eluted with a linear gradient of 0-1.5% ethanol in methylene chloride containing Et₃N (0.1%) and the appropriate fractions were combined and evaporated to yield the purified material as a foam. This material was dissolved in methylene chloride (5 mL) and added dropwise with stirring to pentane (300 mL). The precipitate was collected and dried to yield 173 mg (45%) of pure 21. ³¹P nmr δ (ppm) 29.09 (s), 148.65 (s).

5'-[[5'-O-(Dimethoxytrityl)thymid-3'-yl](2-aminoethyl)phosphonyl]thymidine (24a).

A sample of 16a (70 mg, 0.05 mmol, Rp isomer) was dissolved in anhydrous CH₃CN (800 mL) and treated with ethylenediamine (400 mL). After one hour at room

temperature the reaction mixture was dried under reduced pressure and coevaporated with ethanol (5 × 500 mL) and toluene (2 × 500 mL). The residue was purified on a C₄ semipreparative HPLC column (Waters RCM, 25mm × 10 cm) with a gradient of 35-80% acetonitrile in 0.1 M TEAA.

The deprotected compound, eluting at 16.2 min, was

collected and evaporated to dryness to give 24a, Rp isomer, as a white solid, 46.5 mg (82%).

1H NMR: δ (ppm) 7.49 (s, 1H, H₆), 7.46 (s, 1H, H₆), 6.87-7.37 (m, 13H, trityl), 6.20 (t, 1H, H₁,), 6.15 (t, 1H, H₁,), 5.11 (m, 1H, H₃,), 4.21 (m, 1H, H₃,), 4.03-4.19 (m, 3H, H₃,, H₅,_,5"), 3.90 (m, 1H, H₄,), 3.72 (s, 6H, OCH₃), 3.20-3.27 (m, 2H, H₅,_,5"), 2.71 (m, 2H, CH₂), 2.41 (m, 2H, H₂,_,2"), 2.09 (m, 2H, H₂,_,2"), 1.92 (m, 2H, J=17.5 Hz, CH₂-P), 1.73 (s, 3H, CH₃), 1.41 (s, 3H, CH₃). ³¹P NMR: δ (ppm) 32.754 (s).

An identical procedure was used to prepare the Sp isomer 24b, using (36 mg, 0.03 mmol) of 16b. The

deprotected material was eluted at 14.9 min and these fractions were collected and evaporated to dryness to give 24b as a white solid, 9.3 mg (39%).

¹H NMR: δ (ppm) 7.47 (s, 1H, H₆), 7.46 (s, 1H, H₆), 6.86-7.43 (m, 13H, trityl), 6.20 (t, 1H, H,.), 6.15 (t, 1H, H₁,), 5.09 (m, 1H, H₃,), 4.16 (m, 1H, H₃,), 4.11 (m, 1H, H₄,), 3.94-4.07 (m, 2H, H₅,_,5"), 3.84 (m, 1H, H₄), 3.71 (s, 6H, OCH₃), 3.21-3.28 (m, 2H, H₅,_,5") , 2.74 (m, 2H, CH₂), 2.43 (m, 2H, H₂,_,2"), 2.06 (m, 2H, H₂,_,2"), 1.97 (m, 2H, CH₂-P, J = 18.0 Hz), 1.74 (s, 3H, CH₃), 1.43 (s, 3H, CH₃). ³¹P NMR: δ (ppm) 33.014 (s).

5'-(thymid-3'-yl)[2-aminoethyl)phosphonyl]thymidine (25a, 25b).

A sample of 24b (9.3 mg, 0.01 mmol, Sp isomer was dissolved in 1 mL of 1% dichloroacetic acid in CH₂Cl₂. After one hour at room temperature the reaction mixture was dried under reduced pressure, redissolved in ImL H₂0 and extracted with 3 × 1 mL ethyl acetate. The aqueous solution then was purified on a C₄ semipreparative HPLC column (Waters RCM, 25 mm × 10 cm) with a gradient of 5-70% acetonitrile in 0.1 M TEAA. The detritylated

compound, eluting at 11.8 min, was collected and

evaporated to dryness to give 25b, Sp isomer, as a white solid. ¹H NMR: δ (ppm) 7.67 ( s, 1H, H₆), 7.48 (s, 1H, H₆), 6.18 (m, 2H, H₁,), 5.0 (m, 1H, H₃,), 4.2 (m, 1H, H₃,), 4.12 (m, 2H, H₅,_,5"), 4.01 (m, 1H, H₄,), 3.9 (m, 1H, H₄,), 3.58 (m, 2H, H₅,_,5"), 2.77 (q, 2H, CH₂), 2.11-2.31 (m, 4H, H₂,_,2"), 1.98 (m, 2H, CH₂), 1.79 (s, 3H, CH₃), 1.76 (s, 3H, CH₃). ^3lP NMR: δ 29.71 (s).

An identical procedure was used to prepare the Rp isomer 25a, using (46.5 mg, 0.053 mmol) of the compound 24a. The detritylated material was eluted at 8.48 min. Fractions were collected and evaporated to dryness to give 25a as a white solid. ¹H NMR: d (ppm) 7.67 (s, 1H, H₆), 7.50 (s, 1H, H₆), 6.17 (t, 2H, H₁,), 5.0 (m, 1H, H₃,), 4.23 (m, 1H, H₃,), 4.09-4.19 (m, 2H, H₅,_,5"), 4.0 (m, 1H, H₄,), 3.9 (m, 1H, H₄,), 3.58 (m, 2H, H₅,_,5"), 2.79 (m, 2H, CH₂), 2.07-2.33 (m, 4H, H₂,_, H_2"), 2.0 (m, 2H, CH₂), 1.78 (s, 3H, 5-CH₃), 1.76 (s, 3H, CH₃). ³¹P NMR: δ 29.26 (s). Pyridinium [3-(3,4,5,6-tetrachlorophthaiimido)propyl]phosphonate (27).

The procedure as described for 14 was followed using pyridinium (3-aminopropyl)phosphonate (2g, 6.73 mmol) suspended in DMF (100 mL) and 3,4,5,6-tetrachlorophthalic anhydride (11.54 g, 40.36 mmol). After isolation 3.7 g, (99%) of 27 was obtained.

5'-O-(Dimethoxytrityl)thymidine 3'-[2-(3,4,5,6-tetrachloro phthalimido)propyl]phosphonate (28).

5'-O-(Dimethoxytrityl)thymidine (4.03 g ,7.4 mmol) was dried by coevaporation with pyridine (2 × 5 mL), dissolved in dry pyridine (50 mL), and treated with TPS-Cl (7.13 g, 23.54 mmol) followed by pyridinium salt of 3-phthalimidopropylphosphonate (27, 3.8 g, 6.72 mmol). The resulting suspension was stirred overnight under a dry nitrogen atmosphere, diluted with water (150 mL), and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to dryness and purified by silica column chromatography (120 g) which was eluted with a linear gradient from 0-5% methanol in CH₂Cl₂ containing 0.1% TEA as the solvent. The appropriate fractions were

collected, combined and evaporated to yield 2.5 g (41%) of 28 as a white foam.

5'-[[5'-O-(Dimethoxytrityl)thymid-3'-yl]-[3-(3,4,5,6-tetra chloropthalimido)propyl]phosphonyl]thymidine (29).

Compound 28 (4.0 g, 4.42 mmol) and thymidine (0.67 g, 4.42 mmol) were dried by coevaporation with pyridine (2 × 5 mL), redissolved in dry pyridine (50 mL), and treated with TPS-Cl (2.93 g, 15.49 mmol) followed by tetrazole (0.213 g, 4.87 mmol). The reaction mixture was stirred under dry nitrogen overnight and isolated as described for 16. The fractions eluting at 49-60 min were evaporated to give 29 as a mixture of isomers.

2-Trifluoromethanesulfonyloxyethyl-phthalimide (33):

2-Hydroxyethyl-phthalimide (1.0 g, 4.87 mmol) was dissolved in CH₂Cl₂ (10 mL), cooled to -78°C in a dry ice bath and treated with diisopropylethylamine (1mL, 15.85 mmol), followed by trifluoromethanesulfonic anhydride (0.98 mL, 5.85 mmol) . The reaction mixture was stirred at approx. -65°C under dry nitrogen for 3 hours and then partitioned between cold hexane (100 mL) and 0.5 M HCl (2 × 100 mL). The organic layer was washed once with water (100 mL), followed by 5% aqueous NaHCO₃ (100 mL), water (100 mL), and brine (100 mL), and then dried over sodium sulfate. The solid was removed by filtration and the filtrate was evaporated to dryness to yield 4.14 g of the triflate 33 as a white solid which was used directly without any further purification.

Preparation of 2-Trifluoromethanesulfonyloxyethyltetrachloro-phthalimide (35)

2-Hydroxyethyl-tetrachlorophthalimide (1.0 g, 3.0 mmol) was dissolved in CH₂Cl₂ (30 mL), cooled to -78°C in a dry ice bath and treated with pyridine (0.58 mL, 7.2 mmol), followed by trifluoromethanesulfonic anhydride (0.98 mL, 5.85 mmol). The reaction mixture was stirred at approx. -65°C under dry nitrogen for 3 hours and then partitioned between cold hexane (100 mL) and 0.5 M HCl (2 × 100 mL). The organic layer was washed once with water (100 mL), followed by 5% aqueous NaHCO₃ (100 mL), water (100 mL), and brine (100 mL), and then dried over sodium sulfate. The solid was removed by filtration and the filtrate was evaporated to dryness to yield (0.484 g, 35%) of the triflate 35 as a white solid which was used directly without any further purification. Bis-N,N-diethylamino-2-phthalimidoethyl-phosphine (34).

Bis-diethylamino-H-phosphine (32, 0.95 g, 5.39 mmol) prepared by a published procedure (Phosphorus and Sulfur, 1983, vol. 18, pp. 125-128) is dissolved in THF (2 mL), treated with NaH (151 mg, 6.0 mmol) and the suspension is stirred at 0°C for 1.5 hours. A solution of 33 (1.73 g, 5.39 mmol) in THF (3 mL) is added to the suspension and the mixture was stirred for an additional 2 hours at 0°C. The solution was filtered and the filtrate was evaporated to dryness and used directly for the preparation of 31.

The tetrachloro compound 36 is prepared in the same way.

5'-O-Dimethoxytritylthymidine-3'-(2-phthalimidoethyl)-N,N-di-ethylaminophosphonamidite (31).

5'-Dimethoxytritylthymidine (0.496 g, 0.91 mmol) is dried by coevaporation with pyridine, dissolved in dry methylene chloride (5 mL) under nitrogen, and treated with stirring with tetrazole (31.87 mg, 0.45 mmol) and crude 34 from the previous synthesis. After 1 h at room temp, the mixture is partitioned between 5% aqueous sodium bicarbonate (100 mL) and methylene chloride (2 × 50 mL). The aqueous layer is extracted with methylene chloride (2 × 50 mL) and the combined organic layers are washed with water (2 × 50 mL) and dried over magnesium sulfate. The solids are removed by filtration and the filtrate was concentrated to a gum which is purified by column chromatography on silica gel (20 g). The column is eluted with a linear gradient of 0-5% ethyl acetate in methylene chloride containing Et₃N (0.1%) and the appropriate fractions are combined and evaporated to yield the purified material. This material is dissolved in methylene chloride (5 mL) and added dropwise with stirring to pentane (300 mL). The precipitate is

collected and dried to yield pure material. 5'-O-Dimethoxytritylthymidyl-3'-[2-(3,4,5,6-tetrachlorophthalimido)ethyl]-N-diethylamino-phosphonamidite (37).

The procedure as described above for 31 is followed to yield 5'-O-dimethoxytritylthymidyl-3'-[2-(3,4,5,6-tetrachlorophthalimido)-ethyl]-N-diethylaminophosphonamidite (37).

Example 13

Synthesis of Oligonucleotides.

22a, 22b 26a and 26b were synthesized on a 1 μmol scale using a modified phosphoramidite cycle on a DNA synthesizer with a coupling time of 1 min/cycle. The dimer phosphoramidites (either 20a, 21a, 20b or 21b) were delivered at a concentration of 0.1 M in acetonitrile. For 26a and 26b a standard thymidine phosphoramidite was employed to introduce a phosphodiester linkage at the 5'-terminus. Oligonucleotides 23a and 23b were prepared using conventional methods with the dimer amidite being introduced at the appropriate position. At the conclusion of the syntheses, the synthesis columns containing the tritylated oligonucleotides were dried in vacuo overnight and the supports were removed from the columns,

transferred to screw cap glass vials and treated with ethylenediamine (500 mL) for 30 min at 55°. The

supernatants were collected, evaporated to dryness, and the supports were washed with ethanol (2 × 200 mL) followed by TEAA (0.1 M, 2 × 200 mL) and ethanol (2 × 200 mL). The residues from both the supernatants and washes were combined, dissolved in water (500 mL), filtered through a 0.45 μ membrane and loaded onto a preparative reverse phase C₄ HPLC column. The column was eluted with 0.1 M TEAA/CH₃CN according to the following conditions: 0-5 min 5% CH₃CN; 5-20 min 5-30% CH₃CN; 20-25 min 30-50% B, flow rate of 10 mL/min. The fractions containing product were lyophilized, and detritylation was performed using 0.1 M acetic acid (2 mL) for 30 min at 55°. The solution was lyophilized and the residue was dissolved in TEAA (1 mL) and injected 01 to a C₄ column (Waters RCM 25 mm × 10 cm). The fractions containing pure material were collected and evaporated to dryness and the residue was converted into the sodium form and lyophilized.

Example 14

Thermal Denaturation Experiments

These experiments were carried out using a Gilford Response II temperature controlled spectrophotometer by monitoring the changes in absorbance at 260 nm versus temperature, with a heating rate of 1°/min from 0-70°. Melting curves were obtained both in low salt (150 mM NaCl, 10 mM Na₂HPO₄) and high salt (1 M NaCl, 10mM Na₂HPO₄) conditions at pH 7.0. Transition temperatures were obtained from the first order derivative plot of

absorbance versus temperature. For experiments involving dissociation of triplex structures, 50 mM Tris, 20 mM MgCl₂, 0.1 M NaCl, pH 7 was used as the buffer.

Example 15

Hvdrolvtic stability of 25a and 25b.

The dinucleotides 25a and 25b were incubated at pH 7.1 in TEAA buffer at 37° over 50 h and aliquots were removed at intervals and injected onto a C₄ reverse phase HPLC column. The rate of degradation was determined by measurement of the area remaining under the peak

corresponding to starting material. The half lives as determined by this method were 51 days for 25a and 48 days for 25b.

Example 16

Stability to nucleases

a) S1 nuclease: Solutions of the natural dimer d-TpT and aminoethyl modified dimers 25a and 25b (2 OD₂₆₀) in 100 μL of buffer (50 mM sodium acetate, 250 mM sodium chloride, 1 mM zinc chloride, pH 4.6, containing 50 μG/mL of bovine serum albumin) were equilibrated at 0°C for 15 min, treated with 4 mL S1 nuclease (U.S. Biochemicals, 263 units/μL) and the solutions were stored at 0°C.

Aliquots (5 μL) of the reaction mixture were injected onto an analytical reverse phase HPLC column and eluted with a gradient of 15-80% acetonitrile in 0.1 M TEAA buffer (pH 7.1) and the area under the peak corresponding to starting material was plotted versus time to determine t_1/2 values.

Example 17

Induction of RNAse H Activity

A single-stranded DNA-RNA hybrid target molecule was synthesized consisting of 8-base deoxynucleotide flanks and a central A₁₃ RNA core. This molecule was end labeled and isolated as described above. The target molecule (about 7 fmol, 40,000 cpm) was annealed to 10-, 100- or 1000-fold excess of either 26a, 26b or d-T(pT)₁₂ as a control by heating to 65° for 10 min. in 10 μL GB (20 mM Hepes pH 7.4, 140 mM NaCl, 5 mM KOAc, 5 mM MgCl₂)

followed by slow cooling to room temp. One unit of RNAse H (Promega) was added and the solutions were incubated at 37° for 20 min. Formamide was added to 50% concentration and the solutions were heated (95°, 5 min), and

electrophoresed on a 16% PAGE with 7M urea in TBE. The gels were fixed and dried prior to autoradiography.

Example 18

Serum stability

Ten pmol of d-T (pT) ₁₂ or the alternating aminoethyl oligomers 26a or 26b were 5'-end labelled with T4

polynucleotide kinase in the presence of 400 mCi of

[γ³²P]-ATP (ICN, 7000 Ci/mmol). The labeled compounds were heated (95°, 5 min) in 50% formamide and purified by separation on a 12% PAGE with 7M urea using Tris-borate- EDTA buffer (TBE). The isolated bands were frozen, crushed and extracted with 2mM EDTA, filtered through cellulose acetate (0.45 μ), extracted with

phenol/chloroform and finally precipitated with ethanol. For each time point examined, each oligomer (50,000 cpm) was incubated at 37° in a 5% humidified CO₂

atmosphere with 50 mL of Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% heat-inactivated (56°, 45 min) fetal bovine serum (FBS). Control samples were incubated in plain DMEM. At the end of 0,1, 4 or 24 hours, the samples were mixed with an equal volume of formamide, heated (95°, 5 min), and again separated by 12% urea-PAGE. The gels were fixed, washed and dried before autoradiography (Kodak XAR-5).

DISCUSSION

Synthesis of nucleotide (2-aminoethyl)phosphonates

The strategy for the synthesis of nucleotide (2-aminoethyl)phosphonates initially employed the

phthalimido group for protection of the primary amine of the (2-aminoethyl) phosphonate moiety, since this group was previously employed for the preparation of

(aminomethyl)phosphonate derivatives. Several routes were evaluated for the synthesis of the protected (2-aminoethyl)phosphonate intermediate 9 (Figure 1). An Arbusov reaction of bromoethylphthalimide with trimethyl phosphite under reflux for 72 h did not produce the desired intermediate (2-phthalimidoethyl)phosphonate derivative 10, but an alternate route involving reaction of the triflate ester of hydroxyethylphthalimide 11 with sodium dimethyl phosphite did produce 10 in good yield. Removal of the methyl groups from 10 was accomplished with trimethylsilyl bromide, and the resulting (2-phthalimidoethyl)phosphonic acid 9, as its

triethylammmonium salt, was treated with 5'-dimethoxytrityl-thymidine using 2,4,6- triisopropylbenzenesulfonyl chloride (TPS-Cl) as the coupling agent to give the monomer 12. Reaction of 12 with thymidine and TPS-Cl gave the protected dimer 13 (Figure 2) which was isolated by column chromatography. A variety of conditions for deprotection of the phthalyl group were evaluated. A sample of 13 was detritylated and treatment of the product with ethylenediamine in

acetonitrile for up to 6.5 h at room temp did not produce any cleavage of the phthalyl group. This result is in marked contrast to the results with the

corresponding dinucleotide-(phthalimidomethyl)phosphonate as previously reported (Fathi, R., Huang, Q., Delaney, W. and Cook, A.F. (1994) Bioconjugate Chem. 5:47-57) which could be completely deprotected and isolated in excellent yield upon treatment with ethylenedimine under the same conditions. Other reagents were evaluated for removal of the phthalyl group. Hydrazine has previously been used for the removal of phthalyl protecting groups (Seyferth, D., Marmor, R. S. and Hubert, P. (1971) J. Org. Chem. , 36, 1379-1386) and deprotection of oligonucleotide methylphosphonates (Miller, P., Cushman, C. D. and Levis, J. T. (1991) in Eckstein, F. (ed), Oligonucleotides and Analogs-A Practical Approach, IRL Press, Oxford, pp. 137-154) but this reagent was not satisfactory since

decomposition occurred. One explanation for the

difference in stability between the phthalimidomethyl and phthalimidoethyl groups may be related to the difference in the basicity of the amines from which they were derived. Thus the amine functionality of

(aminomethyl)phosphonic acid is less basic and therefore might be a more effective leaving group than the

aminoethyl analog.

Because of the difficulties experienced with removal of the phthalyl group from 13, other groups were

evaluated for protection of the amino functionality, and aromatic groups were preferred since they provide a lipophilic handle which aids in purification. A benzoyl group was briefly evaluated, but as expected it was not possible to remove this group under mild conditions.

Since halogenated phthalimido groups have been employed for other purposes (Motawia, M. S., Jacobsen, J. P. and Pedersen, E. B. (1989) Chemica Scripta, 29, 51-55 and Motawia, M. S., Nawwar, G. A. M., Andreassen, E. S., Jacobsen, J. P. and Pedersen, E. B. (1987) Liebigs Ann . Chem. , 1111-1114) these were evaluated for protection of the amino group. Reaction of tetrabromophthalic anhydride with (2-aminoethyl) phosphonic acid produced the

tetrabromo derivative 14 which could be isolated as a solid. Reaction of this compound with 5'-dimethoxytritylthymidine using TPS-Cl as the coupling agent did produce the mononucleotide 15, although this reaction was inefficient due to the poor solubility of 14 in the reaction medium. Coupling of 15 with one

equivalent of thymidine gave the dimer as a mixture of isomers 16a and 16b which could be separated by hplc, with no evidence for 3',3'-coupling. After removal of the acid labile dimethoxytrityl group, the tetrabromophthalyl group could readily be removed by treatment with ethylenediamine in acetonitrile. Thus the

introduction of the halogen substituents into the phthalimido group produced a protecting group which is expected to be compatible with other nucleic acid

components.

Although the tetrabromophthalimido group was suitable for protection of the amino function in the synthesis of oligonucleotides, the low yields obtained in the

synthesis of the mononucleotide 14 prompted an evaluation of the corresponding tetrachloro derivatives. Thus 17 was prepared from (2-aminoethyl)phosphonate as described for 14, and in this case conversion to the monomer was much more efficient since 18 could be isolated in 63% yield. The dimer 19 was prepared as described for 16, and deprotection of the tetrachlorophthalyl group could be easily accomplished using ethylenediamine for 10 min at 55°. Thus the tetrachlorophthalimido group appears to be protecting group of choice for this application.

Synthesis of nucleotide (3-aminopropyl)phosphonates

Methods have been developed for the synthesis of (3-aminopropyl)phosphonates in view of their potential interest as antisense or protein binding compounds. These parallelled the procedures for the aminoethyl series and employed the tetrachlorophthalimido group for protection of the amino functionality. Thus the monomer 28 could be prepared by reaction of 5'-dimethoxytrityl-thymidine with the aminopropyl derivative 27, and converted into the protected dimer 29. If required, the isomers of 29 can be separated, converted into the phosphoramidite 30, and incorporated into oligonucleotides. Oligonucleotides containing these aminopropyl residues may have advantages for antisense or protein binding applications.

A phosphonamidite route for the synthesis of aminoalkyl phosphonate derivatives of oligonucleotides.

The routes described above possess the advantage that individual isomers of dinucleotide aminoalkylphosphonates can be prepared and incorporated into oligonucleotides. As can be seen from the hybridization data in Table 1, dramatic differences in the properties of individual isomers can be observed. The disadvantage of the above approach is that it is relatively labor intensive to prepare all of the 32 dimers needed for oligonucleotide synthesis (for the four bases A, C, G and T there are 16 unique dimer sequences, each of can exist as two

isomers). For this reason mononucleotide phosphonamidites of general structure 31 and 27 (figure 5) with aminoalkyl residues attached to every phosphorus atom can be used on the DNA synthesizer to prepare oligonucleotides. The separation of isomers of oligonucleotides prepared by this method is not a practical proposition, but there may be instances where a mixture of isomers can be used for specific purposes.

In this procedure, an H-phosphine compound 32 is reacted with the phthalimido derivative 33 or 35 to produce the phthalimidophosphine 34 or 36, which is then reacted with a protected nucleoside to give the required monomer 31 or 37. Compounds 31 and 37 can be used in the synthesizer to introduce an aminoalkyl moiety at any or all positions in an oligonucleotide. This procedure can also be used to prepare a phosphonamidite of any

nucleoside.

Stereochemistry at phosphorus

The determination of the stereochemistry at

phosphorus was carried out by an adaptation of the 2D-NMR method of Loschner and Engels (Loschner, T. and Engels, J. W. (1990) Nucleic Acids Res. , 18, 5083-5088) These authors determined the stereochemistry about phosphorus of dinucleoside methylphosphonates based upon the level of interactions between the methylphosphonate and the C₃, and C₄, sugar protons. In this work the 2D-ROESY NMR spectra of the partially protected dimers 24a and 24b indicated that the relative interactions of the P-CH₂ and C₄, protons as compared with the C₃, protons was enhanced for one isomer, which was assigned as the Rp isomer 24a, as compared with the Sp isomer 24b. Details of the NMR data will be described elsewhere. This assignment is consistent with data obtained from the hybridization experiments which showed that the oligonucleotide with Rp configuration exhibited a much higher Tm than the

corresponding Sp isomer (see below). Oligonucleotide synthesis

The dimer phosphoramidites 20 or 21 were prepared by a conventional method (Beaucage, S. L. and Caruthers, M. H. (1981) Tet. Lets. 22, 1859-1862) and coupled to produce oligonucleotides 22a and 22b using a modified synthesis cycle for DNA synthesis with a coupling time of 2 min. Under these conditions, coupling yields greater than 96%, as determined by trityl assay, were routinely obtained. Other oligonucleotides were prepared by a combination of additions of standard monomers and dimer amidites. The cleavage from the column and deprotection of the substituted phthalimido groups was accomplished with ethylendiamine in acetonitrile and the

oligonucleotides were purified by conventional hplc methods.

Hybridization Experiments

The alternating sequences 22a and 22b were hybridized to the complementary sequence d-A(pA)₁₂ and the results are shown in Table 1.

Table 1

Hybridization of Modified Oligonucleotides to

Complementary

Sequences d-A (pA)₁₂and r-A (pA)₁₂.

OligoMelting Temp (°C) nucleotide Isomer Target Low Salt^aHigh

Salt^b

d-T(pT)₁₂ n/a d-A(pA)₁₂ 3447

22a Rp d-A(pA)₁₂ 5151

22b Sp d-A(pA)₁₂ 1217 d-T(pT)₁₂ n/a r-A(pA)₁₂ 29.539

22a Rp r-A(pA)₁₂ 34.536.5

22b Sp r-A(pA)₁₂ --

^a150mM NaCl, 10mM Na₂HPO₄, ^b1M NaCl, 10 mM Na₂HPO₄.

As with the aminomethyl series, one isomer hybridized more strongly than its natural counterpart, whereas the other did not form a stable hybrid. It is of interest that under low salt conditions the aminoethyl analog formed a more stable hybrid than the corresponding aminomethyl analog (Tm 51 °C versus 45 °C for the latter), and the dissociation temperature was independent of the salt conditions as has been observed for other oligonucleotides with cationic groups (Letsinger et al ., J. Am. Chem. Soc., 110:4470-4471 (1988)). The relatively high Tm of the Rp isomer versus the Sp isomer is

consistent with data previously obtained for

stereoisomers of oligonucleotide methylphosphonates

(Lesnikowski, Z. J., Jaworska, M. and Stec, W. J. (1990) Nucleic Acids Res. , 18, 2109-2115).

Since the presence of positive charges might potentially increase the likelihood of non-specific hybridization, the stability of hybrids of oligonucleotides 23a and 23b, each possessing one aminoethylphosphonate moiety, with a fully complementary sequence and with a series of

partially mismatched complements was examined. The mismatched bases were introduced opposite to the

aminoethyl group and the results are shown in Table 2.

Table 2

Hybridization of 23a and 23b to Partially Complementary Sequences Possessing a Mismatch Opposite to the

Aminoethyl Group.

Melting Temperature

(°C) of Duplex Oligonucleotide with

Matched or Mismatched Sequence

23a 23b

T(pT)₁₂

d-A(pA)₁₂ 37.5 30

35.5

A(pA)₅pC(pA)₆ 24 17 22

A(pA)₅pG(pA)₆ 24.5 17.5 23

A(pA)₅pT(pA)₆ 24 17.5

22.5

A(pA)₆pC(pA)₅ 24.5 16.5 22

A(pA)₆pG(pA)₅ 24.5 17 23

A(pA)₆pT(pA)₅ 24.5 16.5

22.5

In terms of hybridization to the fully complementary targets, a modest difference was observed for the two isomers, one producing an elevation of 2° versus natural whereas the other caused a marked depression of 5.5°. In all cases the introduction of mismatches produced a substantial depression of melting temperature, i.e.12.5-13.5° for the Sp isomer 23b, 13-13.5° for the Rp isomer 23a, versus 12.5-13.5° for the natural sequence. Thus the introduction of an aminoethyl group does not result in a loss of specificity, and the presence or absence of paired bases appears to be the primary factor for retention of specificity.

Since the ionization of the cationic groups depends on the pH of the medium, a series of hybridization

experiments was carried out with 22a and 22b in which the pH of the solution was varied and these results are summarized in Table 3.

Table 3

Effect of pH on the Tm of d-T(pT)₁₂ or 22a with d-A(pA)₁₂

Dissociation Temperature^a

Duplex pH

5 6 7 8 910

d-A(pA)_{12 +}

d-T(pT)₁₂ 34 34 34 36 3530

22a + d-A(pA)₁₂ 51 51 51 47 4343

^a150mM NaCl, 10mM Na₂HPO₄

For the natural sequences d-T(pT)₁₂ and d-A(pA)₁₂ the dissociation temperature was relatively constant over the pH range of 4-9, with a decrease being observed at pH 10. This latter decrease might be due to the destabilization caused by the ionization of the imino protons of the thymine residues. The hybridization of 22a versus d-A(pA)₁₂ showed a much more dramatic variation with changes of pH, with the Tm's being in all cases substantially higher than the natural duplex. A Tm of 51° was observed over the pH range of 5-7 with a substantial decrease at pH 8-10. These data suggest that at pH 5-7 the ionized aminoethyl groups play an important role in enhancing stability either by interstrand or intrastrand ionic attractions, whereas at higher pH values these attractive forces are absent since the amino groups are unionized. These data are consistent with the pK values reported for (2-aminoethyl) phosphonic acid (Petrov, K. A., Chauzov, V. A. and Erokhina, T. S. (1974) Russian Chem. Reviews, 43, 984-1006).

The hybridization of the alternating-backbone

oligonucleotides 22a and 22b to RNA was also studied, using r-A(pA)₁₂ as the target and the results are also shown in Table 1. Under low salt conditions the Rp isomer 22a exhibited a Tm 5° higher than the natural

counterpart, whereas under high salt conditions the Tm of 22a was actually lower than the natural sequence. As with the hybridization to DNA, the modified oligonucleotides show less salt dependence, as would be expected for a hybrid in which the charge-charge repulsions of the backbone have been reduced. The Sp isomer 22b did not exhibit a detectable Tm under any salt conditions.

Resistance to nucleases

The unprotected dinucleotides 25a and 25b were incubated with SI nuclease which is known to degrade DNA, and the digestion products were exmined by hplc. As expected, the aminoethylphosphonate linkages were found to be

completely resistant under the conditions employed. At the conclusion of the incubation, an aliquot of the natural oligonucleotide d-T(pT)₁₂ was added, and the mixture was analyzed to confirm that the enzyme had retained activity during the entire incubation period. Induction of RNase H activity

It was of interest to determine if the alternating oligomers would induce RNAse H activity when hybridized to a target RNA molecule. For this experiment an

oligonucleotide with an rA₁₃ core and deoxynucleotide flanking regions was synthesized and ³²P-end labelled for use as the target. Cleavage of the RNA region of the target would thus be detected by the appearance of a shorter ³²P-labelled fragment. As described above, the Tm of the alternating Sp isomer 22b is so far below the incubation temperature necessary for the enzyme assay that this molecule was not expected to induce RNAse H activity. The Tm of the Rp isomer 22a, on the other hand, is higher that of the natural oligomer and the

temperature needed for the assay, so that induction of RNase H might be possible with this oligomer. After incubation of samples of 22a, 22b and d-T(pT)₁₃ with labelled target, the products were examined by native PAGE. Analysis of the autoradiogram confirmed the lack of hybridization for the Sp isomer 22b, whereas the expected retardations due to hybridization for the control

oligomer and the Rp isomer 22a were observed. Under the conditions employed the d-T(pT)₁₃ control is effective in inducing RNAse H activity with as little as a 10-fold molar excess over target, whereas the Rp isomer 22b showed no sign of such activity even with a 1000-fold excess of oligonucleotide.

Serum stability

In order to assess the serum stability of

oligonucleotides with alternating

aminoethylphosphonate/phosphodiester linkages,

oligonucleotides 26a and d-T(pT)₁₂ were incubated in conditioned medium containing 10% fetal bovine serum (FBS) and the products were examined by autoradiography. Superior nuclease-stability was observed in the oligonucleotide with the alternating aminoethyl linkages when challenged with serum-containing conditioned medium. Samples of the natural oligonucleotide d-T(pT)₁₂ were incubated in buffer with the addition of serum after 0 hour, 1 hour, 4 hours and 24 hours respective

Substantial degradation was evident after 1 hour and the oligonucleotide was almost completely degraded after 4 hours. Oligonucleotide 26a was incubated under the same conditions in buffer, and in the presence of serum for 0 hour, 1 hour, 4 hours, and 24 hours respectively. Even after 24 hours 26a was essentially undegraded. Thus the natural oligomer showed significant degradation after only one hour whereas the alternating aminoethyl linkages of 26a provide increased stability so that degradation is barely visible after 24 hours. Interestingly, it appears that there was a neighboring group protective effect since there was little evidence of significant

degradation for the 5'- or 3'- terminal or the internal phosphodiester linkages flanked by the aminoethyl

linkages.

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

Claims

WHAT IS CLAIMED IS:

1 . A method for modulating the activity of a target molecule which comprises contacting the target molecule whose activity is to be modulated with an oligonucleotide wherein at least one nucleotide unit of said

oligonucleotide includes a phosphonate moiety having the structure:

wherein X is:

, and wherein R₁ is a hydrocarbon, and each of R₂, R₃ and R₄ is independently hydrogen or a hydrocarbon.

2. The method of claim 1 wherein the target molecule is a protein.

3. The method of claim 2 wherein the protein is a cytokine.

4. The method of claim 2 wherein the protein is selected from the group consisting of basic fibroblast growth factor, gamma interferon and gp30 glycoprotein.

5 . The method of claim 1 wherein R₁ is alkylene.

6 . The method of claim 2 wherein R₁ is an alkylene group having from 1 to about 5 carbon atoms.

7 . The method of claim 6 wherein the alkylene is

selected from the group consisting of methylene, ethylene, propylene, vinylene, substituted vinylene, ethynylene, allyl and substituted allyl.

8 . The method of claim 7wherein the substituted vinylene and substituted allyl are substituted with methyl or ethyl moieties.

9 . The method of claim 1 wherein the oligonucleotide is a pure stereoisomer.

10 . The method of claim 9 wherein the stereoisomer is an R isomer.

11 . The method of claim 9 wherein the stereoisomer is an S isomer.

12 . The method of claim 3 wherein the oligonucleotide comprises an alternating sequence of aminoalkyl

phosphonate and phoephodiester nucleotide units.

13 . The method of claim 1 wherein the oligonucleotide is conjugated to at least one moiety that enhances its uptake into cells.

14 . The method of claim 1 3 wherein the moiety is

selected from the group consisting of polyethylene glycol, polylysine, peptides, acridine, dodecanol, cholesterol, biotin, folate, gluathione and mannose-6- phoephate.

15 . A method for selectively regulating gene expression which comprises contacting the gene to be regulated with a complementary oligonucleotide having at least one nucleotide unit that includes a phosphonate moiety with the formula:

wherein X is:

and wherein R₁ is a hydrocarbon, and R₂, R₃ and R₄ are each independently hydrogen or a hydrocarbon.

16 . The method of claim 15 wherein R₁ is alkylene.

17 . The method of claim 16 wherein R₁ is an alkylene group having from 1 to about 5 carbon atoms.

18 . The method of claim 17 wherein the alkylene is selected from the group consisting of methylene, ethylene, propylene, vinylene, substituted vinylene, ethynylene, allyl and substituted allyl.

19 . The method of claim 18 wherein the substituted nivylene and substituted allyl are substituted with methyl or ethyl moieties.

20 . The method of claim 15 wherein the oligonucleotide is a pure stereoisomer.

21 . The method of claim 20 wherein the stereoisomer is an R isomer.

22 . The method of claim 20 wherein the stereoisomer is an S isomer.

23 . The method of claim 17 wherein the oligonucleotide comprises an alternating sequence of aminoalkyl

phosphonate and phosphodiester nucleotide units.

24 . The method of claim 15 wherein the oligonucleotide is conjugated to at least one moiety that enhances its uptake into cells.

25 . The method of claim 24 wherein the moiety is

selected from the group consisting of polyethylene glycol, polylysine, acridine, dodecanol, cholesterol, biotin, peptides, folate, glutathione and mannose-6- phosphate.

26 . A composition for modulating the activity of a

protein or nucleic acid which comprises an isolated stereoisomer of an oligonucleotide wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the formula:

wherein X is:

, and wherein R₁ is a hydrocarbon, and R₂, R₃ and R₄ are each independently hydrogen or a hydrocarbon.

27 . The composition of claim 2 6 wherein R₁ is alkylene .

28 . The method of claim 27 wherein R₁ is an alkylene group having from 1 to about 5 carbon atoms .

29 . The composition of claim 28 wherein the alkylene is selected from the group consisting of methylene,

ethylene, propylene, vinylene, substituted vinylene, ethynylene, allyl and substituted allyl.

30 . The composition of claim 29 wherein the substituted vinylene and substituted allyl are substituted with methyl or ethyl moieties.

31 . The composition of claim 26 wherein the

oligonucleotide comprises an alternating sequence of aminoalkyl phosphonate and phosphodiester nucleotide units.

32 . The composition of claim 26 wherein the

stereoisomer is an R isomer.

33 . The composition of claim 26 wherein the

stereoisomer is an S isomer.

34 . The composition of claim 18 wherein the

oligonucleotide is conjugated to at least one moiety that enhances its uptake into cells.

35 . The composition of claim 33 wherein the moiety is selected from the group consisting of polyethylene glycol, polydysine, peptides, acridine, dodecanol, cholesterol, biotin, folate, glutathione and mannose-6- phosphate.

36 . A method for detecting a target protein or

carbohydrate in a sample which comprises (i) contacting the sample with an oligonucleotide that selectively binds with the target wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having the formula wherein X is:

and wherein R₁ is a hydrocarbon, and R₂ and R₃ are each independently hydrogen or a hydrocarbon and R₅ is detectable marker; and

(ii) observing any detectable response.

37 . A composition for detecting a target protein or carbohydrate in a sample, which composition comprises an isolated stereoisomer of an oligonucleotide that selectively binds with the target and wherein at least one nucleotide unit of said oligonucleotide includes a phosphonate moiety having, the structure:

wherein X is

and wherein R¹ is a hydrocarbon, R₂ and R₃ are each independently hydrogen or a hydrocarbon and R₅ is a detectable marker.