WO1996039413A1

WO1996039413A1 - Methods and compounds for the synthesis of oligonucleotides and the oligonucleotides thereby produced

Info

Publication number: WO1996039413A1
Application number: PCT/US1996/007430
Authority: WO
Inventors: Radhakrishnan P. Iyer; Dong Yu; Sudhir Agrawal; Weitian Tan; Theresa Devlin; Ivan Habus
Original assignee: Hybridon, Inc.
Priority date: 1995-05-23
Filing date: 1996-05-23
Publication date: 1996-12-12
Also published as: AU5871196A

Abstract

The present invention provides new mononucleotide synthons useful in the synthesis of oligonucleotides having from one to all P-chiral centers that are predominantly and independently in the R or S configuration. The invention also provides methods of synthesizing these synthons, methods of synthesizing oligonucleotides having from one to all P-chiral centers predominantly and independently in the R or S configuration, and such oligonucleotides. Oligonucleotides synthesized with the novel synthons are useful for modulating nucleic acid expression, both in vitro and in vivo, as well as in traditional hybridization assays.

Description

METHODS AND COMPOUNDS FOR THE

SYNTHESIS OF OLIGONUCLEOTIDES AND

THE OLIGONUCLEOTIDES THEREBY PRODUCED

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to methods for the chemical synthesis of oligonucleotides, compounds use ul in the methods, and oligonucleotides thereby produced. Description of the Prior Art

Oligonucleotides and their chemical synthesis have become indespensible tools in modern molecular biology, being used in a wide variety of techniques ranging from PCR to antisense inhibition of nucleic acid expression. Understandably, therefore, there has been an every increasing demand for oligonucleotides having desirable properties such as resistance to nucleolytic attack and increased binding affinities. Furthermore, with the widespread use of oligonucleotides for these varying purposes, there has been an ever increasing demand for fast, inexpensive, and efficient methods of synthesizing oligonucleotides having these desirable properties.

Since Zamecnik and Stephenson, Proc. Natl. Acad Sci. USA 75, 280-284 (1978), first demonstrated virus replication inhibition by synthetic oligonucleotides, there has been much interest in the use of antisense oligonucleotides as agents for the selective modulation of gene expression, both in vitro and in vivo. See, e.g., Agrawal, Trends in Biotech. 10, 152 (1992); Chang and Petit, Prog. Biophys. Molec. Biol. 58, 225 (1992);

and Uhimann and Peymann, Chem. Rev. 90, 543 (1990). Antisense oligonucleotides are constructed to be sufficiently complementary to a target nucleic acid to hybridize with the target under the conditions of interest and inhibit expression of the target. Antisense oligonucleotides may be designed to bind directly to DNA (the so-called "anti-gene" approach) or to viral RNA or mRNA. Id. Expression inhibition is believed to occur by interfering with transcription processing or translation, or inducement of target mRNA cleavage by RNase H.

Antisense oligonucleotides can be used as research tools in vitro to determine the biological function of genes and proteins. They provide an easily used alternative to the laborious method of gene mutation (e.g. , deletion mutation) to selectively inhibit gene expression. The importance of this method is readily appreciated when one realizes that the elucidation of most known biological processes has been determined by deletion mutation.

Antisense oligonucleotides also may be used to treat a variety of pathogenic diseases by inhibiting gene expression of the pathogen in vivo. Oligonucleotide phosphorothioates (PS-oligos) have shown great therapeutic potential as antisense-mediated inhibitors of gene expression (Stein and Cheng, Science 261, 1004 (1993) and references therein) as evidenced by a number of ongoing clinical trials against AIDS and cancer. Agrawal and Tang, Antisense Res. and Dev. 2, 261 (1992) and references therein, and Bayever et al., Antisense Res. Dev. 3, 383 (1993). The synthesis of oligonucleotides for antisense and diagnostic applications is now be routinely accomplished. 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 Peyman, 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 phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites 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. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al. , Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry. Solid phase synthesis of oligonucleotides by the foregoing methods involves the same generalized protocol. Briefly, this approach comprises anchoring the 3 '-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the '-most nucleoside of the nascent, support- bound oligonucleotide. Oligonucleotide synthesis generally begins with coupling, or "loading," of the 3'- most nucleoside of the desired oligonucleotide to a functionalized solid phase support. A variety of solid supports and methods for their preparation are known in the art. E.g. , Pon, "Solid-Phase Supports for Oligonucleotide Synthesis," in Methods in Molec. Biol. , Vol. 20, : Protocols for Oligonucleotides and Analogs, p. 465 (Agrawal, Ed., Humana Press, 1993). Generally, the functionalized support has a plurality of long chain alkyl amines (LCAA) on the surface that serve as sites for nucleoside coupling. Controlled pore glass (CPG) is the most widely used support. It consists of approximately 100-200 μm beads with pores ranging from a few hundred to a few thousand angstroms.

Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 μmol to 1 mmoi amd higher). Padmapriya et al.,

Antisense Res. Dev. 4, 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50, 9255 (1994); Theisen et al., Nucleosides & Nucleotides 12, 1033 (1993); Bonora, Nucl. Acids Res. 21, 1213 (1993); Habus and Agrawal, Nucl. Acids Res. 22, 43 (1994); and Iyer et al., Nucleosides & Nucleotides 14, ** (1995) (in press)) and isolation (Kuijpers et al. Nucl. Acids Res. 18, 5197 (1990); and Reddy et al., Tetrahedron Lett. 35, 4311 (1994)) of oligonucleotides.

Although the phosphotriester method of oligonucleotide synthesis (see, e.g., Koziolkiewicz and Wilk in Methods in Molecular Biology, Vol. 20, pp. 207-220, supra) once dominated oligodeoxynucleotide preparation, there have been no reports of the synthesis of oligonucleotides having multiple phosphotriester linkages. There is still an unfulfilled need for methods of producing such oligonucleotides and for the oligonucleotides themselves.

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 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 oligonucletides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out. Examples of such base-sensitive modified oligonucleotides include, methyl phosphotriester oligonucletides, phosohsphoramides, etc. in other applications of oligonucletides, it is desirable to have oligonucleotides still bound to a solid-support. Such completely deprotected oligonucleotides still bound to the solid support will be useful in a variety of applications such as those involving isolation of transcription factors and other factors or elements that interact with oligonucleotides, solid-phase PCR, investigations into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screening of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols).

PS-oligos synthesized by prior art methods are mixtures of 2" diastereomers, where n is the number of internucleotide phosphorothioates. To date only limited data is available on the comparative biophysical and biological properties of stereodefined phosphorothioates due to non-availability of sufficient quantities of completely "stereoregular" PS-oligos of sufficient length. Stec, Angew-Chem. Int. Ed. Engl. 33, 709 (1994) and references therein; Lesnikowski, J Bioorg- Chem., 21, 127 (1993); Stec and Lesnikowski in Methods in Molecular Biology, Vol. 20, supra, pp. 285-313 and references cited therein; and Tang et al., Nucleosides Nucleotides, in press (1995). Enzymatic synthesis (Tang et al., supra) gives only R_p-phosphorothioates and is not as yet amenable to large-scale work.

The effects of different diastereomers on the efficacy of antisense oligonucleotides for gene modulation remain largely unknown. The potential impact of stereoselective synthesis to augment antisense oligonucleotide efficacy, however, is great. There is a need, therefore, for further research to increase knowledge in this area and to develop methods for large-scale synthesis of "stereoregular" PS-oligos. Concomitantly, there is a need for additional synthetic research tools to aid in this endeavor.

SUMMARY OF THE INVENTION In one aspect of the present invention, a novel nucleoside base protecting group is provided. This protecting group has the general structure XXI:

where n,, n₂, and n₃are independently 0-10 and the nitrogen displayed is the amino moiety of the base.

In a preferred embodiment, compound XXI takes the form N-pent-4-enoyl CH₂ = CH(CH₂)₂CO- (XXII). Compounds XXI and XXII protect the nucleoside base amino moieties by forming amide linkages, as in:

where the nitrogen is the amino moiety of the base B. Compound XXI and the preferred embodiment XXII are particularly advantageously used because they can be removed chemoselectively by treatment with a reagent such as I₂. The use of this mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as alkyl phosphotriesters and other base-sensitive oligonucleotides. this new protecting group is compatible with H-phosphonate chemistry as well as phosphoramidite chemistry. Besides being able to synthesize oligonucleotides bearing "sensitive" functionalities, it can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. With incorporation of this protecting group into nucleoside bases, it is possible to synthesize oligonucleotides still bound to any type of solid support.

Accordingly, in another aspect of the invention, oligonucleotides having 3 or more phosphotriester internucleotide linkages are provided. These oligonucleotides are synthesized according to the method outlined in Figure 2.

In another aspect of the invention, phosphorothioate oligonucleotides bound or unbound to a solid support are provided. This aspect of the invention is is schematically displayed in Figure 3. If free (unbound) oligonucleotide is desired, the oligonucleotide can be synthesized according to the phosphoramidite method using the base protecting groups of the invention. When the full length support bound oligonucleotide is complete, it can be contacted with ammonia for 1 to 2 hours to yield the free, unprotected oligonucleotide.

Where an unprotected, support-bound oligonucleotide is desired, the full length support-bound oligonucleotide is contacted with I₂ in water to cleave the base protecting group and then with anhydrous triethylamine to cleave the β-cyano moiety. Because the base-protecting group can also be used to protect hydroxyl moieties, support-bound branched oligonucleotides can be synthesized using, for example, glycol residues in which one hydroxyl group is protected by DMT and the other by a protecting group according to the invention. Then the DMT group may be selectively removed and an oligonucleotide synthesized from the resulting unprotected hydroxyl. Upon completion of that oligonucleotide, the hydroxyl moiety protected by the protecting group according to the invention can be deprotected with I₂ and water and another, different oligonucleotide synthesized from it. Such an approach is useful in producing combinatorial libraries. The unprotected support bound oligonucleotides of the invention can have phosphodiester, or phosphotriester internucleotide linkages of the form -O-PO(XR)-O- where X is O, NH, or S and R is a C, - C₂₀ alkyl or an aryl moiety.

The resulting unprotected support-bound oligonucleotide is useful for a variety of purposes such as applications involving isolation of transcription factors and other factors or elements that interact with oligonucleotides, solid-phase PCR, investigations into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screening of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols).

The present invention also provides additional methods for the synthesis of oligonucleotides. These methods use phosphoramidite chemistry with novel phosphoramidite reagents, which form another aspect of the invention. The novel synthons and synthetic methods of the invention can be used for the synthesis of diastereomeric mixtures of oligonucleotides and for the synthesis of oligonucleotides enriched in a particular diastereomer. The invention also provides oligonucleotides produced from these compounds by these methods. Oligonucleotides produced using the synthons and methods of the invention are useful for any purpose for which oligonucleotides produced using prior art techniques are used, such as PCR and as inhibitors of nucleic acid expression.

In one aspect of the invention, we provide monomer synthons having the structure:

xm where B is any suitably protected purine or pyrimidine base or derivative thereof and each of the chiral X' have a well defined stereoconfiguration. According to the methods of the present invention, compounds of structure XIII can be made to be diastereomerically enriched or a mixture of diastereomers. Diastereomerically enriched synthons as well as derivatives and analogs thereof are useful in the synthesis of diastereomerically pure oligonucleotides by the phosphoramidite method. They can be used as a substitute for the well-known β-cyanoethyl-protected phosphoramidate. In a preferred embodiment, synthon XIII has the form:

XIV where chiral centers 4 and 5 can be, respectively, R and S, S and R, R and R, or S and S.

In a particularly preferred embodiment of this aspect of the invention, synthon XIII has the form:

VI

In another particularly preferred embodiment, the present invention provides a phosphoramidite monomer synthon Via:

Via

In another aspect of the invention, a method of synthesizing the diastereomerically enriched monomer synthons XIII, XIV, and VI is provided. The method comprises

contacting HO.

\ .

(XV ^•R¹

HN

xvπ

to yield

xvm which, when contacted with a 5 '-protected mononucleoside having an unprotected 3' hydroxyl, yields XIII.

In a preferred embodiment of this aspect of the invention, XVII takes the form:

XJX and yields

XX which can be reacted with a mononucleoside to yield XIV.

In a particularly preferred embodiment of this aspect of the invention, (IR, 2S)-(-)-ephedrine (V) with PC1₃ to yield the chlorophosphoramidite product:

IV

The R_p isomer predominates ( > 95%). Contacting IV with a 5 ' -DMT-protected mononucleoside having a free 3 ' hydroxyl group yields the monomer synthon VI in high yield (84%).

Each of the foregoing reactions is stereoretentive. Thus, particular diastereomers of each of XIII, XIV, and VI can be obtained by starting with the appropriate stereoisomer of XVII, XIX, and V, respectively.

In another particularly preferred embodiment of the invention, the monomer synthon Via is synthesized by contacting:

HO_N

)

HN Me

XJXa

with PC1₃ to yield

IVa which can then be contacted with a 5'-protected mononucleoside having an unprotected 3'- hydroxyl to yield Via.

In another aspect of the invention, thiophosphoramidate monomer synthons enriched in a particular stereoisomer are provided. These compounds have the general structures:

XV XVI

In a preferred embodiment of this aspect of the invention, thiophosphoramidate monomer synthons having ryπ-(VIIIb) and anti- (Villa) conformations are provided:

anti-isomet Λ j-isomer

Villa VUTb

XV, XVI, Villa and VHIb are all made by oxidatively thiolating the monomer synthon precursors XIII, XTV, and VI, respectively, with a sulfurizing agent such as the 3H-1,2- benzodithiole-3-one- 1,1 -dioxide reagent. The result is about 90% retention of configuration. The stereoisomers can be separated by flash chromatography.

In a particularly preferred embodiment of this aspect of the invention, the phosphorothioate monomer synthon VIIIc is provided:

vmc

VIIIc is made by oxidatively thiolating the monomer synthon precursor Via with a sulfurizing agent such as the 3 H-l ,2-benzodithiole-3 -one- 1,1 -dioxide reagent.

In another aspect of the invention, oligonucleotides having one or more P-chiral centers predominantly in the S configuration and methods for their synthesis have been developed. In one embodiment of this aspect of the present invention, these oligonucleotides can be synthesized via the well-known phosphoramidate approach using XIII, XrV, or VI instead of the well known β-cyanoethyl phosphoramidite synthon. The intermediate phosphite linkage may be oxidized with, for example, I₂ and H₂O in THF to yield a phosphodiester linkage, or oxidatively thiolated with a sulfurizing agent, such as the Beaucage reagent, to yield a phosphorothioate linkage. Oligonucleotides synthesized according to this embodiment of the invention will have predominantly S_p configuration ( - 60%) at each internucleotide linkage in which compound XIII, XTV, or VI was employed during synthesis.

In another embodiment of this aspect of the invention, one of XV, XVI, Villa or VHIb is contacted with a nascent oligonucleotide having a free 5 ' hydroxyl group. When either Villa or VIHb is used, the result is an oligonucleotide having a 5 ^' phosphorothioate internucleotide linkage with an R_p:S_p ratio of about 70:30 when Villa is used and 10:90 when VHIb is used. Similar results are obtained from compound XVI when R¹ and R² are both anti- or both syn- with respect to the nucleoside and compound XV when all of the R^j are anti- or syn- with respect to the nucleoside. As noted, each of the foregoing monomer synthons and oligonucleotides can be synthesized using the methods of the present invention to be in enantiomeric excess. One advantage of the methods of the present invention is that the stereochemistry of the precursors is maintained in the products, and, if the reactants are in enantiomeric excess, the products are predominantly in one stereoconfiguration. When stereochemistry is unimportant, however, the present method provides a convenient method of oligonucleotide synthesis using the phosphoramidite method wherein the well-known β-cyanomethol phosphoramidite synthon is replaced by compound Via. The intermediate phosphite linkage may be oxidized with, for example, l₂ and H in THF to yield a phosphodiester linkage, or oxidatively thiolated with a sulfurizing agent, such as the Beaucage reagent, to yield a phosphorothioate linkage.

In an alternative embodiment, compound VIIIc is used to synthesize oligonucleotides having phosphorothioate internucleotide linkages by contacting VIIIc with a nascent oligonucleotide having a free 5 '-hydroxyl group.

Oligonucleotides according to the invention are useful for both in vitro and in vivo applications. For in vitro applications, the present oligonucleotides are useful as research tools in determining gene function by effecting gene modulation and as hybridization probes, for example.

Oligonucleotides according to the invention are also useful for in vivo applications, such as the treatment of pathogen-caused diseases. Oligonucleotides according to the invention can be synthesized to have a sequence sufficiently complementary to a region of a nucleic acid essential for the growth, reproduction, and/or metabolism of the pathogen to inhibit expression of the nucleic acid under physiological conditions.

The foregoing merely summarizes certain aspects of the present invention and is not meant, nor should it be construed, to limit the invention in any way. All patents and other publications cited herein establish the state of the art and are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 displays the synthetic pathway for making the compounds of the present invention. Figure 2 displays synthesis of phosphodiester and phosphotriester oligonucleotides using H-phosphonate chemistry and the base protecting group of the invention.

Figure 3 displays synthesis of free and support-bound unprotected oligonucleotides according to the method and reagents of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect of the present invention, a novel nucleoside base protecting group is provided. This protecting group has the general structure XXI:

where n_{l t} n₂, and n₃ are independently 0-10 and the nitrogen displayed is the amino moiety of the base.

In a preferred embodiment, compound XXI takes the form N-pent-4-enoyl CH₂ =CH(CH₂)₂CO- (XXII). Compounds XXI and XXII protect the nucleoside base amino moieties by forming amide linkages, as in:

where the nitrogen is the amino moiety of the base B. Compound XXI and the preferred embodiment XXII are particularly advantageously used because they can be removed chemoselectively by treatment with a reagent such as I₂. The use of this mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides usch as alkyl phosphotriesters and other base-sensitive oligonucleotides. this new protecting group is compatible with H-phosphonate chemistry as well as phosphoramidite chemistry. Besides being able to synthesize oligonucleotides bearing "sensitive" functionalities, it can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. With incorporation of this protecting group into nucleoside bases, it is possible to synthesize oligonucleotides still bound to any type of solid support.

Accordingly, in another aspect of the invention, oligonucleotides having 3 or more phosphotriester internucleotide linkages are provided. These oligonucleotides are synthesized according to the method outlined in Figure 2. After the complete support- bound oligonucleotide is synthesized according to the H-phosphonate method using the base amino-protecting group of the present invention, the oligonucleotide is contacted carbon tetrachloride, N-methyl imidazole and RXH (where X is O, NH, or S and R is a C C₂₀ alkyl or aryl) and then with iodine in water to remove the protecting group.

Where an unprotected, support-bound oligonucleotide is desired, the full length support-bound oligonucleotide is contacted with I₂ in water to cleave the base protecting group and then with anhydrous triethylamine to cleave the β-cyano moiety.

Because the base-protecting group can also be used to protect hydroxyl moieties. In this aspect of the invention, support-bound branched oligonucleotides can be synthesized using, for example glycol residues in which one hydroxyl group is protected by DMT and the other by a protecting group according to the invention. Then the DMT group may be selectively removed and an oligonucleotide synthesized from the resulting unprotected hydroxyl. Upon completion of that oligonucleotide, the hydroxyl moiety protected by the protecting group according to the invention can be deprotected with I₂ and water and another, different oligonucleotide synthesized from it.

The unprotected support bound oligonucleotides of the invention can have phosphodiester, or phosphotriester internucleotide linkages of the foπn -O-PO(XR)-O- where X is O, NH, or S and R is a C, - C₂₀ alkyl or an aryl moiety. The resulting unprotected support-bound oligonucleotide is useful for a variety of purposes such as applications. involving isolation of transcription factors and other factors or elements that interact with oligonucleotides, solid-phase PCR, investigations into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screeing of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols). The present invention also provides a sructurally novel class of antisense oligonucleotides useful for modulation of nucleic acid expression in vitro and in vivo. The present invention also provides novel methods for synthesizing this class of oligonucleotides using new synthons. The present invention also provides additional methods for the synthesis of oligonucleotides. These methods use phosphoramidite chemistry with novel phosphoramidite reagents, which form another aspect of the invention. The novel synthons and synthetic methods of the invention can be used for the synthesis of diastereomeric mixtures of oligonucleotides and for the synthesis of oligonucleotides enriched in a particular diastereomer. The invention also provides oligonucleotides produced from these compounds by these methods. Oligonucleotides produced using the synthons and methods of the invention are useful for any purpose for which oligonucleotides produced using prior art techniques are used, such as PCR and as inhibitors of nucleic acid expression. In one aspect of the invention, we provide monomer synthons having the structure:

xm wherein R* and R^b, and each R 'are independently H, C, - C₂₀ alkyl, aryl, heterocyclic, C _r

C₂₀ alkoxy, R is a suitable protecting group, such as DMT, n is 1-3, i is 1-n, X* is C, O, S, or N, such that if n > 1 the identity of each X* (i.e. , each of X ' . . . X 5 is independent of the identity of every other X^s and the identity of each substituent R ' (i.e., R ¹ . . . R ⁿ) is independent of every other R', each R' is covalently bound to the corresponding X ' (e.g. , X'-R^l . . . Xⁿ-Rⁿ), the X' are arranged consecutively such that X ^l is bound to the N and X" is bound to the O, and B is any suitably protected, modified or unmodified, purine or pyrimidine base. As used herein, the term "aryl" means a polyaromatic-ring structure having from 1 to 5 linearly or angularly fused aromatic rings, such as phenyl and naphthyl. As used herein the term "heterocyclic" means a 5 or 6 membered ring having from 1 to 5 heteroatoms (i.e. , N, S, or O) that may be located at any position within the ring. Furan and thiophene are examples of heterocyclic moieties encompassed by this definition. Compound XIII is synthesized according to the methods of the present invention (infra) to be predominantly in one stereoconfiguration.

The stereochemistry of the product XIII depends on the stereochemistry of the starting material. Synthesis of XIII from its precursor is accomplished in a stereorentive manner, infra.

In a preferred embodiment of this aspect of the invention, R* is H, n is 2, and X ' and X² are each C, which has the structure XIV:

XTV In this embodiment, the configurations at carbons 4 and 5 can be, respectively, R and S, S and R, R and R, or S and S, each of which can be obtained in pure form.

In a particularly preferred embodiment of this aspect of the present invention, n is 2, X¹ and X ² are each C, R ' is methyl, R is phenyl, R ' is H, R ^b is methyl, and the compound has the R,, configuration as shown in structure VI:

VI

All of the four diastereomers of VI (in which carbons 4 and 5 are in the (R,S), (S,R),

(R,R), and (S,S) configurations) can be made from one of the stereoisomers of precursor rV, infra.

Via In addition, the present method provides a method of synthesizing oligonucleotides according to the phosphoramidite method using the phosphoramidite Via. The method

comprises the synthon Via with a nascent oligonucleotide having a free 5 '-hydroxyl group. The chemistry is otherwise the same as standard phosphoramidite chemistry. Myriad suitable base protecting groups are known to those skilled in the art. E.g. ,

Sonveaux in Methods in Molecular Biology, v. 26: Protocols for Oligonucleotide Conjugates pp. 1-72 (S. Agrawal, Ed., Humana Press (1994)) and references cited therein. Similarly, numerous modified bases are known to those skilled in the art. E.g. , Meyer, Methods in Molecular Biology, v. 26, supra, pp. 73-92 and references cited therein. Synthons XIII, XTV, and VI and derivatives thereof are useful in the synthesis of oligonucleotides by the phosphoramidite method, as discussed more fully below. It can be used as a substitute for the well-known β-cyanoethyl-protected phosphoramidate:

xπ

Furthermore, the synthetic protocol for incorporating XIII, XIV, and VI in a nascent oligonucleotide is the same as that for the β-cyanoethyl-protected phosphoramidates.

E.g. , Beaucage in Methods in Molecular Biology, Vol. 20, Protocols for Oligonculeotides

and Analogs, supra, pp. 33-61 and references cited therein. For simplicity, as used herein the term "nascent oligonucleotide" means a solid support-bound nucleotide chain having at least one nucleotide.

In another aspect of the invention, a method of synthesizing the diastereomerically enriched monomer synthon XIII is provided. In this aspect of the invention PC1₃ is reacted with a compound of structure XVII:

^H0\ . .

(XV~ R¹

HN' it xvπ to yield

xvm where each of X¹, R', R^b, and n are defined the same as described for compound XIII.

XVIIIis contacted with a 5 '-protected mononucleoside having an unprotected 3 '-hydroxyl to yield XIII. Compounds XIII and XVIII are obtained from their precursors (XVIII and XVII, respectively) a stereoretentive manner, i.e. , the stereoconfiguration of the precursor is maintained in the reaction.

Compound XIV is obtained in a similar manner by contacting XIX

XIX with PC1₃ to yield

XX wherein each of R¹ and R² are the same as R ' in compound XIII and R ^b is the same as in compound XIII. Compound XIV is obtained by contacting XX with a 5 '-protected mononucleoside having an unprotected 3 '-hydroxyl. As before, the reactions are stereoretentive, and the products of each reaction, XIV and XX, retain the same stereoconfiguration as their precursor, XX and XIX, respectively.

In a preferred embodiment of this aspect of the invention, a method of synthesizing the diastereomerically enriched monomer synthon VI is provided. The method comprises contacting (IR, 2S)-(-)-ephedrine (V) with PC1₃ at between -100 and 40 °C for between one and 40 hours. In a preferred embodiment, the two compounds are allowed to react in N-methyl morpholine and toluene at -78 °C for 3 hours and then at 22 °C for 12 hours. Other suitable solvents are benzene, tetrahydrofuran, ether, and dioxane. The result is about a 75 % yield of the chlorophosphoramidite product:

IV The R_p isomer predominates ( > 95%). Contacting IV with a 5 '-DMT-protected mononucleoside having a free 3 '-hydroxyl group yields the monomer synthon VI in high yield (84%). In a preferred embodiment, the mononucleoside and IV are allowed to react in ethyl ether and triethylamine as a scavenger of HCl liberated during the reaction. Other scavengers such as pyridine and 2,6-lutidine can also be used. The reaction can be conducted at temperatures ranging from between -100 and 40 °C for between 1 and 40 hours. In a preferred embodiment, the mixture is allowed to react at -78 °C for 3 hours and then at 22 °C for 12 hours. Other suitable solvents such as benzene, tetrahydrofuran,

ether, and dioxane can also be used. Compound IV is fairly stable, undergoing no apparent decomposition (as evaluated by ³¹P-NMR) after being stored at -5 °C for several days.

The other stereoisomers of ephedrine (1S,2R; 1S,2S; and 1R,2R) (V) are also commercially available and can be used in place of (lR,2S)-(-)-ephedrine (V) to obtain the other diastereomers of IV:

HO

)

HN Me

XDta with PC1₃ to yield

Cl *,

> & '

Me' 3 4

IVa which can then be contacted with a 5'-protected mononucleoside having an unprotected 3'- hydroxyl to yield Via. The same reaction conditions as described previously can be used.

XV

XVI wherein the definitions of B, R, R\ R^a, R^b, R¹, R², X ', i, and n are the same as described previously for compound XIII. Monomer synthons XV and XVI are obtained in the predominant configurational stereoisomer by stereoretentive oxidative thiolation of the phosphorous of compounds XIII and XIV, respectively. Oxidative thiolation of a particular stereoisomer of XIII or XIV (provided above) results in approximately 90% conversion to the corresponding thiophosphoramidate stereoisomer. In a preferred embodiment of this aspect of the invention, the anti- isomer of synthon VIII is provided. The syn- (Vlllb) and anti- (Villa) forms of the synthon VIII according to this aspect of the invention have the following structures:

anti-isomeτ .sryn-isomer

Villa and Vlllb are made by oxidatively thiolating the monomer synthon VI. The result is a 90:10 anti:syn mixture.

vπic

VIIIc is made by oxidatively thiolating the monomer synthon precursor Via Any suitable method of oxidative thiolation may be used, such as elemental sulfur. E.g. , Stec et al., J Am. Chem. Soc. 106, 6077 (1984). Preferably, the thiophosphoramidate monomer synthons are synthesized by contacting the phosphoramidite precursors with the Beaucage reagent, 3H-l ,2-benzodithiol-3-one-l, l-dioxide:

I according to the method of Iyer et al. , /. Am. Chem. Soc. 112, 1253 (1990) and Iyer et al. ,

/. Org Chem. 55, 4693 (1990). In a preferred embodiment, reagent I is used as a 2% solution in acetonitrile and the mixture is allowed to react for 30 seconds at about room temperature. All of the various diastereomers (e.g. , Villa and Vlllb) are easily separated by conventional chromatography or crystallization. In another aspect of the invention, oligonucleotides having from one to all nucleotide P-chiral centers independently predominantly in the S configuration and methods for synthesizing them are provided. As used herein, the term "predominantly" means more than half. In one embodiment of this aspect of the present invention, these oligonucleotides can be synthesized via the well-known phosphoramidate approach (e.g. , Beaucage in Methods in Molecular Biology, Vol 20, Protocols for Oligonculeotides and Analogs, supra, pp. 33-61 and references cited therein) using XIII in place of XII. In a

preferred embodiment of this aspect of the invention, XIV is used in place of XII. In a particularly preferred embodiment, VI is used.

In brief, a nascent oligonucleotide having a free 5 ' hydroxyl is contacted with XIV, XV, or VI in the presence of tetrazole. A phosphate linkage is thereby formed. The phosphite linkage may then be oxidized with, for example, I₂ and H₂O in THF to yield a phosphodiester linkage or oxidatively thiolated with I to yield a phosphorothioate linkage. Phosphorothioate oligonucleotides synthesized according to this embodiment of the invention have predominantly S_p configuration (-60%) at each internucleotide linkage in which compound XIV, XV, or VI was employed during synthesis. When stereochemistry is unimportant, however, the present method provides a convenient method of oligonucleotide synthesis using the foregoing synthetic methods with compound Via. In an alternative embodiment, compound VIIIc is used to synthesize oligonucleotides having phosphorothioate internucleotide linkages by contacting VIIIc with a nascent oligonucleotide having a freee 5 '-hydroxyl group. In another embodiment of this aspect of the invention, oligonucleotides having one or more phosphorothioate internucleotide linkages that are independently predominantly in the R or S configuration are provided. In this embodiment, one of the stereoisomers of XV or XVI is contacted with a nascent oligonucleotide having an unprotected 5 ' hydroxyl group. In a preferred embodiment, Villa or VIHb is used, resulting in an oligonucleotide having a 5 ' phosphorothioate internucleotide linkage with an R,:S_P ratio of about 70:30

(starting with Villa) or 10:90 (starting with Vlllb). Similar results are obtained from compound XVI when R¹ and R² are both anti- or both syn- with respect to the nucleoside and compound XV when all of the R' are anti- or syn- with respect to the nucleoside.

The antisense oligonucleotides of the present invention may be designed to incorporate a number of additional features that have been demonstrated to increase efficacy. For example, they may be designed to be "self-stabilized," i.e. , having a first region sufficiently complementary to a second region to allow for intramolecular hybridization, thereby rendering the oligonucleotide less susceptible to nucleolytic attack. Such oligonucleotides are described in PCT International Application Publication No. WO 94/01550. All of the foregoing methods can be used with RNA and DNA and with any solid support. See, e.g., Pon in Methods in Molecular Biology, Vol. 20, pp. 465-496.

Alternatively, the presently disclosed oligonucleotides may be designed to be

"fold-back triplex forming," i.e., having a first region complementary to a target nucleic acid and a second region having a sequence that allows for triplex formation by Hoogsteen base pairing between it and the duplex formed by the first region and the target nucleic acid, as described in PCT International Application Publication No. WO 94/17091.

Oligonucleotides according to the invention are useful for both in vitro and in vivo applications. For in vitro applications, the present oligonucleotides are useful as research tools in determining gene function. Because they can be prepared to be complementary to a particular sequence, the present oligonucleotides can be used to selectively inhibit expression of a target gene. The present oligonucleotides thus provide an attractive and easily used alternative to the laborious method of gene inhibition by mutation (e.g. , deletion mutation). The significance of this will be appreciated when one realizes that the elucidation of most biological pathways now known has been determined by deletion mutations. Oligonucleotides according to the invention are also useful in standard hybridization assays.

The oligonucleotides of the present invention are also useful as therapeutic agents for diseases or physiological conditions involving expression of specific genes. Oligonucleotides useful for treating a disease or condition will have a nucleotide sequence sufficiently complementary to the target nucleic acid to bind to the target nucleic acid under physiological conditions. As used herein, the terms "complementary" and "sufficiently complementary" are used interchangeably and, when used to describe the sequence of an antisense oligonucleotide, mean that the oligonucleotide sequence is such that the oligonucleotide inhibits expression of the target nucleic acid under the conditions of interest (e.g. , in vitro experimental conditions or physiological conditions). In general, oligonucleotides according to the invention will have a sequence complementary to a nucleic acid (e.g., a gene or mRNA) that is essential to a biological process. As elaborated more fully below, such processes include reproduction and metabolic processes of pathogens and other disease-causing infectious agents. Alternatively, the biological process can be a naturally occurring one whose inhibition is desirable, e.g. , spermatogenesis in men and ovulation in women desiring contraception. The oligonucleotides of the invention can also be complementary to a gene or other nucleic acid whose expression causes or is involved in a diseased or otherwise abnormal state of the organism.

Because of their efficacy at gene modulation, the presently claimed oligonucleotides are also useful for treating diseases arising from genetic abnormalities that cause under- or over-expression of a gene. For diseases in which an abnormal gene is expressed or a normal gene is over-expressed, for example, the presently claimed oligonucleotides may be designed to target the abnormal or normal gene directly, or, in the alternative, to target the gene encoding the protein that promotes expression of the abnormal or normal gene. Conversely, where a normal gene is under-expressed, one may design an oligonucleotide that suppresses expression of a gene encoding a protein that suppresses expression of the normal gene.

In many cases the target nucleic acid sequence will be a viral nucleic acid sequence. The use of antisense oligonucleotides to inhibit various viruses is well known and has been reviewed in Agrawal, Trends in Biotechnology 10, 152 (1992). Viral nucleic acid sequences that hybridize to effective antisense oligonucleotides have been described for many viruses, including human immunodeficiency virus type I (U.S. Patent No. 4,806,463), Herpes simplex virus (U.S. patent No. 4,689,320), Influenzavirus (U.S. Patent No. 5,194,428), and Human papilloma virus (Storey et al., Nucleic Acids Res. 12, 4109 (1991)). Sequences hybridizing to any of these nucleic acid sequences can be used, as can nucleotide sequences complementary to nucleic acid sequences from any other virus. Additional viruses that have known nucleic acid sequences against which an antisense oligonucleotide according to the invention can be prepared include, but are not limited to, Foot and Mouth Disease Virus (See Robertson et al. , /. Virology 54, 651 (1985); Harris et al., J. Virology 36, 659 (1980)), Yellow Fever Virus (see Rice et al. , Science 229, 726 (1985)), Varicella-Zoster Virus (see Davison and Scott, J. Gen. Virology 67, 2279 (1986), Cucumber Mosaic Virus (see Richards et al. , Virology 89, 395 (1978)), Hepatitis B Virus (see Raney and McLachlen, in Molecular Biology of Hepatitis B Virus (CRC Press, 1991)), Hepatitis C Virus (see Miller and Purcell, Proc. Natl. Acad. Sci. USA

87, 2057 (1990); Proc. Natl Acad. Sci. USA 89, 4942 (1992); J. General Virology 74, 661 (1993)), and Respitory Syncitial Virus (see Collins, in The Paramyxo Viruses, Chapter 4, pp. 103-162 (David W. Kingsbury, Ed., 1991)).

Alternatively, the oligonucleotides of the invention can have a nucleotide sequence complementary to a nucleic acid sequence of a pathogenic organism. The nucleic acid sequences of many pathogenic organisms have been described, including the malaria organism, Plasmodiumfalciparum and many pathogemc bacteria. Examples of pathogenic eukaryotes having known nucleic acid sequences against which oligonucleotides of the present can be prepared include, but are not limited to Trypanosoma brucei gambiense and Leishmania (see Campbell et al., Nature 311, 350 (1984)), and Fasciola hepatica (see Zurita et al., Proc. Natl. Acad Sci. USA 84, 2340 (1987)). Antifungal oligonucleotides can be prepared having a nucleotide sequence that is complementary to a nucleic acid sequence from, e.g. , the chitin synthetase gene, and antibacterial oligonucleotides according to the invention can be prepared using, e.g. , the alanine racemase gene.

In yet another embodiment, the oligonucleotides can have a nucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state. The nucleic acid sequences of several such cellular genes have been described, including prion protein (Stahl and Prusiner, FASEB J. 5, 2799 (1991)), the amyloid-like protein associated with Alzheimer's disease (PCT International Application Publication No. WO 95/09236), and various well-known oncogenes and proto-oncogenes, such as c-myb, c-myc, c-abl, and n-ras.. In addition, oligonucleotides that inhibit the synthesis of structural proteins or enzymes involved largely or exclusively in speπnatogenesis, sperm motility, the binding of the sperm to the egg or any other step affecting sperm viability may be used as contraceptives for men. Similarly, contraceptives for women may be oligonucleotides that inhibit production of proteins or enzymes involved in ovulation, fertilization, implantation or in the biosynthesis of hormones involved in those processes. Hypertension can be controlled by oligonucleotides that suppress the synthesis of angiotensin converting enzyme or related enzymes in the renin angiotensin system; platelet aggregation can be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis; deposition of cholesterol in arterial wall can be inhibited by suppression of the synthesis of fatty acyl co-enzyme A: cholesterol acyl transferase in arteriosclerosis; inhibition of the synthesis of cholinephosphotransferase may be useful in hypoiipidemia.

There are numerous neural disorders in which oligonucleotides of the present invention can be used to reduce or eliminate adverse effects of the disorder. For example, suppression of the synthesis of monoamine oxidase can be used in Parkinson's disease; suppression of catechol O-methyl transferase can be used to treat depression; and suppression of indole N-methyl transferase can be used in treating schizophrenia.

Suppression of selected enzymes in the arachidonic acid cascade (which leads to prostaglandins and leukotrienes) may be useful in the control of platelet aggregation, allergy, inflammation, pain and asthma. Suppression of the protein expressed by the multi-drug resistance (mdr) gene, which is responsible for development of resistance to a variety of anti-cancer drugs and is a major impediment in chemotherapy may prove to be beneficial in the treatment of cancer. Nucleotide sequences complementary to nucleic acid sequences from any of these genes can be used for the oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to any other cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.

Antisense regulation of gene expression in plant cells has been described in U.S. Patent No. 5,107,065, and the antisense oligonucleotides of the invention can potentially be applied in this context as well. Since the nucleotide sequence of the oligonucleotide can be adapted to form

Watson-Crick base pairs with essentially any gene, the therapeutic spectrum of the

oligonucleotides of the invention should be very broad. Still, certain diseases are of

particular interest. For example, a variety of viral diseases may be treated by oligonucleotides having one or more S-triesterphosphorothioates internucleotide linkages, including AIDS, ARC, oral or genital herpes, papilloma warts, flu, foot and mouth disease, yellow fever, chicken pox, shingles, HTLV-leukemia, and hepatitis. Among fungal diseases treatable by oligonucleotides according to the invention are candidiasis, histoplasmosis, cryptococcocis, blastomycosis, aspergillosis, sporotrichosis, chromomycosis, dematophytosis and coccidioidomycosis. The method can also be used to treat rickettsial diseases (e.g. , typhus, Rocky Mountain spotted fever), as well as sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum.

A variety of parasitic diseases can be treated by oligonucleotides of the present invention, including amebiasis, Chagas' disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocystis carini pneumonia; also worm (helminthic diseases) such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode infections. Malaria can be treated by oligonucleotides of the present invention, regardless of whether it is caused by P. falciparum, P.vivax, P. orale, or P. malaria. The infectious diseases identified above can all be treated with oligonucleotides according to the invention because the infectious agents and their gene sequences for these diseases are known, and, thus, oligonucleotides according to the invention can be prepared having a nucleotide sequence that hybridizes to a nucleic acid sequence that is an essential

nucleic acid sequence for the propagation of the infectious agent, such as an essential gene. As used herein, an essential gene or nucleic acid is one that is required for a biological process and without which the biological process does not occur.

The following examples are provided for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any way.

EXAMPLES

Unless otherwise stated, all chemicals recited in the following Examples were

obtained from Aldrich of Milwaukee, WI.

Example 1 Stereoselective Synthesis of a Mononucleotide Synthon

The chlorophosphoramidite, (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-l ,3,2-

oxazaphospholidine (TV) was obtained by mixing 8.14 g of lR,2S-ephedrine (V) and 10.4

ml of N-methyl morpholine in 250 ml of toluene under argon and cooling to -78 °C. 4.3 ml of PC1₃ in 10 ml of toluene was added over a period of 15 minutes. The mixture was

kept at -78 °C for 1 hour and then allowed to warm to room temperature over a period of

16 hours, the insoluble salt precipitate was filtered under argon. The precipitate was

washed with 3 x 25 ml of toluene. The combined washings and filtrate were concentrated

in vacuo in a rotary evaporator to remove toluene. Vacuum distillation of the residue gave

a colorless liqued boiling at 0.1 mm Hg at 95 °C to give ca. 9 g (80% yield) of the

product. This procedure is similar to that described previously. Sun et al., J. Chem. Soc.

Perkin Trans. I p. 3183 (1994) and references therein and Carey et al., J. Chem. Soc.

Perkin Trans. I, p. 831 (1993).

³¹P-NMR examination of the resulting crude reaction mixture revealed the presence of- a predominant isomer ( > 95%) at δ 169.4 ppm and a minor component (< 5%) at δ

161 ppm. Upon vacuum distillation of the reaction mixture (95-97 °C at 0.1 mm Hg), a

colorless liquid was obtained, which solidified to a white crystalline mass upon cooling to -78 °C (isolated yields of 75 %). Carey et al. , supra, reported a b. p. of 160 °C at 0.1

mm Hg. NMR analysis gave the following results: ^3,P-NMR (CDC1₃) (TMP external

standard) δ 169.1 ppm; 'H-NMR (CDC1₃) δ (ppm) 0.71 (3H, d, J=6.3 Hz), 2.69 (3H, d,

³Jp._H = 15.1 Hz, N-CHj), 3.63 (IH, ddq, J = 1.3, 5.5, ³J _P._H=7.6 Hz, H-4), 5.85 (IH, dd,

j = 5.5 Hz, ³J_P._H — 1.2 Hz), 7.15 (5H, m, -Ph). These spectral features are in agreement

with values reported by Sun et al. and Carey et al. , supra, and lead to the assignment of

structure IV as being the R isomer in which the chlorine atom is disposed trans relative

to the C-Ph and C-Me substituents in the phospholidine ring. IV could be stored as a solid in a desiccator at -5 °C for several days with no apparent decomposition (as evaluated by

³¹P-NMR). Upon addition of water to IV, the H-phosphonate VII was obtained as a

mixture of disstereomers (R_p:S_p, 55:45 ³¹P-NMR).

2.16 g of 5'-0-dimethoxytrityl thymidine was dissolved in a mixture of anhydrous

ether (20 ml) and anhydrous triethyiamine (5 ml). The solution was added gradually (10

min) to 1.2 g of the chlorophosphoamidite (IV) at room temperature and the solution

stirred at room temperature for 6 hours. The reation mixture was poured into 200 ml of ice-cold water. It was then extracted with ethylacetate (3 x 200 ml). The combined organic layer was washed with water. The organic layer was evaporated to dryness to give

3 g (84 % yield) of VI as a white foamy material.

Synthesis of XIII and XIV is conducted according to the same protocol.

The ³¹P-NMR spectrum of VI has a signal at δ 140 ppm, corresponding to a single

P-epimer. In analogy with substitution reactions of VI involving carbon-, oxygen-, and nitrogen-based nucleophiles (Sun et al. and Carey et al. , supra), which gave substitution

products with overall retention of configuration, VI can be formulated as having the

structure with R_p configuration (Fig. l). This hitherto unreported nucleoside

phosphoramidite VI is a white solid and is stable when stored dry at 0 - 5 ^βC. The NMR

and mass spectral features of VI are as follows: ³¹P-NMR (CDC1₃) (TMP ext. standard) δ

169 ppm; Η-NMR (CDC1₃) δ (ppm) 0.61 (3H, d, J = 6.5 Hz), 1.41 (3H, s, T-CH ), 2.42

(2H, m, H-2'), 2.63 (3H, d, ³J_P._H = 12 Hz, N-CH₃), 3.37 (IH, dd, J = 10.6, 2.6 Hz, H-5'), 3.46 (IH, dd, J = 10.6, 2.6 Hz, H-5'), 3.52 (IH, ddq, J = 6.9, 6.5 Hz, ³J_P.„ = 2.4 Hz, H-4), 3.76 (6H, s, -OCH₃), 4.08 (IH, m, H-4'), 4.91 (IH, m, H-3'), 5.56 (IH,

dd, J = 6.9 Hz, ³J_P._H(J) = 1.84 Hz, H-5), 6.41(1H, dd, J = 6.7, 6.7 Hz, H-l'), 6.85 (4H, m, -Ph), 7.25 (14H, m, -Ph), -7.6 (IH, s, H-6), 9.1 (IH, s, -NH). FAB-MS (m/z) = 736

Oxidative sulfurization of the phosphoramidite VI with thiolsulfonate I (R.I.

Chemicals, Costa Mesa, CA) according to Iyer et al. , J. Am. Chem. Soc. 112, 1253

(1990), and Iyer et al., J. Org. Chem. 55, 4693 (1990) gave the thiophosphoramidates

Villa: Vlllb (90: 10, 81 % yield) (isomer ratio based on ³¹P-NMR. The NMR and mass

spectral features were as follows: Villa, ³¹P-NMR (CDC1₃) δ (ppm) 79.0; 1H -NMR

(CDC1₃) δ (ppm) 0.78 (3H, d, J = 6.6 Hz, -CH ) 1.41 (3H, s, T-CH3) 2.55 (2H, m, H-2 '), 2.70 (3H, d, ³J_P._H = 12.5 Hz, -NCH₃), 3.36 (IH, dd, J = 10.5, 2.3 Hz, H-5 '), 3.56 (IH, dd, J = 10.5, 2.2 Hz, H-5 ') 3.76 (IH, ddq, J = 6.6, 6.1 ³J_P._H = 12.3 Hz, H-4),

3.78 (6H, s, -OCH₃), 4.28 (IH, m, H-4 '), 5.57 (IH, m, H-3 '), 5.62 (IH, dd, J = 6.1 Hz, ³Jp._H(5) = 2.8 Hz, H-5), 6.48 (IH, dd, J = 9.0, 5.6 Hz, H-l '), 6.85 (4H, m, -Ph), 7.26

(14H, m, -Ph), 7.62 (IH, s, H-6) 8.90 (IH, s, -NH). FAB-MS (m/z) 769, C₄₁H₄₄N₃O_gPS.

The predominant isomer, VIII A (which is easily separated from Vlllb by flash

chromatography), has been assigned the configuration indicated in Fig. 1. The assignment

of configurations for Villa and Vlllb is based on the generally accepted notion that P(III)

oxidations proceed with high stereoselectivity and with overall retention of configuration.

E.g. , Beaucage and Iyer, Tetrahedron 48, 2223 (1992), and Bentrude et al., J. Am. Chem.

Soc. Ill, 3981 (1989).

Example 2 Synthesis of Nucleotide Dimers Using

Diastereomerically Enriched Monomer Synthons

Having obtained the nucleoside phosphoramidite VI in preparative-scale reactions,

the stage was set for its use in solid-phase coupling with CPG-bound nucleoside. Thus,

contacting a solution of VI in acetonitrile with CPG-T (10 mmoi) for a period of two

minutes in the presence of tetrazole as an activator followed by oxidation with the

thiolsulfonate I resulted in efficient formation of the product IX with a coupling efficiency

of greater than 95 % (as evaluated by "trityl yields"). Iyer et al., J. Am. Chem. Soc,

supra, and Iyer et al., / Org. Chem., supra. Following synthesis, the CPG-bound product was heated with aqueous ammonium hydroxide (28% , 55 °C, 1 hr). Examination of the

products by reverse-phase HPLC revealed that the dinucleoside phosphorothioate X had been formed as a mixture of diastereomers (R_p:S_p, 40:60). Interestingly, the commonly used cyanoethylphosphate deprotection strategy (28% aq. NH₄OH, 55 °C) was found to be sufficient to cleave the chiral phosphate appendage in IX and generate the

phosphorothioate X. The lack of high stereoselectivity in the formation of X is consistent

with other reports wherein epimerization of the phosphorous center (in the case of

stereoisomerically pure phosphoramidites) is observed when acidic type activators, e.g. ,

tetrazole, are used in conjunction with phosphoramidite methodology in the synthesis of

deoxyribonucleoside phosphorothioates. Stec, supra, and Beaucage, supra.

Example 3

Synthesis and Purification of Oligonucleotides

Oligonucleotides are synthesized on a 1 mmoi scale following the standard protocol

by using an automated synthesizer (e.g. , Millipore 8700 DNA Synthesizer, Bedford, MA).

Where a predominantly R_p configuration is desired, the phosphoramidite VI is used by dissolving it in dry acetonitrile at a concentration of 50 mg/ml. For phosphorothioate

oligonucleotides, the iodine oxidation step is replaced by sulftirization with

3H-l,2-benzodithiol-3-one- 1,1 -dioxide (Beaucage reagent). \ytι et al. , J. Org. Chem. SS,

4693 (1990). Two-hour treatment with ammoniun hydroxide at room temperature is

carried out to cleave the oligomer from the support and to deprotect nucleoside bases. Oligonucleotides are purified by reverse-phase HPLC and/or PAGE, and desalted by using C-1 SEP-PAK cartridges. Example 4

Stereoselective Synthesis of a Mononucleotide Phosphorothioate

Treatment of Villa and Vlllb with sodium methoxide in methanol at ambient

temperature overnight followed by heating with NH₄OH (28% NH₄OH for 1-2 hr at 55 °C gave the phosphorothioate:

XI in 90% yield with moderate to high stereoselectivity (as monitored by ³¹P-NMR and

HPLC). The R,,:S_P ratio of XI obtained from Villa was 70:30, whereas the ratio of

isomers obtained from Vlllb was 10:90. Configurations were assigned using the criteria

reported for dinucleoside phosphorothioates by Iyer et al., Bioorg. Med. Chem. Lett. 4, 2471 (1994).

Example 5

Stereospecific Phosphorothioate Synthesis

Diazabicyclononane (DBU) (296 mg, 1.95 mmoi) is dissolved in anhydrous THF

(1.5 ml) and added to 3'-0-t-butyl dimethylsilyl thymidine (46 mg, 0.129 mmoi) at 0 °C

for 20 minutes. This solution is added slowly to the solution of Villa (50 mg, 0.065

mmoi) and the contents stirred for 30 minutes at room temperature. The reaction mixture is allowed to warm to room temperature and stirred for 12 h. The solution is evaporated to remove solvent and treated with ammonium hydroxide (28% , 1 ml) and heated for 4 h at 55 °C. The solution is evaporated to dryness. Chromatographic purification affords

45 mg (70 % yield) of 5'-0-DMT-3'-0-TBDMS TT dimer with R_p:S_p ratio of 70:30.

Example 6 Cellular Uptake

Human T cell and leukemia cell line H9 are used in this study. They are cultured in RPMI media supplemented with 10% fetal bovine serum (heat inactivated to 56 °C for

30 minutes to inactivate the nucleases), 2 mM glutamine, 100 ml streptomycin, 100 U/ml penicillin and 6 x 10"⁵ M of 2-mercaptoethanol in an air incubator (37 °C, humidified by

5% CO₂-95% O₂).

Fiuorescein labeling of oligonucleotides

Fiuorescein is conjugated to the 5 ' end of the oligonucleotides by either an automated DNA synthesizer or by a manual procedure using a "FLUORESCEIN-ON" phosphoramidite. The efficiency of fiuorescein labeling is determined by using a spectrofluorometer (excitation 488 nm, emission 520 nm).

Cell uptake

The concentrations of the fiuorescein labeled and unlabelled oligonucleotides in the samples are measured by a spectrofluorometer and UV spectroscopy and adjusted to be the same by adding the corresponding unlabelled oligonucleotides. Labelled oligonucleotides

(0.2 OD/100 ml) are added to the cells (5 x 10⁶ cells/ml, 0.5 ml) and set to culture. After 4 hours of culture, aliquots of cell culture mixtures are removed, washed, and resuspended in Hank's balanced salt solution (HBSS) supplemented with 0.1 % BSA and 0.1 % sodiun

azide. Propidium iodide (final concentration 10 μl/ml) is used to distinguish viable cells

from dead cells. Flow cytometric data on 5,000 viable cell is acquired in list mode on Epics XL (Coulter, Hialeah, FL), and data are analyzed by Epics XL (version 1.5 software) after gating on living cells by forward scatter versus side scatter and propidium iodide staining.

The results demonstrate that oligonucleotides according to the invention are taken up by cells.

Example 7

Inhibition of HIV- 1 Replication The following assays are used to measure the ability of the oligonucleotide of the invention to inhibit HIV-1 replication.

Svncvtia Assay

The ability of the oligonucleotides of the invention to inhibit HIV-1 replication, and thus syncytia formation, in tissue culture is tested in T cell cultures according to the method of Agrawal and Sarin, Advanced Drug Delivery Rev. 6, 251 (1991). Briefly, CEM cells 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 and the infected cells are divided among walls of 24 well plates. To the infected cells, an appropriate concentration (from stock solution) of oligonucleotide is added to obtain the required concentration in 2 ml medium. The cells

are then cultured for three days. At the end of three days, infected cells are examined

visually for syncytium formation or stained with trypan blue or CTT for cytopathic effect

determination. The results demonstrate that oligonucleotides according to the invention inhibit syncitia formation. p24 Expression Assay

HIV expression can be determined by measuring the level of viral protein p24

expression in CEM cells essentially as described by Agrawal and Sarin, supra. Briefly,

cells are pelleted and then resuspended in phosphate saline at a concentration of about

10⁶/ml. The cells are spotted on toxoplasmosis slides, air dried, and fixed in methanol/acetone (1: 1) for 15 min at room temperature (RT). The slides are next

incubated with 10% normal goat serum at RT for 30 min and washed with phosphate

buffered saline (PBS). Anti-p24 monoclonal antibody is added to each well, and the slides

are incubated in a humid chamber at 37°C. The slides are labelled with goat anti-mouse IgG for 30 min and then washed in PBS overnight. The percentage of cells fluorescing in oligonucleotide-treated and untreated cells is compared.

The results demonstrate that oligonucleotides according to the invention substantially and significantly reduce p24 expression. Cvtn arrπc Effect ( F^

HIV-induced cytopathic effect is determined by measuring the decrease in the

number of viable cells after infection. The cells are counted by adding MTT or trypan

blue dye to the cells and determining how many cells (dead) take up the dye. The assay

is done in triplicate.

The results demonstrate that oligonucleotides according to the invention will reduce the viral cytopathic effect.

Reverse Transcriptase Assay

This assay is performed essentially as described in Agrawal and Sarin, supra.

Supernatants from virus-infected cultures in the presence and absence of oligonucleotide

are collected and virus particles precipitated with poly(ethyleneglycol). The virus pellet is suspended in 300 μl of buffer containing 50 mM Tris-HCl (pH 6.8), 5 mM dithiothreitol

(DTT), 250 mM KCl, and 25 % Triton X-100. Reverse transcriptase activity in the

solubilized pellet is assayed in a 50 μl reaction mixture containing 50 mM Tris-HCl (pH

7.8), 5 mM DTT, 100 mM KCl, 0.01 % Triton X-100, 5 μg dtl5.rAn as template primer, 10 mM MgC12, 15 μM [³H]dTTP (15 Ci/mmol), and 10 μl of the disrupted virus suspension. After incubation for 1 hr at 37°C and subsequent addition of 50 μg yeast tRNA, the incorporation into the cold trichloroacetic acid-insoluble DNA fraction is assayed by counting in a β scintillation counter. The results demonstrate that oligonucleotides according to the invention inhibit

reverse transcriptase. Example 8

4-Pentenoic anhydride required for the preparation of the protected nucleoside is

synthesized by a modification of the literature report. Ellervik, U and Magnusson, G.

Acta Chemica Scandinavica, 1993, 47:826-828 as given below:

4-Pentenoic acid (25 g, 250 mmoi) is dissolved in 200 ml of dichloromethane and

the solution cooled in ice-bath. Triethyl amine (35 ml, 250 mmoi) is added slowly to the

cooled solution. Next, N,N-bis[oxo-3-oxazolidinyl]phosphorodiamidic chloride (prepared by the literature procedure, J. Cabre-Castellvi et al., Synthesis, 616-620, 1981) was added

to the above reaction mixture. The reaction mixture is allowed to warm to room

temperature and stirred for 2-3 h. The reaction slurry was filtered and the residue was washed with dichloromethane (ca. 100 ml). The combined filtrate and washings were

combined and evaporated to give a pale yellow liquid. Vacuum distillation at 90-93 °C/6 mm Hg gave 4-pentenoic anhydride as a colorless liquid ca. 20 g (85% yield).

Example 9 Preparation of N-pent-4-enovl 2'-deoxv adenosine (dA Npr :

2'-Deoxy adenosine (Mallinkckrodt) (2.5 g, 10 mmoi) is dried by repeated evaporation from anhydrous pyridine and is suspended in 50 ml of anhydrous pyridine. Trichloromethylsilane (64. ml, 50 mmoi) is added and the reaction is stirred for about 1 h. Then, 4-pentenoic anhydride (4g, 20 mmoi) is added and the contents stirred. After 15 min triethyl amine (3 ml) was added and the contents stirred for 2-3 h. The reaction

slurry was cooled to 0-5 °C and 10 ml of water was added. After 5 min., 28% NH4OH (10 ml) was added. The resulting clear solution was evaporated to dryness. Water (150 ml)

was 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.

Same general procedure can be employed for the preparation of dG and dC

protected nucleosides.

Example 10

Preparation of 5 ' -Q-DMT-N-4-pent-4-enoy 1-2 ' -deoxy adenosine- ' -H-phosphonate

(triethvl ammonium saltl

The title compound was prepared by adopting a procedure as described by Froehler in Protocols for Oligonucleotides and analogs, Agrawal, S. Ed., pp. 63-80 as given below:

To 544 mg (1.63 mmoi) of dA(N-pr) in 20 ml of anhydrous pyridine is added 1.108

g (3.3 mmoi) of dimethoxytritylchloride. The reaction mixture is stirred at room

temperature for 12 h. The reaction mixture is evaporated to dryness. The residue is chromatographed over silica gel 60 and eluted with CH₂C12:CH₃OH:(Et)3N to give 0.73 of 5'-O-DMT-N-4-pent-4-enoyl-2'-deoxyadenosine as a white foamy material.

To a stirred solution of 1,2,4 triazole (0.944 g, 13.3 mmoi) and triethyl amine (5.5 ml, 30 mmoi) in anhydrous CH₂C1₂ (40 ml) is added PC1₃ (0.35 ml, 3.9 mmoi) at room temperature under argon. After 30 min, the reaction mixture is cooled to 0°C and 5'- DMT-protected nucleoside (500 mg, 0.88 mmoi) in 15 ml CH₂C1₂ is added dropwise over

10-15 min at 0°C and allowed to warm to room temperature. The reaction mixture is

poured into 1M triethylammoniumbicarbonate (TEAB) (75 ml, pH 8.5) with stirring. The mixture is transferred to a separatory funnel and the phases are separated. The aqueous

phase is extracted with methylene chloride and the combined organic phase washed with

1M TEAB (1 x 50 ml). The organic layer is dired over sodium sulfate and evaporated to

dryness. The solid product thus obtained is purified by chromatography over silica gel. Elution with CH₂Cl₂:CH₃OH:(Et)₃N (18: 1: 1) gave 0.065 g of the title compound.

Other H-phosphonate nucleosides were similarly prepared in overall yields ranging from 70-90% .

Similarly nucleoside 5'-O-DMt-3'-6-cyanoethyl-N,N-diisopropylphosphoramidites

and 5 ' -O-DMT-3 ^* )-methyl-N , N-diisopropylphosphoramidites were prepared using standard protocols as described by Beaucage, S.L., in Protocols for Oligonucleotides and Analogs, Agrawal, S. Ed., pp. 33-61.

Example 11

Protocol for removal of the protecting group after dinucleoside synthesis (Eg. Tg-dimerϊ:

The support bound synthesized by H-phosphonate methodology (Froehler ref. above) is treated with a solution of 2% 1₂ in (pyridine: water, 98:2) for 30 min. This procedure completely removes the base protecting groups. This additional step is not necessary if one is making Po oligos using H-phosphonate methodology because simultaneous oxidation and deprotection can be achieved by a single 1₂ protocol using the

reagent specified above.

Example 12 Protocol for preparation of phosphate methylated oligonucleotide feg.. TG rP-OMeΗ:

The support-bound H-phosphonate oligonucleotide is treated with a 10% solution

of MeOH (ROH) in CCl₄/N-methylimidiazole/Et₃N, 9/0.5/0.5 for 40 min. The solid support is washed with acetonitrile dried and then treated with the iodine solution as above

to remove the base-protecting group. The CPG-bound oligonucleotide is next treated with

a solution of K₂CO₃ in methanol (0.05 M) for 4-6 h. to cleave the oligo from the support.

The solution is evaporated to dryness and the oligonucleotide can be purified by reverse

phase HPLC to give the pure phosphatemethylated dimer.

Similar protocol can be employed for longer oligos except that the time of

deprotection and oxidation have to be increased.

Other backbone modified oligonucleotides can be prepared by using ROH (for alkyl

phosphotriesters), RNH₂ (for phosphoramidates).

Example 13 Protocol for preparation of phosphorothioates (free and support-houndl:

Following the preparation of phosphorothioate oligonucleotide by phosphoramidite methodology, but using the new nucleobase protecting group, the support-bound oligo is treated with the iodine solution, as above, to remove the base-protecting group and then with anhydrous triethylamine to remove the phosphate protecting group. Finally cleavage

from the support is achieved by treatment with K₂CO₃ solution as above.

Claims

We claim:

1. A mononucleotide synthon having the structure:

wherein R* and R^b, and each R' are independently H, C, - C₂₀ alkyl, aryl, heterocyclic, C _r

C₂₀ alkoxy, R is a protecting group, n is 1-3, i is 1-n, X' is C, O, S, or N, such that if n

> 1 the identity of each X' is independent of the identity of every other X "and the identity

of each substituent R' is independent of every other R ', each R 'is covalently bound to the

corresponding X', the X' are arranged consecutively such that X ' is bound to the N and

Xⁿ is bound to the O, and B is any protected, modified or unmodified, purine or

pyrimidine base.

2. A mononucleotide synthon according to claim 1 having the structure:

XTV A mononucleotide synthon according to claim 2 having the structure:

4. A mononucleotide synthon according to claim 3 having the structure and

stereoconfiguration:

5. A mononucleotide synthon having the structure:

XV wherein R^a and R^b, and each R'are independently H, C, - C₂₀ alkyl, aryl, heterocyclic, C _r

of each substituent R' is independent of every other R ', each R 'is covalently bound to the corresponding X', the X' are arranged consecutively such that X ' is bound to the N and

X° is bound to the O, and B is any protected, modified or unmodified, purine or

pyrimidine base.

6. A mononucleotide synthon according to claim 5 having the structure:

XVI

7. A mononucleotide synthon according to claim 6 having the structure and configuration:

Villa

8. A mononucleotide synthon according to claim 6 having the structure and configuration:

9. A method of synthesizing the mononucleotide synthon according to claim 1

comprising contacting a compound of structure:

xvπ with PC1₃ to yield

xvm

and then contacting XVIII with a 5'-protected monnucleoside having an unprotected 3'-

hydroxyl.

10. A method according of synthesizing a mononucleotide synthon according to claim 2 comprising contacting a compound having structure:

XIX with PC1₃ to yield

XX and then contacting XX with a 5'-protected monnucleoside having an unprotected 3'- hydroxyl.

11. A method of synthesizing the compound according to claim 3 comprising contacting

ephedrine with PC1₃ to yield the chlorophosphoramidite:

and then contacting the chlorophosphoramidite with a 5 '-protected mononucleoside having an unprotected 3 '-hydroxyl.

12. A method of synthesizing the compound according to claim 4 comprising contacting

(lR,2S)-(-)-ephedrine with PC1₃ to yield the chlorophosphoramidite:

IV and then contacting IV with a 5 '-protected mononucleoside having an unprotected 3'-

hydroxyl.

13. A method of synthesizing the compound according to claim 5 comprising contacting

compound XIII with oxidative thiolation agent.

14. A method according to claim 13, wherein the oxidative thiolation agent is 3H-1.2- benzodithiol-3 -one- 1 , 1 -dioxide .

15. A method of synthesizing the compound according to claim 6 comprising contacting the compound XIV with oxidative thiolation agent.

16. A method according to claim 15, wherein the oxidative thiolation agent is 3H-1,2- benzodithiol-3-one- 1 , 1 -dioxide .

17. A method of synthesizing the compound according to claim 7 comprising contacting compound VI oxidative thiolation agent.

18. A method according to claim 17, wherein the oxidative thiolation agent is 3H-1.2- benzodithiol-3-one-l , 1 -dioxide.

19. An oligonucleotide having from one to all nucleotide P-chiral centers independently predominantly in the S configuration. 20. An oligonucleotide having from one to all phosphorothioate internucleotide linkages

that are independently predominantly in the R or S configuration.

21. A method of synthesizing an oligonucleotide according to claim 19 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound XIV.

22. A method of synthesizing an oligonucleotide according to claim 19 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound XV.

23. A method of synthesizing an oligonucleotide according to claim 19 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound VI.

24. A method of synthesizing an oligonucleotide according to claim 20 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound XV.

25. A method of synthesizing an oligonucleotide according to claim 19 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound XVI.

26. A method of synthesizing an oligonucleotide according to claim 19 comprising contacting a nascent oligonucleotide having a free 5 'hydroxyl with compound Villa or Vlllb. 27. A nucleoside base amino protecting group having the structure:

where n,, n₂, and n₃ are independently 0-10, and the arrow indicates the point of linkage to the amino moiety.

28. The nucleoside base amino protecting group according to claim 27 having the structure:

and the arrow indicates the point of linkage to the amino moiety.

29. An oligonucleotide comprising from 3 to all internucleoside linkages being phosphotriester linkages of the form:

where X is O, N, or S and R is a C, - C₂₀ alkyl group or an aryl group.

30. A method of synthesizing a phosphotriester oligonucleotide comprising synthesizing a support bound oligonucleotide according to the H-phosphonate method using the base protecting according to either of claims 27 or 28, then contacting the support bound 31. A method of sythesizing an unprotected support bound oligonucleotide comprising

synthesizing a support bound oligonucleotide according to the phosphoramidite method

using the base protecting according to either of claims 27 or 28, contacting the support

bound oligonucleotide with iodine in water, and then contacting the support bound

oligonucleotide with anhydrous triethylamine.

32. A method of screening for compounds that preferentially bind to oligonucleotides

having a particular sequence comprising contacting an composition containing the compound that preferentially binds to oligonucleotides having a particular sequence with

a support bound oligonucleotide according to claim 31, wherein the support-bound

oligonucleotide has the sequence to which the compound preferentially binds.

33. The method of claim 32 wherein the compound is a nucleic acid, a protein, or a

transcription factor.

34. An unprotected solid support-bound oligonucleotide.

35. A combinatorial library comprising the oligonucleotide according to claim 34.

36. A method of screening compounds that preferentially bind to oligonucleotides

having a particular sequence comprising contacting the compounds with a combinatorial

library according to claim 35.

37. A mononucleotide synthon according to claim 2, wherein R^b is methyl and R ^l and

R² are both H. 38. A mononucleotide synthon according to claim 6, wherein R^b is methyl and R ' and

R² are both H. 9. A method according to claim 10, wherein R^b is methyl and R ' and R ²are both H.