WO1998005675A2 - Radiolabeled o-methyl phosphotriester and o-methyl phosphorothioate internucleoside linkages and oligonucleotides containing the same - Google Patents

Abstract

The invention provides oligonucleotides containing methyl phosphotriester linkages and processes for making and methods for using such oligonucleotides. The preparation of radiolabeled oligonucleotides is also disclosed. The radiolabel is a 14C-methyl group attached to an oxygen atom of an internucleoside phosphate group.

Description

RADIOLABELED O-METHYL PHOSPHOTRIESTER AKD O-METHYL PHOSPHOROTHIOATE INTERNUCLEOSIDE LINKAGES AND OLIGONUCLEOTIDES CONTAINING THE SAME

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to synthetic oligonucleotides and to their use in molecular biology applications and in the antisense therapeutic approach.

Summary of the Related Art

Oligonucleotideε have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e . g . . Methods in Molecular Biology, Vol 20 : Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues : A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Pey- man, supra . Agrawal and Iyer, Curr. Op . in Biotech . 6,: 12 (1995) ; and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993) . Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol . 72.: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34 : 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides . These early approaches have largely given way to the more efficient phosphora- midite and H-phosphonate approaches to synthesis . Beaucage and Carrutherε, Tetrahedron Lett. 21- 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramiditeε in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.

Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett . 2Ji: 3539-3542 (1987) , teaches synthesis of oligonucleotide methylphoεphonates using phoεphoramidite chemistry. Connolly et al., Biochemistry 23 : 3443 (1984), discloses synthesis o£ oligonucleotide phospho- rothioates using phosphoramidite chemistry. Jager el al . , Biochemistry 21_ : 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al . , Proc. Watl. Acad . Sci . USA £5: 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.

The routine synthesis of oligonucleotides is presently carried out using various N-acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine) , and benzoyl for adenine and cytosine. After the synthesis of the oligonucleotides is carried out using either phosphoramidite chemistry or H-phosphonate chemistry, the protecting groups are removed by treatment with o ammonia at 55-60 C for 5-10 hours. Using these protecting groups,

PO oligonucleotides and other modified oligonucleotides can be synthesized. But in certain instances where modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection iε being carried out.

This limitation in the oligonucleotide synthesis procedure has resulted in the inability to synthesize certain modified oligonucleotides that may have considerable utility. For example, oligonucleotides containing methyl phosphotriester intemucleotide linkages could have many beneficial properties, because the methyl phosphotriester group is nonionic, but is similar in size and molecular shape to the phosphodiester linkage. Such nonionic methyl phosphotriester linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides. For example, Galbraith et al . , Antisense Research and Development 4.: 201-206 (1994) disclose complement activation by oligonucleotides. Henry et al . , Pharm. Res. ϋ: PPDM8082 (1994) discloses that oligonucleotides may potentially interfere with blood clotting.

The art has recognized the desirability of incorporating methyl phosphotriester intemucleotide linkages into oligonucleotides and many attempts have been made to make and use such oligonucleotides. However, these attempts have subsequently been discovered to be unsuccessful. Miller et al., J. Am. Chem. Soc. 93: 6657-6665 (1971), discloses alleged methylphosphotriester DNA synthesis by methylation of the phosphate using p-toluenesulphonyl chloride and methanol. Moody et al., Nucl. Acids Res. 2 _: 4769- 4783 (1989), discloses regiospecific inhibition of DNA duplication by oligonucleotides synthesized according to the method of Miller et al . . Buck et al . . Science 248: 208-212 (1990), discloses that oligonucleotides according to Moody et al . inhibit viral infectiv- ity of HTV-1. However, Buck et al . . Science 2 l- 125-126 (1990), retracts the earlier Buck et al. report and discloses that oligonucleotides synthesized according to this method do not contain methyl phosphotriester inte ucleotide linkages. The difficulty in synthesizing oligonucleotides having methyl phosphotriester intemucleotide linkages is due to the lability of the methyl ester bond under the oligonucleotide synthesis conditions used in the steps of deprotecting the nucleoside bases and cleaving the oligonucleotides from the solid support. Alul et al., Nucl. Acids Res. i£: 1527-1532 (1991), addressed the problem of cleaving the oligonucleotide from the solid support by introducing an oxalyl-type linker that can be cleaved under conditions that preserve the methyl ester bond. However, the problem of base deprotection was not addressed, so they were only able to synthesize methyl phosphotriester-linked thymidines, which lack an exocyclic amino group and thus do not require deprotection. Kuijpers et al . , Nucl. Acids Res. .18: 5197-5205 (1990), attempted to address the deprotection problem by treating the nucleoside bases for 43 hours in potassium carbonate/ methanol. Unfortunately, NMR analysis of their oligonucleotides revealed that considerable demethylation had occurred, resulting oligonucleotides having a mixture of methylphosphotriester and phosphodiester linkages. Similarly, Vinogradov et al.. Tetrahedron Lett. 3_4_: 5899-5902 (1993), attempted to solve the problem by using an isopropoxyacetyl group to protect the nucleoside bases, but found that at least 35-40% demethylation still occurred. Most recently, Hayakawa et al., J. Org. Chem. 60: 925-930 (1995), claimed to have synthesized a decamer oligonucleotide containing a single methyl phosphotriester intemucleotide linkage. However, NMR data supporting th s claim was absent. Moreover, the method employed utilized costly and toxic palladium, which could contaminate the oligonucleotide product and render it unsuitable for therapeutic applications. In addition, the method was not shown to be able to introduce multiple methylphosphotriester linkages into the oligonucleotide.

There is therefore, a need for oligonucleotides containing methyl phosphotriester inte ucleotide linkages, as well as for new methods for synthesizing such oligonucleotides. Ideally, such oligonucleotides should be easy to synthesize and should be capable of containing numerous other beneficial modifications. Moreover, there is a need to be able to radiolabel such oligonucleotides, so that they can be followed in the body. Ideally, such labeling should utilize isotopes other than the commonly used ³²P or ³⁵S, which should also allow for multiple labeling studies, e. g. , to follow the metabolic fate of the oligonucleotides .

BRIEF SUMMARY OF THE INVENTION

The invention provides oligonucleotides containing O-methyl phosphotriester or phosphorothioate intemucleotide linkages and processes for making and methods for using such oligonucleotides . The oligonucleotides according to the invention are easy to synthesize and can conveniently be made to contain numerous other beneficial modifications. In addition, the invention provides methods for radiolabeling such oligonucleotides using ^⁴C as the isotopic label, and further provides oligonucleotides labeled in such manner .

In a first aspect, the invention provides oligonucleotides containing O-methyl phosphotriester or phosphorothioate inte ucleotide linkages having the structure I:

(I) wherein "Nucl* represents the 3' position of a first nucleoside, •Nuc2" represents the 5' position of a second nucleoside, C* represents ¹²C or ¹⁴C, and X represents sulfur or oxygen. As used hereafter, the term 'methyl phosphotriester* is intended to include both O-methyl phoεphotriesterε and O-methyl phosphorothioates, as in (I), above The linkage provides the benefit of having a molecular size that is similar to that of a natural phosphodiester linkage, but at the same time having nonionic character. Such an intemucleoside linkage should confer upon an oligonucleotide a reduction in polyanion-mediated side effects and should also improve cellular uptake of the oligonucleotide. It also provides a convenient location for incorporation of a radiocarbon label.

Oligonucleotides according to this aspect of the invention have from one to about all intemucleotide linkages in the form of methyl phosphotriester linkages. In embodiments of oligonucleotides according to this aspect of the invention that have fewer than all methyl phosphotriester intemucleoside linkages, the other inte ucleoside linkages may be any of the known inter- nucleoside linkages, or may be any intemucleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the process according to the invention is compatible.

Oligonucleotides containing such a mixture of intemucleoside linkages are referred to herein as mixed backbone oligonucleotides. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, the intemucleoside linkages that are not methyl phosphotriester linkages are selected from the group consisting of phosphodiester, alkylphoεphonate, carbamate and phosphorothioate intemucleoside linkages. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, several adjacent nucleosides comprising one region of the oligonucleotide are connected by methyl phosphotriester linkages, and several other adjacent nucleosides comprising another region of the oligonucleotide are connected by a different type of intemucleoside linkage. These preferred oligonucleotides are referred to herein as "chimeric" oligonucleotides .

Oligonucleotides according to the invention are useful for a variety of purposes. For example, the labeled oligonucleotides according to the invention can be used as probes in conventional nucleic acid hybridization assays . Oligonucleotides according to the invention can also be used as antisense "probes" of specific gene function by being used to block the expression of a specific gene in an experimental cell culture or animal system and to evaluate the effect of blocking such specific gene expression. In this use, oligonucleotides according to the invention are preferable to traditional "gene knockout" approaches because they are easier to use and can be used to block specific gene expression at selected stages of development or differentiation. Finally, oligonucleotides according to the invention are useful in the antisense therapeutic approach. In this use, oligonucleotides according to the invention should have reduced polyanion-mediated side effects and improved cellular uptake. Radiolabeled

oligonucleotides in this use can be followed in the body to assess their metabolic fate and provide information on optimal dosing.

In a second aspect, the invention provides a simple process for synthesizing an oligonucleotide containing from one to about all methyl phosphotriester intemucleoside linkages. In one embodiment, this process comprises condensing in the presence of lH-tetrazole a methoxy-3 '-0- (phosphoramidite) -5 '-0-(4, 4'- dimethoxytriphenyl)methyl nucleoside with another nucleoside, wherein at least one of the nucleosides has a nucleoside base- protecting group, to produce adjacent nucleosides coupled by a phosphite linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group, oxidizing the internucleotidic phosphite linkage, and chemoselectively removing the nucleoside base-protecting group with a chemoselective removing agent, without demethylating the methyl phosphotriester linkage(s) . A chemoselective removing agent means an agent that iε capable of removing a base protecting group according to the invention. In certain preferred embodiments, the chemoselective removing agent is selected from the group consisting of halogens, especially Br2, CI2 and ∑2, any of which are preferably taken up in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or as an N-halosuccinimide. In alternative embodiments, non-chemoselective reagents may be used, such as aqueous ammonium hydroxide, alcoholic ammonia, alkali carbonates in organic solvents, primary or secondary amines, alkali hydroxides, or any amidolytic reagent, i.e., an agent capable of hydrolyzmg an amide linkage Another embodiment comprises condensing in the presence of a suitable activator, such as pivaloyl chloride, a nucleoside H-phosphonate or thio-H- phosphonate with another nucleoside, wherein at least at least one of the nucleosides has a nucleoside base protecting group, to produce adjacent nucleosides coupled by an H-phosphonate or thio- H-phosphonate linkage, wherein at least one of the nucleosides has a nucleoside base protecting group, oxidizing the H-phosphonate linkage in carbon tetrachloride/pyridine/methanol or in carbon tetrachloride/N-methyl imidazole/triethylamine/methanol to produce an O-methylphosphotriester or O-methylphosphorothioate linkage, then chemoselect vely removing the nucleoside base protecting group with a chemoselective removing agent, without demethylating the O-methylphosphotriester or O-methylphosphorothioate linkage, as described previously, and most preferably in 12/pyridine/ methanol. In this latter embodiment, the methyl phosphotriester linkage can be conveniently labelled by using ¹⁴C-methanol.

In preferred embodiments, the process accord ng to this aspect of the invention iε carried out on a solid support and in most preferred embodiments further comprises the step of cleaving the oligonucleotide from a solid support without demethylating the methyl phosphotriester linkage(ε) . This process allows for synthesis of oligonucleotides containing methyl phosphotriester intemucleoside linkages, because the process utilizes a new nucleoside base protecting group that can be chemoselectively removed, in contrast to the harsh deprotecting conditions utilized by known methods, which would demethylate the sensitive methyl phosphotriester linkage. Importantly, the process according to the invention is compatible with and can be used in conjunction with any of the well known oligonucleotide synthetic chemistries, including the H-phosphonate, phosphoramidite and phosphotriester chemistries. Consequently, the process according to the invention can be used to synthesize oligonucleotideε having methyl phosphotrieεter linkages at some intemucleoside positions and other linkages at other intemucleoside positions .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a scheme for a preferred embodiment of a process for synthesis of an oligonucleotide containing O-methyl phosphotriester intemucleoside linkages. In this scheme, a) is IH-tetrazole; b) is t-BuOOH (1 M in toluene); c) is DCA/DCM; d) is 12 (2% in Pyr/MeOH 98/2); e) is 3H-benzodithiol-3-one 1,1-dioxide; and f) is anhyd K₂C0₃/MeOH (0.05 M) .

Figure 2 shows results of ^Ip-NR (D2O, 85% H3PO4 as external reference) and -^H-NMR (D2O) for a trinucleotide chimera according to the invention.

Figure 3 shows results of 31p_NR for two nonanucleotide chimeras according to the invention (panels A and B) , and for a phoεphodieεter-phosphorothioate chimera of identical sequence (panel C) .

Figure 4 shows results of polyacrylamide gel electrophoresis for two nonanucleotide chimeras according to the invention (first two lanes) , and for a phosphodiester-phosphorothioate chimera of identical sequence (last lane) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to synthetic oligonucleotides and to their use in molecular biology applications and in the antisense therapeutic approach. The patents and publications identified in this specification are within the knowledge of those skilled in this field and are hereby incorporated by reference in their entirety.

The invention provides oligonucleotides containing methyl phoεphotriester linkages and processes for making and methods for using such oligonucleotides . The oligonucleotides according to the invention are easy to synthesize and can conveniently be made to contain numerous other beneficial modifications.

In a first aspect, the invention provides oligonucleotides having from one to about all inte ucleotide linkages in the form of a methyl phosphotriester inte ucleoside linkage having the structure I:

(I)

wherein "Nucl" represents the 3' position of a first nucleoside, "Nuc2" represents the 5' position of a second nucleoside, C* is Q or 1⁴C, and X is sulfur or oxygen. This structure is similar in molecular size and shape to the natural phosphodiester intemucleoside linkage, and as such, should not contribute significantly to any steric constraints to the oligonucleotide. Accordingly, this intemucleoside linkage should not have a significant effect on the ability of an oligonucleotide to hybridize with a complementary nucleic acid. The linkage provides the benefit of having nonionic character. Such an inte ucleoside linkage should confer upon an oligonucleotide a reduction in polyanion-mediated side effects and should also improve cellular uptake of the oligonucleotide.

For purposes of the invention, the term "oligonucleotide" includes polymers of two or more deoxyribonucleotide or 2 ' -O- substituted ribonucleotide monomers, or any combination thereof. Such monomers may be coupled to each other by any of the numerous known intemucleoside linkages. In certain preferred embodiments, these inte ucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/ or having additional εubstituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane. For purposes of the invention the term "2'-0-εubεtituted" means substitution of the 2' position of the pentoεe moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubst tuted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy grou ( to produce a ribonucleoside) , an amino or a halo group, but not with a 2'-H group.

Oligonucleotides according to the invention will preferably have from about 12 to about 50 nucleotides, most preferably from about 17 to about 35 nucleotides. Preferably, such oligonucle- otideε will have a nucleotide sequence that is complementary to a genomic region, a gene, or an RNA transcript thereof The term complementary means having the ability to hybridize to a genomic region, a gene, or an RNA transcript thereof under physiological conditions. Such hybridization is ordinarily the result of base- specific hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridization. As a practical matter, such hybridization can be inferred from the observation of specific gene expression inhibition. The gene sequence or RNA transcript sequence to which the modified oligonucleotide sequence is complementary will depend upon the biological effect that is sought to be modified. In some cases, the genomic region, gene, or RNA transcript thereof may be

from a virus. Preferred viruses include, without limitation, human immunodeficiency virus (type 1 or 2), influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus, cytomegalovirus, respiratory syncytial virus, influenza virus, hepatitis B virus, hepatitis C virus and papillo a virus. In other cases, the genomic region, gene, or RNA transcript thereof may be from endogenous mammalian (including human) chromosomal DNA. Preferred examples of such genomic regions, genes or RNA transcripts thereof include, without limitation, sequences encoding vascular endothelial growth factor (VEGF) , beta amyloid, DNA methyltransferase, protein kinaεe A, ApoE4 protein, p-glycoprotein, c-MYC protein, BCL-2 protein, protein kinase A and CAPL. In yet other cases, the genomic region, gene, or RNA transcript thereof may be from a eukaryotic or prokaryotic pathogen including, without limitation, Plasmodiurn alciparum, Plasmodium malarie, Plasmodium ovale, Schiεtoso a spp . , and Wycσbaσterium tuberculosis.

In embodiments of oligonucleotides according to this aspect of the invention that have fewer than all methyl phosphotriester inte ucleoside linkages, the other inte ucleoside linkages may be any of the known inte ucleoside linkages, or may be any inter- n cleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the

process according to the invention is compatible . In certain preferred embodiments, the other intemucleoside linkages are phosphodiester or phosphorothioate linkages. In the case of phosphorothioate inte ucleoside linkages, the linkages may be phosphorothioate mixed enantiomers or stereoregular phosphorothioateε (see Iyer et al . , Tetrahedron Asymmetry 6.: 1051-1054 (1995).

Oligonucleotides containing such a mixture of intemucleoside linkages are referred to herein as mixed backbone oligonucleotides. In some preferred embodiments of mixed backbone oligonucleotides according to the invention, several adjacent nucleosides comprising a first region of the oligonucleotide are connected by methyl phosphotriester linkages, and several other adjacent nucleosides comprising a second region of the oligonucleotide are connected by a different type of intemucleoside linkage. These preferred oligonucleotides are referred to herein as "chi- meric" oligonucleotides or "chimeras" . In certain particularly preferred chimeric oligonucleotides according to the invention, the oligonucleotide comprises a methyl phosphotriester region and a phosphorothioate and/or phosphodiester region. In this context, a "methyl phosphotriester region" is a region within an oligonucleotide of from about 2 to about 15 contiguous nucleosides linked to each other through methyl phosphotriester linkages according to the invention, I. A "phosphorothioate region' is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphorothioate linkages A "phosphodiester region" is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphodiester linkages In most preferred chimeric oligonucleotides according to the invention, the oligonucleotide comprises a phosphorothioate or phosphodiester region flanked on either side by a methyl phosphotriester region, or alternatively, a methyl phosphotriester region flanked on either side by a phosphorothioate or phoεphodiester region. In one preferred embodiment the nucleoεideε of one or more of the methyl phoεphotriester region, the phosphodieεter region and/ or the phosphorothioate region are 2 '-o-substituted ribonucleotides, as defined above herein. Preferred chimeric oligonucleotides according to the invention are further characterized by having the ability to activate RNaseH.

Oligonucleotides according to the invention are useful for a variety of purposes. For example, they can be labelled with a reporter group and used as probes in conventional nucleic acid hybridization assays. They can also be used as antisense "probes* of specific gene function by being used to block the expression of a specific gene in an experimen al cell culture or animal system and to evaluate the effect of blocking such specific gene expression. This is accomplished by administering to a cell or an animal an oligonucleotide according to the invention that has a nucleotide sequence that is complementary to a specific gene that iε expressed in the cell or animal to inhibit the expression of the specific gene, and observing the effect of inhibiting the expression of the specific gene. In this use, oligonucleotides according to the invention are preferable to traditional "gene knockout" approaches because they are easier to use and can be used to block gene specific gene expression at selected stages of development or differentiation.

Finally, oligonucleotides according to the invention are useful in the antisense therapeutic approach. In this use, oligonucleotides according to the invention should have reduced polyanion-mediated side effects and improved cellular uptake. For therapeutic use, oligonucleotides according to the invention may optionally be formulated with any of the well known pharmaceutically acceptable carriers or diluents . This formulation may further contain one or more additional oligonucleotides according to the invention. Alternatively, this formulation may contain one or more other antisense oligonucleotide, such as an oligonucleotide phosphorthioate, a RNA/DNA hybrid oligonucleotide, or a chimeric oligonucleotide containing known inte ucleoside linkages, or it may contain any other pharmacologically active agent.

Therapeutic use of oligonucleotides according to the invention is for treating a disease caused by aberrant gene expression. This is accomplished by administering to an individual having the disease a therapeutically effective amount of an oligonucleotide

according to the invention, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein such aberrant expression causes the disease. In this context, aberrant gene expression means expression in a host organism of a gene required for the propagation of a virus or a prokaryotic or eukaryotic pathogen, or inappropriate expression of a host cellular gene. Inappropriate host cellular gene expression includes expression of a mutant allele of a cellular gene, or underexpresεion or overexpresεion of a normal allele of a cellular gene, such that diεeaεe results from such inappropriate host cellular gene expression. Preferably, such administration should be parenteral, oral, sublingual, transdermal, topical, intranaεal or intrarectal. Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease. When administered syεtemically, the therapeutic compoεition iε preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.01 micromolar to about 10 micromo- lar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may desirable to administer simultaneously, or sequentially a therapeutically

effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode

In a second aspect, the invention provides a simple process for synthesizing an oligonucleotide containing from one to about all methyl phosphotriester intemucleoside linkages In one embodiment, this process comprises condensing in the presence of IH-tetrazole a methoxy-3 '-0- (phosphoramidite) -5 '-O- (4,4' - dimethoxytriphenyl)methyl nucleoside with another nucleoside, wherein at least one of the nucleosides has a nucleoside base- protecting group, to produce adjacent nucleosides coupled by a phosphite linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group, oxidizing the internucleot dic phosphite linkage, and chemoselectively removing the nucleoside base-protecting group with a chemoselective removing agent, without demethylating the methyl phosphotriester linkage (s) In a preferred embodiment, the internucleotidic phoεphite linkage is oxidized using t-butyl hydroperoxide, most preferably in toluene, to yield an O-methyl phosphotriester linkage. However, other non- iodine-based oxidizing agents are known in the art and may be used for th s purpose (see e.g., Beaucage and Iyer, Tetrahedron 48: 2223 (1992) . In another preferred embodiment, the internucleotidic phosphite linkage is oxidized using 3H-benzodithiol-3- one 1,1-dioxide to yield an S-methyl phosphotriester linkage. See Figure 1. A chemoselective removing agent means an agent that is capable of removing a base protecting group according to the invention. In certain preferred embodiments, the chemoselective removing agent is selected from the group consisting of halogens, especially Br2, CI2 and I2, any of which are preferably taken up in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or as an N-halosuccinimide. In alternative embodiments , non-chemoselective reagents may be used, such as aqueous ammonium hydroxide, alcoholic ammonia, alkali carbonates in organic εolventε, primary or secondary amines, alkali hydroxides, or any amidolytic reagent, i.e., an agent capable of hydrolyzing an amide linkage. A preferred alkali carbonate in an organic solvent iε potaεεium carbonate in methanol, most preferably at a concentration of about 0.05 M.

Another embodiment comprises condensing in the presence of a suitable activator, such as pivaloyl chloride, a nucleoside H- phosphonate or thio-H-phosphonate with another nucleoside, wherein at least at least one of the nucleosides has a nucleoside base protecting group, to produce adjacent nucleosides coupled by an H- phoεphonate or thio-H-phosphonate linkage, wherein at least one of the nucleosides has a nucleoside base protecting group, oxidizing the H-phosphonate linkage in a suitable halogen source, such as CCI4, l2» or an N-halosuccirianu.de, together with a suitable non- nucleophilic base, such as pyridine, DBU, N-methylimidazole, or KOtBu to produce an O-methylphosphotriester or O- methylphosphorothioate linkage, then chemoselectively removing the nucleoside base protecting group with a chemoselective removing agent, without demethylating the O-methylphosphotriester or 0- methylphosphorothioate linkage, as described previously, and most preferably in 12/pyridine/methanol . Preferred combinations of halogen source and non-nucleophilic base include carbon tetrachloride/pyridine/methanol or in carbon tetrachloride/N- methyl imidazole/triethylamine/methanol. When using carbon tetrachloride/pyridine/methanol in thiε embodiment, the preferred ratios are about 9/1/1 and the incubation time iε preferably about 10 minutes. When using carbon tetrachloride/N-methyl imidazole/ triethylamine/methanol in thiε embodiment, the preferred ratios are about 9/0.5/0.5/1.1 and the incubation time is preferably about 10 minutes. In this embodiment, the methyl phosphotriester linkage can conveniently be labelled by using ¹⁴c-methanol .

In preferred embodiments, the proceεs according to this aspect of the invention is carried out on a solid εupport and in moεt preferred embodiments further comprises the step of cleaving the oligonucleotide from a solid support without demethylating the methyl phosphotriester linkage(ε) . This process allows for synthesis of oligonucleotides containing methyl phosphotriester inte ucleoside linkages, because the process utilizes a new nucleoside base protecting group that can be chemoselectively removed, in contrast to the harsh deprotecting conditions utilized

by known processes, which would cleave the sensitive methyl phosphotriester linkage. The new nucleoside base protecting group has the general

wherein nj_, 2 and n are each independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R3 shown may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R^ R₂ and R₃ are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, eεter, carboxyl, or aldehyde group, and wherein dotted lineε represent alternative exocyclic or endocyclic double bonds. In a preferred embodiment, a is hydrogen when n^ is 0 and iε carbon or nitrogen when n^ iε 1-10, b iε hydrogen when n^ and n2 are both 0 and iε carbon or nitrogen when either or both n^ and n2 are 1-10, c is hydrogen when n₂ is 0 and is carbon or nitrogen when is 1-10, and e is hydrogen when n₃ iε 0 and is carbon or nitrogen when n₃ is 1-10. In a particularly preferred embodiment, compound XI has nj_, n₂ and n values of 0, and a, b, c ,d and e are each hydrogen, and the protecting group takes the form N-pent-4-enoyl, i.e., CH2 - CH(CH2)2^{C0 -} (III) - Compounds II and III protect the nucleoside base amino moieties by f orming amide linkages , as in :

where the nitrogen displayed is the protected amino moiety of the nucleoside base B. The chemoselective removal of the nucleoεide baεe protecting group is accomplished by using a chemoselective removing agent. In certain preferred embodiments, the chemoselective removing agent is selected from the group consisting of halogens, especially Br2, Cl₂ and I₂, any of which are preferably in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or as an N-haloεuccinimide . Cleavage of the oligonucleotide from the solid support without demethylating the methyl phosphotriester intemucleoside linkage is preferably carried out by treating the support bound oligonucleotide using anhydrous K₂C0₃, most preferably in an aprotic solvent such as methanol.

Importantly, the process according to the invention iε compatible with and can be used in conjunction with any of the well known oligonucleotide synthetic chemistries, including the H- phosphonate, phosphoramidite and phosphotriester chemistries. Consequently, the process according to the invention can be used to synthesize oligonucleotides having methyl phosphotriester linkages at some intemucleoside positions and other linkages at other intemucleoside positions. In one preferred embodiment, synthesis is carried out on a suitable solid support using either H- phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H- phoεphonate chemiεtry for εome cycleε and phosphoramidite chemistry for other cycles) . Suitable solid supports include any of the standard solid supports used for solid phase oligonucleotide synthesis, such as controlled-pore glass (CPG) . (See, e.g., Pon, Methods in Molec. Biol . ,20,: 465 (1993)). Synthesis on such a solid support begins with coupling a nucleoside synthon according to the invention to a nucleoside that is covalently linked to the solid support (i.e., linked to a functionality on the solid support, preferably an amino or hydroxyl functionality) . More generally, the process according to the invention can be used with any of the chemistries commonly used for oligonucleotide synthesis, whether in solution phase or in solid phase.

The versatility of chemical synthetic approach of the process according to the invention makes the process according to the invention suitable for the synthesis of any of a broad class of compounds, all of which are referred to herein as "oligonucleotides", as previously defined herein.

The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting n nature .

Example 1 Preparation of N-Pent-4-enovl (PNT) 2 ' -deoxy adenosine (dA Nor):

2 ' -Deoxyadenosine (Mallinkckrodt) (2.5 g, 10 mmol) was dried by repeated evaporation from anhydrous pyridine and was suspended in 50 ml of anhydrous pyridine. Trichloromethylεilane (64. ml, 50 mmol) was added and the reaction stirred for about 1 h. Then, 4-pentenoic anhydride (4g, 20 mmol) was added and the contents stirred. After 15 mm triethyl amine (3 ml) was added and the contents stirred for 2-3 h. The reaction εlurry was cooled to 0-5 C and 10 ml of water was added. After 5 min., 28% NH4OH (10ml) was added. The resulting clear solution was evaporated to drynesε . Water (150 ml) waε added and the reaction mixture was extracted with ethylacetate : ether (50 ml, 1:1) . The aqueous layer was separated and concentrated to a small volume. Upon leaving at room temperature, a white precipitate of the title compound was obtained. Filtration and drying gave ca. 3.5 g of pure title compound. Several experiments repeating the above procedure, using larger scale of operation, gave the title compound in 85-90% yield.

The same general procedure can be employed for the preparation of dG and dC protected nucleosides. Example 2 Preparation of 5 ' -0-DMT-N-4-pent-4-enoyl-nucleoside svnthons

The PNT nucleosides prepared according to Example 1 were then employed in the synthesis of beta-cyanoethyl- (CEPNT) and methoxy- (MEPNT) ' -O- (phosphoramidite) -5 ' -O- (4 , -dimethoxytriphenyl) methyl) [DMT] monomers according to standard procedures. See Beau- cage, in Protocols for Oligonucleotides and Analogs; S. Agrawal, Ed.; Humana Press: Totowa, NJ (1993); Vol. 20, pp. 33-61. The nucleoside phosphoramidites were fully characterized and the following spectral data was obtained. MEPNT ( A) . White foam; overall yield of 70-72%

31p-NMR ⁽CDCL₃₎. -_ld 147.04, 146.90 ppm (ca. R_p:S_p, 1:1 mixture) 1H-NMR (CDCL₃): 8.61 (IH, ε), 8.55 (IH, br) , 8.17 (IH, s), 7.42- 7.19 (9H, m) , 6.82-6.75 (4H, m) , 6.48 (IH, dd, J=2.9, 6.4 Hz), 5.93 (IH, ddt, J=6.5, 10.3, 17 Hz) 5.13 (IH, dd, J=17.0, 1.4 Hz), 5.04 (IH, dd, J=1.4, 10.3 Hz), 4.82-4.70 (IH, m) , 4.38-4.28 (IH, m) , 3.8 (6H, ε), 3.58 (2H, m) , 3.49 (2H, , ³J_P__H=18.1 Hz, J=6.8 Hz), 3.35 (3H, d, ³J_P_H=13.4 HZ), 3.0 (2H, t, J=7.4 Hz), 2.87 (IH, m), 2.66 (IH, m) , 2.53 (2H, ) , 1.17 (12H, dd, J=6.8 Hz, ⁴Jp__H-2.4 Hz)

FAB-MS: Calcd for C43H53N6O7P, 797 (M+H)⁺; Found m/z 797. HEPNT (dC) . Pale yellow foam; overall yield of 74-76% 31p_nM <CDC1₃): ^Λld, 147.49, 146.81 ppm (ca. Rp:Sp, 1:1 mixture). iH-NMR (CDC1₃): ^Λld 10.0 (IH, br) , 8.24 (IH, d, J=7.4 Hz), 8.18 (IH, d, J=7.4 Hz), 7.40-7.08 (9H, m) , 6.84-6.76 (4H, m) , 6.17 (IH, dd, J=6.3, 5.1 Hz), 5.78 (IH, ddt, J=6.4, 10, 16.9 Hz), 5.02 (IH, dd, J=1.4, 17,3 HZ), 4.94 (IH, dd, J=1.4, 10.2 Hz), 4.62-4.54 (IH, m) , 4.08 (IH, ) , 3.61 (6H, s), 3.56-3.40 (4H, m) , 3.26 (3H, d, 3j_p__H=13.2 Hz), 2.88-2.57 (3H, m) , 2.40-2.34 (2H, m) , 2.24-2.18 (IH, ) , 1.02 (12H, d, J=6.7 Hz).

FAB-MS: Calcd. for C₄₂H5₃ 4θ8P, 773 (M+H)⁺; Found m/z , 773. MBPNT <dG) White foam; overall yield of 70-72%

31^p-NMR (CDC1₃): ^Λld 146.78, 146.74 ppm (ca. Rp:Sp, 1:1 mixture) i-H-NMR (CDC1₃): ^Λld 8.02 (IH, br) , 7.92 (IH, s) , 7.80 (IH, ε) , 7.43-7.20 (9H, m) , 6.80-6.69 (4H, m) , 6.20 (IH, dd, J=5.6, 7.9 Hz), 5.68 (IH, m) , 4.96 (IH, dd, J=1.5, 17.1 Hz), 4.94 (IH, dd, J=1.5, 9.3 Hz), 4.72-4.63 (IH, m) , 4.14-4.07 (IH, m) , 3.63 (6H, s), 3.57-3.36 (4H, m) , 3.29 (3H, d, ³Jp__H=13.2 Hz), 3.08 (2H, m) , 2.84-2.76 (IH, m) , 2.59-2.46 (IH, m) , 2.24 (2H, m) , 1.02 (12H, d, J=6.7 Hz)

FAB-MS: Calcd for C₄₃H₅₃N6θ₈P, 813 (M+H)⁺; Found m/z, 813 CEPNT (αλ) . White foam; overall yield of 70-71% 31p-NMR (CDCI3); ^Ald 146.9, 146.81 ppm (ca. Rp:Sp, 1:1 mixture) IH-NMR (CDCI3): ^Λld 8.60 (IH, br) , 8.58 (IH, s) , 8.15 (IH, s), 7.40-7.25 (9H, m) , 6.81-6.70 (4H, m) , 6.43 (IH, dd, J=2.4, 6.6 Hz), 5.90 (IH, ddt, J=6.5, 10.3, 16.9 Hz), 5.1 (IH, dd, J=1.5, 17.1 Hz), 5.02 (IH, dd, 1.5, J=10 Hz), 4.78 (IH, m) , 4.30 (IH, ) , 4.20-4.07 (2H, m, 3.74 (6H, s), 3.66-3.54 (2H, ) , 3.48 (2H, m) , 3.40-3.31 (2H, m) , 2.98 (2H, t, J=7.3 Hz), 2.6 (IH, m) , 2.53-2.41 (3H, m) , 1.16 (12H, d, J-6.6 Hz). FAB-MS: Calcd for C45H54N₇O₇P, 836.3900 (M+H)⁺; Found, m/z , 836.3899.

CEPNT (dC) . Yellow foam; overall yield 72-75%

31p-NMR (CDCI₃); ^Λld 147.42, 146.81 ppm (ca. R_p:S_p, 1:1 mixture) IH-NMR (CDCI3): ^Λld 9.75 (lH,br), 8.20 (IH, d, J=7.3 Hz), 7.43- 7.20 (9H, m) , 7.24 (IH, d, J=7.3 Hz), 6.75-6.56 (4H, m) , 6.22 (IH, t, J=6.1 Hz), 5.8 (IH, ddt, J=6.3, 10.2, 16.6 Hz), 5.05 (IH, dd, J=1.4, 17.1 Hz), 4.98 (IH, dd, J=1.4, 10.3 Hz), 4.60 (IH, m) , 4.23-4.12 (3H, m) , 3.76 (6H, ε) , 3.66-3.33 (6H, m) , 2.58 (2H, t, J=6.6 Hz), 2.41 (3H, m) , 2.3 (IH, m) , 1.1 (12H, d, J=6.3 Hz). FAB-MS; Calcd for C44H54N5O8P, 812.3788 (M+J)⁺; Found /z, 812.3798.

CEPNT (dG) . White foam; overall yield of 70-72%

31p-NMR (CDCI3) : ^Λld 146.89, 146.83 ppm (ca. Rp-.S_p, 1:1 mixture). 3-H-NMR (CDCI3): ^Λld 8.04 (IH, br) , 7.95 (IH, s), 7.82 (IH, s) , 7.43-7.25 (9H, m) , 6.82-6.69 (4H, m) , 6.25 (IH, dd, J=5.6, 7.8 Hz), 5.70 (IH, ) , 5.00 (IH, dd, J=1.5, 17 Hz), 4.95 (IH, dd, J=1.5, 9.5 Hz), 4.70-4.60 (IH, m) , 4.15-4.06 (3H, m) , 3.65 (6H, s), 3.58-3.20 (6H, m) , 2.60 (2H, t, J=6.6 Hz), 2.45 (IH, ) , 2.28 (3H, ) , 1.09 (12H, d, J=6.4 Hz).

FAB-MS: Calcd for C45H54 7O8P, 852.3850 (M+H)⁺, Found m/z, 852.3869.

Example 3 Phosphoramidite solid Phase coupling of nucleoside svnthons . introduction of the methyl phosphotriester linkage and removal of base protecting groups Methoxy- (MEPNT) 3 ' -O- (phosphoramidite) -5 ' -O- (4, 4- dimethoxy ripheny1) methyl) [DMT] monomers were coupled in a standard lH-tetrazole-mediated phosphoramidite coupling reaction to form the dinucleoside phosphites. The dinucleoside phosphites were then oxidized using t-butyl hydroperoxide (IM in toluene) to yield the protected O-methyl phosphotriester, or 3H-benzodithiol- 3-one 1,1-dioxide to yield the protected S-methyl phosphotriester. Subsequent exposure to iodine reagent (2% I₂ in pyridine/ MeOH, 98/ 2) at room temperature for 30 minutes completely removed the base protecting groups to give CPG-bound dinucleoside methyl phosphotriesters. Cleavage from the support using anhydrous K2CO₃ (0.05 M in MeOH) at room temperature for eight hours gave free dinucleoside methyl phosphotriesters in 95-97% yield as Rp and p diastereomeric mixtures. The products were analyzed by HPLC (see Iyer et al, Bioorg. Chem. £: 1 (1995)).

Example 4 H-phosphonate solid phase coupling of nucleoside svnthons , introduction of the methyl phosphotriester linkage and removal of base protecting groups PNT-protected nucleosides were prepared as described in Example 1 above. The PNT-protected nucleosides were then converted to the corresponding H-phosphonates using standard procedures , and the resultant PNT-protected nucleoside H- phosphonates were then coupled to the 5' position of a nucleoside attached to a CPG solid support under standard H-phosphonate coupling conditions. Froehler, B.C. in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs, Agrawal, s., Ed., Humana Preεε, Totowa, NJ, 1993, Vol. 20, pp. 33-61. Following synthesis of the H-phosphate dimer, the CPG was removed from the column and treated for 15 minutes with a mixture of carbon tetrachloride, N-methylimidazole, triethylamine and l⁴CH₃OH ( : : : ). The CPG was then treated with 0.05 M potassium carbonate in methanol under argon for eight hours. Thiε treatment waε sufficient to ensure both the removal of the PNT group and the cleavage of the dinucleotide from the support. Neutralization followed by HPLC purification gave the l*C-methyl phosphotriester. Reverse phase HPLC analysis of the dimer was performed on an instrument equipped with a photodiode array UV detector and a radiomatic flow scintillation analyzer . The presence of a phosphodieter impurity waε found to be less than 2% by HPLC analysis of the crude reaction mixture. A specific activity of 0.8 microcurie/micromole was obtained. The labeled methyl phosphotriester after purification was essentially 100% radiochemically pure. The purified methylphosphotriester waε treated with potassium carbonate overnight with no loss of specific activity, thus demonstrating that demethylation does not occur during the deprotection/cleavage step.

Example 4 Synthesis of Chimeric Oligonucleotides The CEPNT and MEPNT monomers were used to prepare chimeric trinucleotides having one phosphodiester or phosphorothioate intemucleoside linkage and one O- or S-methyl phosphotriester intemucleoside linkage under conditions as descibed in Example 3. Syntheεis was carried out on a solid support using conventional succinyl-linked nucleoside loading. The phosphodiester or phoεphorothioate intemucleoside linkage was assembled using the CEPNT monomer and the O- or S-methyl phosphotriester intemucleoside linkage was assembled using the MEPNT monomer. The trimers thus obtained, a mixture of four diastereomerε, were characterized by ³^-P-NMR and ^-H-NMR and by MALDI-TOF mass spectroscopy. Typical NMR results are shown, for one tri er in Figure 2. In the ³¹P-coupled 3-H-NHR, the OCH₃ protons appeared as four sets of doublets, indicating the presence of the four diastereomers . The MALDI-TOF mass spectrum revealed the expected molecular ion at 911.7 (Na⁺ form) for the species containing the phosphorothioate and S-methylphosphotrieεter linkages.

This strategy was extended to the synthesis of support-bound nonanucleotide chimeras incorporating four phosphorothioate intemucleotide linkages and either four S- or O- methylphosphotriester intemucleotide linkages. In each case, 31p-NMR analysis proved that the methylphosphotriester and phosphorothioate εegmentε were present in the correct relative proportion, as shown, for example, in Figure 3. In addition, these chimeras exhibited slower mobility on polyacrylamide gel electrophoresis than a phosphodiester-phosphororthioate chimera of identical sequence, as shown in Figure 4. These results demonstrate that the mild deprotection conditions according to the invention allow the syntheεis of any chimeric oligonucleotide containing these base-εenεitive intemucleotide linkages .

Examle 5 Relative nuclease resistance of oligonucleotides containing ethyl phosphotriester linkages Oligonucleotides containing either all methyl phosphotriester intemucleoside linkages or a mixture of methyl phosphotriester intemucleoside linkages and phosphorothioate or phosphodiester inte ucleoside linkages in various chimeric configurations were synthesized according to Example 3 or 4. Oligonucleotide phosphodiesters and phosphorothioates were synthesized according to standard procedures. To test the relative nuclease resistance of these oligonucleotides the oligonucleotides were treated with snake venom phos- phodiesteraεe (SVPD). About 0.2 A26O units of oligonucleotide was disεolved in 500 microliters buffer (40 mM NH4CO3 , pH 7.0 , 20 mM MgCl₂) and mixed with 0.1 units SVPD. The mixture was incubated at 37 C for 420 minutes. After 0, 200 and 420 minutes, 165 micro- liter aliquots were removed and analyzed using ion exchange HPLC. Oligonucleotides containing methyl phosphotriester intemucleoside linkages exhibited greater nuclease resistance than oligonucleotides containing exclusively phosphodieεter or phosphorothioate intemucleoside linkages.

Example 5 Duplex stability of oligonucleotides containing methvl phosphotriester intemucleoside linkages Oligonucleotides containing either all methyl phosphotriester intemucleoside linkages or a mixture of methyl phosphotriester intemucleoside linkages and phosphorothioate or phosphodiester intemucleoside linkages in various chimeric configurations were synthesized using the process described in Example 3 or 4. Oligonucleotide phosphodiesters and phosphorothioates were synthesized according to standard procedures. The oligonucleotides are tested for their ability to form duplexes with complementary oligodeoxyribonucleotides and oligoribonucleotides . In separate reactions, each oligonucleotide is mixed with an equivalent quantity (0.2 A₂60 units) of its complementary oligonucleotide in 150 mM NaCl, lOmM Na Pθ , ImM

EDTA (pH 7.0). The mixture is heated to 85°C for 5 minutes, then o o cooled to 30 C. The temperature is then increased from 30 C to o o

80 C at a rate of 1 C per minute and A₂60 i-^s recorded as a function of temperature. Oligonucleotides according to the invention are expected to form duplexes with complementary oligodeox- yribonucleotideε or oligoribonucleotideε at temperatures well above physiological temperatures.

Example 7 Inhibition of HIV-1 by oligonucleotides containing methyl phosphotriester intemucleoside linkages Oligonucleotides containing either all methyl phosphotrieεter intemucleoside linkages or a mixture of methyl phosphotrieεter intemucleoside linkages and phosphorothioate or phosphodiester inte ucleoside linkages in various chimeric configurations are synthesized according to the process described in Examples 3 or 4. Oligonucleotide phosphodiesterε and phosphorothioateε are synthesized according to standard procedures . The oligonucleotides have a previously described sequence that is complementary to a portion of the gag gene of HXV-l (see Agrawal and Tang, Antisense Research and Development 2.- 261-266(1992)). Oligonucleotides are tested for their ability to inhibit HIV- 1 in a tissue culture syεtem. H9 lymphocytes are infected with HIV-1 virions (0.01-0.1 TCID₅₀/cell) for one hour at 37°C. After one hour, unadsorbed virions are washed away and the infected cells are divided among wells of 24 well plates. To the infected cells, an appropriate concentration (from stock solution) of oligonucleotide is added to obtain the required concentration (0.1 -10 micromolar) in 2 ml media. The cells are then cultured for four dayε . At the end of four dayε, inhibition of HIV-1 is assesεed by observing or measuring reductions in syncytium formation, p24 expression and reverse transcriptase activity. All of the tested oligonucleotides according to the invention are expected to show significant reductions in these parameters without significant cytotoxicity .

Claims

What is claimed is.

1 An oligonucleotide having from one to about all intemucleoside linkages in the form of a methyl phosphotriester mter- nucleoside linkage having the structure I.

X

II

Nucl-0-P-0-Nuc2

I

OC*H₃

wherein "Nucl" represents the 3' position of a first nucleoside, "Nuc2" represents the 5' position of a second nucleoside, C* is 12c or l^C, and x is sulfur or oxygen.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide has from about 12 to about 50 nucleotideε.

3. The oligonucleotide according to claim 2, wherein the oligonucleotide haε from about 17 to about 35 nucleotideε.

4. A mixed backbone oligonucleotide containing at least one *-*C- methyl phosphotriester inte ucleoside linkages and at least one intemucleoside linkage that iε not a ¹⁴C-methyl phosphotriester inte ucleoside linkage.

5. The mixed backbone oligonucleotide according to claim 4, wherein the intemucleoside linkage (s) that are not ¹⁴C-methyl phosphotriester linkages are selected from the group consisting of unlabeled methyl phosphotriester, phosphodiester and phoεphorothioate intemucleoside linkageε .

6. A method of synthesizing an oligonucleotide having at least one methyl phosphotriester inte ucleotide linkage, the method comprising condensing in the presence of a suitable activator, a nucleoside H-phoεphonate or thio-H-phoεphonate with another nucleoside, wherein at leaεt at leaεt one of the nucleosides has a nucleoside base protecting group, to produce adjacent nucleosides coupled by an H-phosphonate or thio-H-phosphonate linkage, wherein at least one of the nucleosides has a nucleoside base protecting group; oxidizing the H-phosphonate linkage in a suitable halogen source, together with a suitable non-nucleophilic base to produce an O-methylphosphotriester or O-methylphosphorothioate linkage; then chemoselectively removing the nucleoside base protecting group with a chemoselective removing agent, without demethylating the O-methylphosphotriester or O-methylphosphorothioate linkage.

7. The process according to claim 6, wherein the nucleoside base

protecting group has the general

where nj_, n2, and n are independently 0-10, the ring structures shown may be aromatic or heterocyclic, the nitrogen displayed is the protected amino moiety of the nucleoside base, and R^ and R₂ are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group.

8. The process according to claim 6 , wherein the internucleotidic phosphite linkage is oxidized using t-butyl hydroperoxide to yield an O-methyl phosphotriester linkage.

9. The process according to claim 6 , wherein the chemoselective removing agent is a halogen in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms.

10. The process according to claim 6 , wherein the halogen is Cl₂ or Br2.

11. The procesε according to claim 7, wherein n^, n₂ and n₃ are each 0.

12 The process according to claim 11, wherein the chemoselective removing agent is a halogen in water, or in pyridme/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms

13. The process according to claim 11, wherein the halogen is Cl or Br₂ •

14. The process according to claim 11, wherein the internucleotidic phosphite linkage is oxidized using 3H- benzodithiol-3-one 1,1-dioxide to yield an S-methyl phosphotriester linkage