RU2824141C2 - Novel bicyclic nucleosides and oligomers derived therefrom - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I) and oligonucleotides obtained therefrom. In particular, present invention relates to a compound of formula (I)

where one of T₁ and T₂ is OR₁ or OR₂; and the other of T₁ and T₂ is OR₁ or OR₂; where R₁ is H or a hydroxyl-protective group, wherein said hydroxyl-protective group is independently selected in each case from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4''-trimethoxytrityl (TMTr), 9-phenylxanthine-9-yl (pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX), and R₂ is a phosphorus-containing moiety, wherein said phosphorus-containing moiety is a phosphoramidite moiety of formula (X)

where R₅ is C₁-C₉ alkyl substituted with cyano; R₆ and R₇ are independently C₁-C₉ alkyl; or together with the nitrogen atom to which they are attached form a heterocyclic ring, where said heterocyclic ring is selected from pyrrolidinyl, piperidinyl or morpholinyl, where said heterocyclic ring is optionally substituted with C₁-C₃ alkyl; and where Bx is a purine base or a pyrimidine base.

EFFECT: novel oligonucleotides are obtained, which can be used as an antisense agent for preventing or treating a disease, where said disease is muscular dystrophy, preferably Duchenne muscular dystrophy.

24 cl, 12 tbl, 59 ex, 12 dwg

Description

The present invention relates to new bicyclic nucleosides and oligomers obtained therefrom. In particular, the present invention relates to a compound of formula (I), to an oligomer containing at least one compound of formula (IV), to said compounds or an oligomer according to the present invention for use as a medicament for preventing, treating or diagnosing a disease and to a pharmaceutical composition containing at least one compound or at least one oligomer according to the present invention. The invention also relates to the in vitro use of an oligomer according to the present invention for binding to a target nucleic acid sequence and to a method for the solid-phase synthesis of an oligomer according to the present invention.

LEVEL OF TECHNOLOGY

Antisense therapy has become an important platform for the development of innovative drugs. Oligonucleotide analogues that exhibit strong and sequence-specific binding to single-stranded RNA or double-stranded RNA and are resistant to enzymatic degradation are possible candidates for therapeutic use as inhibitors or modulators of protein expression. Chemically modified nucleosides are incorporated into antisense compounds to enhance their properties, such as nuclease resistance, pharmacokinetics, or affinity for the target RNA.

In recent years, the main direction of development in the field of synthetic biology has been the development of antisense systems with improved activity and efficiency, obtained without using known molecular components found in nature. The main requirement for solving this problem is the development of artificial genetic polymers, often called xeno-nucleic acids (XNA), that perform the function of natural DNA or RNA (Herdewijn et al., Chem. Biodiversity 2009, 6, 791). It is expected that the availability of such systems and their introduction into living organisms will give them new properties that are of interest in the field of biotechnology and medicine.

The potential for use as alternative genetic material has been explored for only a few candidates among the vast number of nucleic acid modifications that have emerged over the past 30 years. In one attempt to incorporate non-naturally occurring components into the natural genetic apparatus with minimal chemical changes, it was shown that thymidine in the genome of E. coli strains can be replaced by 5-chlorouridine in an evolutionary process until a minimal residual thymidine content is achieved (Marliere et al., Angewandte Chemie, Int. Edition 2011, 50, 7109). In another approach, unnaturally occurring nucleic acids such as 1,5-anhydrohexitol nucleic acid (HNA; Hendrix et al., Chem. Eur. J. 1997, 3, 110) and cyclohexene nucleic acid (CeNA; Nauwelaerts et al., Nucleic Acids Res. 2005, 33, 2452), fluoroarabinooligonucleotides (FANA; Wilds et al., J. Nucleic Acids Res. 2000, 28, 3625), arabinonucleic acids (ANA; Damha et al., J. Am. Chem. Soc. 1998, 120, 12976), threose nucleic acids (TNA; Schoning et al., Science 2000, 290, 1347) and locked nucleic acid (LNA; Koshkin et al., J. Am. Chem. Soc. 1998, 120, 13252; Obika et al., Tetrahedron Lett. 1998, 39, 5401), can be transcribed from DNA and reverse transcribed into DNA by DNA polymerases.

In an alternative approach, the backbone structure of RNA and DNA is altered by changing the attachment site of the internucleoside phosphate unit to the sugar from the 3' to the 2' oxygen atom (2',5'-DNA or 2',5'-RNA). While 2',5'-RNA is a naturally occurring biopolymer that was first discovered in bacteria in the form of 2',5'-polyadenylates, 2',5'-DNA does not occur in nature (Trujillo et al., Eur. J. Biochem. 1987, 169, 167). The base pairing and replication properties of both polymers have been studied previously. 2',5'-RNA was shown to bind to complementary RNA but not to DNA. Duplexes with RNA are slightly less stable than pure RNA duplexes, and duplexes of pure 2',5' RNA sequences exist but are even less stable (Wasner et al., J. Biochemistry 1998, 37, 7478). Furthermore, primer template extension experiments have shown that extended fragments containing up to four 2',5'-linked nucleotides in the template can be reverse transcribed into DNA by polymerases or reverse transcriptases even in the absence of significant affinity of 2',5' DNA for native DNA (Sinha et al., J. Am. Chem. Soc. 2004, 126, 40).

For entropic stabilization of complex formation with complementary natural nucleic acids, a bicyclic DNA analogue was developed, which differs from natural DNA by the presence of an additional ethylene bridge located between the C(3') and C(5') centers. The sites of attachment of the linker phosphodiester units are the same as in natural nucleic acids, i.e. at the 3'- and 5'-ends. Such a change in the carbon skeleton provides a closed conformation of the sugar, which leads to the fact that in bicyclodeoxynucleosides there is an increased preorganization of single strands for duplex formation. Decameras of bicyclodeoxyadenosine and bicyclothymidine bind to natural complements of RNA and DNA, as well as to each other, with the formation of double and triple helical structures. Compared with natural DNA, duplex formation is associated with reduced enthalpy and entropy of base pairing and compensates for the free energy of duplex formation (Bolli et al., Nucleic Acids Res. 1996, 24, 4660).

However, after more than three decades of research in the field of antisense therapy, clinical applications remain limited by low biological stability, poor pharmacokinetic properties and off-target toxicity of this class of compounds.

Thus, there remains a need for antisense compounds that are resistant to enzymatic degradation in vivo and exhibit strong and sequence-specific binding to nucleic acids.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect, the invention provides a compound of formula (I):

where one of T ₁ and T ₂ is OR ₁ or OR ₂ ;

and the other of T ₁ and T ₂ is OR ₁ or OR ₂ ; where

R ₁ is H or a hydroxyl protecting group, and

R ₂ is a phosphorus-containing moiety; and

Bx is a nitrogenous base.

According to a second aspect, the invention provides an oligomer comprising at least one compound of formula (IV)

wherein, independently, in each of said at least one compound of formula (IV), one of _T3 or _T4 is a nucleoside linker group;

the other of _T3 and _T4 is _OR1 , _OR2 , a 5'-terminal group, a 7'-terminal group, or a nucleoside linker group, where _R1 is H or a hydroxyl protecting group, and _R2 is a phosphorus-containing moiety; and Bx is a nitrogenous base.

According to a third aspect, the present invention provides a compound of formula (I), (II) or (III) according to the invention or an oligomer according to the invention for use as a medicament for the prevention or treatment of a disease.

According to a further aspect, the present invention provides an oligomer according to the invention, preferably an oligomer of formula (V) according to the invention for use as a medicament for the prevention, treatment or diagnosis of a disease, wherein said disease is muscular dystrophy, and preferably said disease is Duchenne muscular dystrophy.

According to a further aspect, the invention provides a pharmaceutical composition comprising at least one compound selected from formulae (I), (II) or (III), or at least one oligomer according to the invention.

According to a further aspect, the oligomer according to the invention is used in vitro to bind to a target nucleic acid sequence.

According to a further aspect, the invention provides a method for solid-phase synthesis of the oligomer according to the invention.

The invention described herein provides novel compounds wherein the position of a group used for linking to other moieties, such as a nucleoside linker group, is shifted compared to compounds known in the art. Thus, the compounds of the invention are or can be linked via the 5' end and 7' end of the bicyclic sugar to other compounds such as nucleosides or nucleotides (FIG. 1C and FIG. 1D). In contrast, according to the prior art, nucleosides or nucleotides are linked via the 3' and 5' ends, both in the case of known nucleosides or nucleotides containing a bicyclic sugar (FIG. 1B) and in natural DNA or RNA (FIG. 1A).

As a consequence of said shift of the linker group, the geometry of the oligomer backbone in the compounds according to the invention changes. Said change in the geometry of the backbone in turn leads to a change in the stacking of the nitrogenous bases in the compounds according to the invention compared to the helices of natural nucleic acids. Consequently, the oligomers according to the invention adopt helical conformations that differ significantly from natural DNA. Furthermore, taking these facts into account, it can be expected that the oligomers according to the invention that form duplex structures will have a geometry that differs from traditional canonical duplexes.

Despite the changes in the backbone geometry and the stacking of the nitrogenous bases, the inventors have surprisingly found that the oligomers of the invention form base pairs with natural DNA and RNA bases, and the duplexes formed from the oligomers of the invention have a thermal stability that is in the same range as that of natural DNA duplexes. Therefore, the oligomers of the invention have the structural and base pairing properties necessary to be considered as new xeno nucleic acids that can perform the function of natural DNA. In addition, the oligomers of the invention have a comparable base pairing selectivity and an even more pronounced mismatch recognition ability compared to similar natural DNA. Improved mismatch recognition generally reduces potential side effects and is thus an attractive property for a potential antisense agent.

DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1: A) α-monocyclic DNA; B) bicyclic (bc-)DNA; C) 7',5'-β-bc-DNA, i.e. the proposed compound of formula (III); D) 7',5'-α-bc-DNA, i.e. the proposed compound of formula (II).

FIG. 2: Comparison of 7',5'-β-bc-DNA, i.e., the proposed compound of formula (III), shown on the left, and 7',5'-α-bc-DNA, i.e., the proposed compound of formula (II), shown on the right.

FIG. 3: X-ray structure of a) 5'-O-n-nitrobenzoyl-7',5'-α-bc-T, b) 5'-O-acetyl-7',5'-α-bc-GAc. Non-polar hydrogen atoms are omitted for clarity.

FIG. 4: Insertion of 7',5'-α-bc-DNA with reversed polarity into β-DNA.

FIG. 5: UV melting curves (260 nm) of oligonucleotide ON21 (SEQ ID NO: 21) with fully modified parallel (oligonucleotide ON22; SEQ ID NO: 22) and antiparallel (oligonucleotide ON23; SEQ ID NO: 23) complement, parallel DNA and parallel RNA and their comparison with the corresponding duplex of natural DNAs. Total strand concentration: 2 μM in 10 mM _{NaH2PO4 ,} 150 mM NaCl, pH 7.0.

FIG. 6: CD spectra of duplexes a) DNA⋅RNA, b) ON21⋅RNA and c) ON21⋅DNA, d) ON21⋅ON22 at 20°C. Experimental conditions: Total strand concentration 2 μM in 10 mM NaH ₂ PO ₄ , 150 mM NaCl, pH 7.0.

FIG. 7: Fragment of a gel image, a) Control experiment with DNA. DNA degradation reaction, after b) 1 hour, c) 2 hours, d) 4 hours, e) 24 hours; f) Control experiment with ON21 oligonucleotide; ON21 degradation reaction, after g) 1 hour, h) 2 hours, i) 4 hours, j) 24 hours.

FIG. 8: Results of complement activation C3; PO denotes phosphate nucleoside linkers, PS denotes phosphorothioate nucleoside linkers, bc-DNA denotes 7',5'-α-bc-DNA backbone, tc-DNA denotes tricyclic backbone. Each measurement was performed in at least 3 replicates. The sequence is similar for 5 ON.

FIG. 9: Comparison of mRNA expression levels after incubation with PS-α-bc-DNA or PS-tc-DNA. The sequence is similar for the two ONs. Abbreviations are as indicated for FIG. 8.

FIG. 10: X-ray structure of 7'-O-p-nitrobenzoyl-7',5'-β-bc-T. Hydrogen atoms have been omitted for clarity.

FIG. 11: UV melting curves (260 nm) of the 7',5'-β-bc-DNA homoduplex and their comparison with the corresponding natural DNA duplex. Total strand concentration:

2 µM in 10 mM NaH ₂ PO ₄ , 150 mM NaCl, pH 7.0.

FIG. 12: CD spectra of three duplexes a) ON13⋅ON14, b) ON13⋅DNA and c) DNA⋅DNA at temperatures from 10 to 80°C. Experimental conditions: Total strand concentration 2 μM in 10 mM NaH ₂ PO ₄ , 1 M NaCl, pH 7.0, 10°C.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains.

The terms "amino protecting group,""amino protecting group," or "amino-protecting group," as used interchangeably herein, are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wilts, ^3rd edition, John Wiley & Sons, 1999, Greene's Protective Groups in Organic Synthesis, P.G.M. Wuts, ^5th edition, John Wiley & Sons, 2014, and Current Protocols in Nucleic Acid Chemistry, eds. S.L. Beaucage et al. 06/2012, and in particular in section 2. "Amino-protecting groups" suitable for the present invention include and are typically and preferably independently in each case selected from methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfo)fluorenyl methyl carbamate, 2,7-di-tert-butyl [9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilyl ethyl carbamate (Teos), 2-phenyl ethyl carbamate (hZ), 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz) and 2,4,6-trimethylbenzylcarbamate; as well as formamide, acetamide, benzamide.

The terms "hydroxyl protecting group", "hydroxyl group protecting group" or "hydroxyl-protecting group", as used interchangeably herein, are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, ^3rd edition, John Wiley & Sons, 1999; Greene's Protective Groups in Organic Synthesis, P.G.M. Wuts, ^5th edition, John Wiley & Sons, 2014, and in Current Protocols in Nucleic Acid Chemistry, ed. by S.L. Beaucage et al. 06/2012, and in particular in section 2. In a particular embodiment, the "hydroxyl protecting groups" according to the present invention comprise and are typically and preferably independently in each case selected from acetyl, benzoyl, benzyl, β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMTr), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), a silyl ether such as tert-butyldiphenylsilyl ether (TBDPS), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers; simple methyl ethers, simple ethoxyethyl ethers (EE).

In a preferred embodiment, the "hydroxyl protecting groups" of the present invention comprise and are typically and preferably independently in each occurrence selected from acetyl, tert-butyl, tert-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triphenylsilyl, triisopropylsilyl, benzoyl formate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, 4-monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr) and 4,4',4'-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, bonila, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

In some embodiments, the hydroxyl protecting group is independently in each instance selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4''-trimethoxytrityl (TMTr), 9-phenylxanthine-9-yl (pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In preferred embodiments, the hydroxyl protecting group is independently selected in each instance from triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4"-trimethoxytrityl (TMTr), 9-phenylxanthine-9-yl (pixyl), and 9-(p-methoxyphenyl)-xanthine-9-yl (MOX). In further preferred embodiments, the hydroxyl protecting group is independently selected in each instance from trityl, 4-monomethoxytrityl, and 4,4'-dimethoxytrityl. In a particularly preferred embodiment, said hydroxyl protecting group is independently in each case selected from triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4"-trimethoxytrityl (TMTr), 9-phenylxanthine-9-yl (pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In a more preferred embodiment, the hydroxyl protecting groups according to the present invention are acetyl, dimethoxytrityl (DMTr), tert-butyldimethylsilyl (TBDMS), tri-isopropylsilyloxymethyl (TOM) or tert-butyldiphenylsilyl ether (TBDPS). In yet another particularly preferred embodiment, said hydroxyl protecting group is independently in each case selected from 4,4'-dimethoxytrityl (DMTr) or 4-monomethoxytrityl. In yet another particularly preferred embodiment, said hydroxyl protecting group is 4,4'-dimethoxytrityl (DMTr).

The term "phosphorus-containing moiety" as used herein refers to a moiety containing a phosphorus atom in the P ^III or P ^V valence state, represented by formula (VII)

where W is O, S or Se, or W is an electron pair;

R ₃ and R ₄ independently of one another are H, halogen, OH, OR ₅ , NR ₆ R ₇ , SH, SR ₈ , C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ aminoalkyl; where R ₅ is C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; acetyl; a hydroxyl protecting group; wherein _R6 and _R7 , independently of one another, represent hydrogen, _C1 - _C9 alkyl optionally substituted with cyano, nitro, halogen, _C2 - _C6 alkenyl, _C3 - _C6 cycloalkyl, _C1 - _C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, preferably said heterocyclic ring being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, and said heterocyclic ring being optionally substituted with _C1 - _C3 alkyl; and _R8 is a thiol protecting group; and the wavy line denotes the point of attachment to the oxygen atom in said _OR2 group. If W is O, S or Se, then said P atom in said phosphorus-containing moiety is in the ^PV valence state. If W is an electron pair, then said P atom in said phosphorus-containing moiety has the ^PIII valence. The moiety of formula (VII) includes any possible stereoisomers. Furthermore, said moieties represented by formula (VII) include salts thereof, wherein, as a rule, preferably, said salts are formed as a result of treatment with inorganic bases or amines and, as a rule, preferably, are salts obtained as a result of the reaction, wherein the OH or SH groups (independently of each other) are said R ₃ and R ₄ . Preferred inorganic bases or amines that form salts with OH or SH groups are well known in the art and, as a rule, preferably, are trimethylamine, diethylamine, methylamine or ammonium hydroxide. Said phosphorus-containing moieties included in the present invention, if appropriate, are also abbreviated as "O ^- HB ⁺ ", wherein said HB ⁺ refers to the resulting cation.

In a preferred embodiment, in the "phosphorus-containing moiety" R ₃ and R _4, independently of one another, are H, OH, OR ₅ , NR ₆ R ₇ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ aminoalkyl; wherein R ₅ is C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen; aryl, C ₁ -C ₆ alkylene aryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen; acetyl; a hydroxyl protecting group; R ₆ and R _7, independently of one another, are hydrogen, C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen; aryl optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; and _R8 is a thiol protecting group; and the wavy line represents the point of attachment to the oxygen atom in said _OR2 group.

The term "phosphorus-containing moiety" as used herein includes and is generally preferably independently in each instance selected from a moiety derived from phosphonates, phosphite triester, monophosphate, diphosphate, triphosphate, phosphate triester, phosphate diester, thiophosphate ester, dithiophosphonate ester, or phosphoramidites.

Thus, in a preferred embodiment, said OR ₂ is independently in each case selected from phosphonates, phosphite triester, monophosphate, diphosphate, triphosphate, phosphate triester, phosphate diester, thiophosphate ester, dithiophosphate ester or phosphoramidites, and preferably said OR ₂ is a phosphoramidite or phosphate triester, more preferably a phosphoramidite.

In a preferred embodiment, the phosphorus-containing moiety is derived from a phosphonate represented by formula (VII), wherein W is O, R ₃ is selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ aminoalkyl, and R ₄ is OH or O ^- HB ⁺ ; and the wavy line denotes the point of attachment to the oxygen atom in said OR ₂ group. In another embodiment, the phosphorus-containing moiety of formula (VII) is an H-phosphonate, wherein W is O, R ₃ is hydrogen, and R ₄ is OH or O ^- HB ⁺ ; and preferably, said O ^- HB ⁺ is HNEt ₃ ⁺ . In a further embodiment, the phosphorus-containing moiety of formula (VII) is an alkyl phosphonate, wherein W is O, R ₃ is alkyl, and R ₄ is OH or O ^- HB ⁺ ; and preferably, said O ^- HB ⁺ is HNEt ₃ ⁺ . More preferably, the phosphorus-containing moiety of formula (VII) is a methyl phosphonate, wherein W is O, R ₃ is hydrogen, and R ₄ is OH or O ^- HB ⁺ ; and preferably, said O ^- HB ⁺ is HNEt ₃ ⁺ . In another embodiment, the phosphorus-containing moiety of formula (VII) is a phosphonocarboxylate, wherein R ₃ or R ₄ are independently a carboxylic acid. Preferably, said phosphonocarboxylate is phosphonoacetic acid or phosphonoformic acid. In a further embodiment, the phosphorus-containing moiety of formula (VII) is 2-aminoethyl phosphonate.

In a preferred embodiment, R ₃ and R ₄ in the phosphorus-containing moiety of formula (VII) independently of one another are H, OH, halogen, OR ₅ , NR ₆ R ₇ , SH, SR ₈ , C ₁ -C ₄ alkyl, preferably C ₁ -C ₂ alkyl, C ₁ -C ₄ haloalkyl, preferably C ₁ -C ₂ haloalkyl, C ₁ -C ₄ alkoxy, preferably C ₁ -C ₂ alkoxy, C ₁ -C ₄ haloalkoxy, preferably C ₁ -C ₂ haloalkoxy, C ₁ -C ₄ aminoalkyl, preferably C ₁ -C ₂ aminoalkyl; where R ₅ is C ₁ -C ₆ alkyl, preferably C ₁ -C ₃ alkyl, each of which, independently from the others, is optionally substituted with cyano, nitro, halogen, NHC(O)C ₁ -C ₃ alkyl, NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, C ₁ -C ₃ alkylene aryl, C ₁ -C ₃ alkylenediaryl, each of which, independently from the others, is optionally substituted with cyano, nitro, halogen, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl, C ₁ -C ₄ haloalkoxy, NHC(O)C ₁ -C ₃ alkyl, NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; acetyl; a hydroxyl protecting group; and R ₆ and R ₇ independently of one another represent hydrogen, C ₁ -C ₆ alkyl, preferably C ₁ -C ₄ alkyl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, C ₂ -C ₄ alkenyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy; aryl optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with C ₁ -C ₃ alkyl; and R ₈ represents a thiol protecting group; and the wavy line indicates the point of attachment to the oxygen atom in the indicated OR ₂ group.

In another preferred embodiment, R ₃ or R ₄ in the phosphorus-containing moiety of formula (VII) are independently at each occurrence and of each other halogen, preferably chlorine, or OR ₅ , wherein R ₅ is a hydroxyl protecting group. Additional phosphorus-containing moieties useful in the invention are described in Tetrahedron No. 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

The term "phosphorus-containing moiety" as used herein preferably refers to a group R ₂ containing a phosphorus atom in the valence state P ^III or P ^V , represented independently in each case by formula (VIII), formula (IX) or formula (X)

wherein Y is O, S or Se, and Y is preferably O or S, more preferably Y is O; and R ₅ and R _5' independently in each occurrence and from each other are hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; a hydroxyl protecting group; wherein _R6 and _R7 , independently of one another, represent hydrogen, _C1 - _C9 alkyl optionally substituted with cyano, nitro, halogen, _C2 - _C6 alkenyl, _C3 - _C6 cycloalkyl, _C1 - _C3 alkoxy; aryl, preferably phenyl, optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, preferably said heterocyclic ring being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, wherein said heterocyclic ring is optionally substituted with _C1 - _C3 alkyl; and _R8 is a thiol protecting group; and the wavy line denotes the point of attachment to the oxygen atom in said _OR2 group.

In a preferred embodiment, said phosphorus-containing fragment R ₂ is represented by formula (VIII)

wherein Y is O, S or Se, wherein Y is preferably O or S, most preferably Y is O; and R ₅ and R _5' independently in each occurrence and from each other are hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C 1 -C 3 alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; a hydroxyl protecting group; P(O)( _OR9 )( _OR9' ), P(O)OP(O)( _OR9 )( _OR9' ); wherein R ₉ and R _9' independently in each occurrence and from each other represent hydrogen, C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, C ₁ -C ₆ alkylene aryl, C ₁ -C ₆ alkylene diaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl, C ₁ -C ₄ haloalkoxy, -NHC(O)C ₁ -C ₃ alkyl, NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; a hydroxyl protecting group; and the wavy line indicates the point of attachment to the oxygen atom in the indicated OR ₂ group.

In a preferred embodiment, R ₅ and R _5' in formula (VIII) independently in each occurrence and from each other represent hydrogen, C ₁ -C ₆ alkyl, preferably C ₁ -C ₃ alkyl, C ₁ -C ₄ alkoxy, preferably C ₁ -C ₂ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, preferably phenyl, _C1 - _C4 alkylenearyl, _C1 - _C4 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; a hydroxyl protecting group.

In a preferred embodiment, R ₅ and R _5' in formula (VIII) independently of each other are C ₁ -C ₄ alkyl or aryl, preferably phenyl. In another preferred embodiment, R ₅ and R _5' in formula (VIII) independently of each other are methyl or ethyl. In a further preferred embodiment, R ₅ and R _5' in formula (VIII) independently of each other are phenyl or benzyl. In another preferred embodiment, R ₅ and R _5' independently at each occurrence and of each other are hydrogen or a hydroxyl protecting group, preferably a hydroxyl protecting group. In a preferred embodiment, in formula (VIII) R ₅ and R _5' independently at each occurrence and of each other are hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which is independently of the others optionally substituted with cyano, nitro, halogen; aryl, C ₁ -C ₆ alkylene aryl, each of which independently of the others is optionally substituted with cyano, nitro, halogen; or a hydroxyl protecting group. As a rule, preferably, said phosphorus-containing moiety R ₂ represented by formula (VIII) is referred to herein as a "phosphate moiety".

In a preferred embodiment, said phosphorus-containing fragment R ₂ is represented by formula (IX)

where Y is O, S or Se, and Y is preferably O or S, most preferably Y is O; and

R ₅ is independently in each occurrence hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which is independently from the others optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, C ₁ -C ₆ alkylene aryl, C ₁ -C ₆ alkylene diaryl, each of which is independently from the others optionally substituted with cyano, nitro, halogen, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl, C ₁ -C ₄ haloalkoxy, -NHC(O)C ₁ -C ₃ alkyl, NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; hydroxyl protecting group;

R ₆ and R ₇ independently of one another represent hydrogen, C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy; aryl, preferably phenyl, optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with C ₁ -C ₃ alkyl; and the wavy line denotes the point of attachment to the oxygen atom in said OR ₂ group. Typically, preferably, said phosphorus-containing moiety R ₂ represented by formula (IX) is referred to herein as a "phosphoramidate moiety" or, used interchangeably, as a "phosphoramidate moiety".

In a preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X)

where R ₅ is hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, independently of one another optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl, a hydroxyl protecting group; and

R ₆ and R ₇ independently of one another represent hydrogen, C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy; aryl, preferably phenyl, optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with C ₁ -C ₃ alkyl, and the wavy line denotes the point of attachment to the oxygen atom in said OR ₂ group. Typically, preferably said phosphorus-containing moiety R ₂ represented by formula (X) is referred to herein as a "phosphoramidite moiety" or as used interchangeably as a "phosphoramidite moiety".

In a preferred embodiment of formula (IX), said Y is O; said R ₅ is independently in each occurrence hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which is independently of the others optionally substituted with cyano, nitro, halogen; aryl, C ₁ -C ₆ alkylene aryl, each of which is independently of the others optionally substituted with cyano, nitro, halogen; a hydroxyl protecting group; where R ₆ and R ₇ are independently of one another hydrogen, C ₁ -C ₉ alkyl, optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl; aryl, optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; an amino protecting group; and the wavy line indicates the point of attachment to the oxygen atom in the indicated OR ₂ group.

In a preferred embodiment of formula (X), said R ₅ independently in each occurrence represents hydrogen, C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which is independently of the others optionally substituted with cyano, nitro, halogen; aryl, C ₁ -C ₆ alkylene aryl, each of which is independently of the others optionally substituted with cyano, nitro, halogen; a hydroxyl protecting group; where R ₆ and R ₇ independently of one another represent hydrogen, C ₁ -C ₉ alkyl, optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl; aryl, optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; an amino protecting group; and the wavy line indicates the point of attachment to the oxygen atom in the indicated OR ₂ group.

In a particularly preferred embodiment, said phosphorus-containing moiety R ₂ is independently in each case selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety.

In a further preferred embodiment, said R ₅ is independently in each occurrence hydrogen, C ₁ -C ₆ alkyl, preferably C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, each of which is independently optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C4 alkylenearyl, _C1 - _C4 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; a hydroxyl protecting group; wherein _R6 and _R7 , independently of one another, represent hydrogen, _C1 - _C6 alkyl optionally substituted with cyano, nitro, halogen, _C2 - _C4 alkenyl, _C3 - _C6 cycloalkyl, _C1 - _C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with _C1 - _C3 alkyl; and the wavy line denotes the point of attachment to the oxygen atom in said _OR2 group.

In a further preferred embodiment, said R ₅ is C ₁ -C ₃ alkyl optionally substituted with cyano, chloro, fluoro or bromine; aryl, C ₁ -C ₃ alkylene aryl, C ₁ -C ₃ alkylenediaryl, each of which is independently optionally substituted with cyano, nitro, chloro, fluoro, bromine, C ₁ -C ₂ alkoxy, C ₁ haloalkyl. In a more preferred embodiment, said R ₅ is C ₁ -C ₃ alkyl, optionally and preferably substituted with cyano, chloro, fluoro or bromine; preferably substituted with cyano. In an even more preferred embodiment, said R ₅ is C ₂ alkyl substituted with cyano, preferably said R ₅ is -CH ₂ CH ₂ -CN.

In a further preferred embodiment, said R ₅ is C ₁ -C ₄ alkyl, preferably methyl or ethyl; aryl, preferably phenyl or benzyl; chloride or a hydroxyl protecting group. In a further preferred embodiment, said R ₅ is methyl or a hydroxyl protecting group.

In a further preferred embodiment, said R ₅ is C ₁ -C ₆ alkoxy optionally substituted with cyano, chloro, fluoro or bromine.

In a further preferred embodiment, said R ₆ and R ₇ independently of each other are H or C ₁ -C ₃ alkyl; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, wherein said heterocyclic ring is optionally substituted with methyl. In a further preferred embodiment, said R ₆ and R ₇ independently of each other are C ₁ -C ₃ alkyl, alkoxy or aryl, wherein aryl is preferably phenyl or benzyl, optionally substituted with cyano, nitro, chloro, fluoro, bromine. In a further preferred embodiment, said R ₆ is hydrogen and R ₇ is (i) C ₁ -C ₉ alkyl or (ii) aryl, (i) or (ii) optionally substituted with cyano, nitro, halogen, aryl, wherein preferably R ₇ is C ₁ -C ₃ alkyl, phenyl or benzyl.

In a further preferred embodiment, said R ₆ and R ₇ are independently selected from methyl, ethyl, isopropyl or isobutyl. In a more preferred embodiment, said R ₆ and R ₇ are independently selected from isopropyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is (i) C ₁ -C ₉ alkyl; (ii) aryl, preferably phenyl; or (iii) said (i) or said (ii) optionally substituted with cyano, nitro, halogen, aryl; and said R ₆ and R ₇ independently of one another are C ₁ -C ₉ alkyl, preferably isopropyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein R ₅ is C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, C ₁ -C ₆ alkylene aryl, C ₁ -C ₆ alkylene diaryl, independently of each other optionally substituted with cyano, nitro, halogen, C ₁ -C ₄ alkoxy, C 1 -C ₄ haloalkyl, C _{1 -C 4} haloalkoxy, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; and R ₆ and R ₇ independently of one another are C ₁ -C ₉ alkyl optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy, phenyl optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with C ₁ -C ₃ alkyl; and the wavy line denotes the point of attachment to the oxygen atom in said OR ₂ group.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is C ₁ -C ₉ alkyl, optionally substituted with cyano, nitro, chloro, fluoro, bromine, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl; aryl, C ₁ -C ₆ alkylene aryl, C ₁ -C ₆ alkylene diaryl, which are independently of each other optionally substituted with cyano, nitro, chloro, fluoro, bromine, C ₁ -C ₄ alkoxy, C ₁ -C ₄ haloalkyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is C ₁ -C ₃ alkyl, optionally substituted with cyano, chloro, fluoro and bromine; aryl, C ₁ -C ₃ alkylene aryl, C ₁ -C ₃ alkylene diaryl, which are independently of each other optionally substituted with cyano, nitro, chloro, fluoro, bromine, C ₁ -C ₂ alkoxy, C ₁ haloalkyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is C ₁ -C ₃ alkyl, 2-cyanoethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, -(CH ₂ ) _n NHC(O)CF ₃ , where n = 3-6; phenyl, C ₁ -C ₃ alkylenephenyl, benzhydryl, which are independently of each other optionally substituted with cyano, nitro, chlorine, fluorine, bromine, C ₁ -C ₂ alkoxy, -CF ₃ .

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is methyl, ethyl, 2-cyanoethyl, preferably 2-cyanoethyl (CH ₂ ) ₂ CN).

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₆ and R ₇ independently of one another are C ₁ -C ₃ alkyl or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein said heterocyclic ring is selected from pyrrolidine, piperidine, morpholine, wherein said heterocyclic ring is optionally substituted with C ₁ -C ₃ alkyl, and further preferably said heterocyclic ring is optionally substituted with methyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein R ₆ is the same as R ₇ , and R ₆ and R ₇ are isopropyl or methyl.

In another particularly preferred embodiment, said phosphorus-containing moiety R ₂ is represented by formula (X), wherein said R ₅ is methyl, ethyl, 2-cyanoethyl, preferably 2-cyanoethyl, and R ₆ is the same as R ₇ , and R ₆ and R ₇ are isopropyl or methyl.

Each alkyl moiety, alone or as part of a larger group such as alkoxy or alkylene, is a straight or branched chain and is preferably _C1 - _C6 alkyl, more preferably _C1 - _C3 alkyl. Examples include methyl, ethyl, n-propyl, prop-2-yl (isopropyl; which is interchangeably abbreviated herein as iPr or Pri, particularly in the depicted chemical formulas), n-butyl, but-2-yl, 2-methylprop-1-yl or 2-methylprop-2-yl. Examples of alkoxy include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy. According to the present description, alkoxy may include additional substituents, such as halogen atoms, to form haloalkoxy moieties.

Each alkylene moiety is a straight or branched chain and is, for example, -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH(CH ₃ )-CH ₂ - or -CH(CH ₂ CH ₃ )-.

Each alkenyl moiety, alone or as part of a larger group such as alkenyloxy or alkenylene, is a straight or branched chain and is preferably _C2 - _C6 alkenyl, more preferably _C2 - _C4 alkenyl. Each moiety may have the (E)- or (Z)-configuration. Examples include vinyl and allyl. The compound according to the present invention containing an alkenyl moiety may thus include, if possible, said compound containing the alkenyl moiety in the (E)-configuration, said compound containing said alkenyl moiety in the (Z)-configuration, and mixtures thereof in an arbitrary ratio.

Each alkynyl moiety, alone or as part of a larger group such as alkynyloxy, is a straight or branched chain and is preferably _C2 - _C6 alkynyl, more preferably _C2 - _C4 alkynyl. Examples are ethynyl and propargyl.

The halogen is fluorine, chlorine, bromine or iodine, preferably chlorine. In a preferred embodiment, the halogen substituent is chlorine.

Each haloalkyl moiety, alone or as part of a larger group such as haloalkoxy, is an alkyl group substituted by one or more identical or different halogen atoms. Examples include difluoromethyl, trifluoromethyl, chlorodifluoromethyl, and 2,2,2-trifluoroethyl.

The term "aryl" as used herein refers to a monovalent aromatic hydrocarbon radical containing 6-14 carbon atoms ( _C6 - _C14 ), derived by the removal of one hydrogen atom from one carbon atom in a parent aromatic ring system, and to said aryl optionally independently substituted with one or more substituents, typically preferably one or two substituents, as described below. Aryl includes bicyclic radicals containing an aromatic ring fused to a saturated, partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring. The aryl groups are optionally independently substituted with one or more substituents, typically preferably one or two substituents, wherein _{said substituents are independently in each occurrence selected from C1-C4 alkyl, halogen, CF3, OH, C1-C3 alkoxy, NR2OR21, C6H5, C6H5 substituted with halogen, C1-C3 alkyl, C1-C3 alkoxy , NR2OR21 , wherein R20 , R21 are independently in each occurrence H , C1 - C3 alkyl .} Exemplary aryl groups include, but are not limited to, radicals derived from benzene (phenyl), substituted phenyls, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthalene, etc. The term "aryl" as used herein preferably refers to phenyl optionally substituted with 1-3 R ₂₂ , wherein R ₂₂ is independently in each occurrence halogen, -OH, C ₁ -C ₃ alkyl optionally substituted with one or two OH, C ₁ -C ₂ fluoroalkyls, C ₁ -C ₂ alkoxy, C ₁ -C ₂ alkoxy-C ₁ -C ₃ alkyls, C ₃ -C ₆ cycloalkyls, -NH ₂ , NHCH ₃ or N(CH ₃ ) ₂ .

If a group is said to be optionally substituted, it preferably optionally contains 1 to 5 substituents, more preferably optionally 1 to 3 substituents, even more preferably optionally 1 or 2 substituents. If a group is said to be optionally substituted and said group contains more than one optional substituent, said more than one substituent may be the same or different in each instance.

The term "nitrogenous base" as used herein, abbreviated as _Bx , refers to unmodified and naturally occurring nitrogenous bases, as well as modified or non-naturally occurring nitrogenous bases and synthetic mimetics thereof. A nitrogenous base is any heterocyclic base that contains one or more atoms or groups of atoms that can form hydrogen bonds with a heterocyclic base of a nucleic acid.

In one embodiment, the nitrogenous base is a purine base or a pyrimidine base, wherein preferably said purine base is a purine or a substituted purine, and said pyrimidine base is a pyrimidine or a substituted pyrimidine. More preferably, the nitrogenous base is (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi) 5-methyluracil (MeU), or a derivative of (i), (ii), (iii), (iv), (v), or (vi). The terms "derivative of (i), (ii), (iii), (iv), (v), or (vi)" and "derivative of a nitrogenous base" are used interchangeably herein. Derivatives of (i), (ii), (iii), (iv), (v) or (vi) and derivatives of nitrogenous bases, respectively, are known to those skilled in the art and are described, for example, in Sharma V.K. et al., Med. Chem. Commun., 2014, 5, 1454-1471, and include, but are not limited to, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, alkyladenine such as 6-methyladenine, 2-propyladenine, alkylguanine such as 6-methylguanine, 2-propylguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, alkynylpyrimidine bases such as 5-propynyl-(-C=C- _CH3 )-uracil, 5-propynyl-(-C=C- _CH3 )-cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil; 8-substituted purine bases such as 8-halo-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl-adenine or -guanine, 5-substituted pyrimidine bases such as 5-halo-, in particular 5-bromo-, 5-trifluoromethyl-uracil or -cytosine; 7-methylguanine, 7-methyladenine, 2-E-adenine, 2-aminoadenine, 8-azaguanine and 8-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, hydrophobic bases, mixed bases, extended bases or fluorinated bases. In certain embodiments, the nitrogenous base includes, but is not limited to, tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, or 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). The term "nucleobase derivative" also includes derivatives in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, or 2-pyridone. Additional nitrogenous bases according to the invention include, but are not limited to, those known in the art (e.g., U.S. Patent No. 3,687,808; Swayze et al., The Medicinal Chemistry of Oligonucleotides, in Antisense a Drug Technology, Chapter 6, pp. 143-182 (Crooke, S. T., ed., 2008); The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, Int. Ed., 1991, Vol. 30 (6), pp. 613-623; Sanghvi, Y. S., Antisense R ₆ search and Applications, Crooke, S. T. and Lebleu, B., eds., CRC Press, 1993, pp. 273-302).

Preferred nitrogenous base derivatives include methylated adenine, guanine, uracil and cytosine and nitrogenous base derivatives, preferably (i), (ii), (iii) or (iv), wherein the corresponding amino groups, preferably exocyclic amino groups, are protected with acyl protecting groups or dialkylformamidino, preferably dimethylformamidino (DMF), and further include nitrogenous base derivatives such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-ioduracil, 2,6-diaminopurine, azacytosine and pyrimidine analogues such as pseudoisocytosine and pseudouracil.

In a further preferred embodiment, said nitrogenous base derivative is selected from methylated adenine, methylated guanine, methylated uracil and methylated cytosine and from a nitrogenous base derivative of (i), (ii), (iii) or (iv), wherein the corresponding amino groups, preferably exocyclic amino groups, are protected by a protecting group.

In a further preferred embodiment, said nitrogenous base derivative is selected from methylated adenine, methylated guanine, methylated uracil and methylated cytosine and a nitrogenous base derivative of (i), (ii), (iii) or (iv), wherein the corresponding amino groups, preferably exocyclic amino groups, are protected with acyl protecting groups or dialkylformamidino, preferably dimethylformamidino (DMF).

In a further preferred embodiment, said nitrogenous base derivative is selected from a nitrogenous base derivative of (i), (ii), (iii) or (iv), wherein the corresponding amino groups, preferably exocyclic amino groups, are protected by a protecting group.

In a further preferred embodiment, said nitrogenous base derivative is a nitrogenous base derivative of (i), (ii), (iii) or (iv), wherein the exocyclic amino groups are protected with acyl protecting groups or dialkylformamidino, preferably dimethylformamidino (DMF).

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₁ , wherein each Rn is independently selected from C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₆ -C ₁₀ aryl-C ₁ -C ₁₀ alkylene or C ₆ -C ₁₀ aryloxy-C ₁ -C ₁₀ alkylene, and said dialkylformamidino protecting group is =C(H)-NR ₁₂ R ₁₃ , wherein R ₁₂ and R ₁₃ are independently selected from C ₁ -C ₄ alkyl.

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₄ , wherein each R ₁₄ is independently selected from C ₁ -C ₄ alkyl; phenyl; phenyl substituted with halogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₄ alkoxy; benzyl; benzyl substituted with halogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₄ alkoxy; or phenyloxy-C ₁ -C ₂ alkylene optionally substituted with halogen, C ₁ -C ₆ alkyl, C ₁ -C ₄ alkoxy; and said dialkylformamidino protecting group is =C(H)-NR ₁₂ R ₁₃ , where R ₁₂ and R ₁₃ are independently selected from C ₁ -C ₄ alkyl.

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₅ , wherein each R ₁₅ is independently selected from C ₁ -C ₄ alkyl; phenyl; phenyl substituted with halogen, C ₁ -C ₄ alkyl, C ₅ -C ₆ cycloalkyl, C ₁ -C ₄ alkoxy; benzyl; benzyl substituted with halogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy; or phenyloxymethylene (CH ₂ -OC ₆ H ₅ ), wherein phenyl is optionally substituted with halogen, C ₁ -C ₄ alkyl, C ₅ -C ₆ cycloalkyl, C ₁ -C ₄ alkoxy; and said dialkylformamidino protecting group is =C(H)-NR ₁₂ R ₁₃ , wherein R ₁₂ and R ₁₃ are independently selected from C ₁ -C ₄ alkyl.

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₆ , wherein each R ₁₆ is independently selected from C ₁ -C ₃ alkyl; phenyl; phenyl substituted with C ₁ -C ₃ alkyl, methoxy; benzyl; benzyl substituted with C ₁ -C ₃ alkyl, methoxy; or phenyloxymethylene (CH ₂ -OC ₆ H ₅ ), wherein C ₆ H ₅ is optionally substituted with C ₁ -C ₃ alkyl, methoxy; and said dialkylformamidino protecting group is =C(H)-NR ₁₂ R ₁₃ , wherein R ₁₂ and R ₁₃ are independently selected from C ₁ -C ₄ alkyl.

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₇ , wherein each R ₁₇ is independently selected from C ₁ -C ₃ alkyl; phenyl; phenyl substituted with C ₁ -C ₃ alkyl, methoxy; benzyl; benzyl substituted with C ₁ -C ₃ alkyl, methoxy; or phenyloxymethylene (CH ₂ -OC ₆ H ₅ ), wherein C ₆ H ₅ is optionally substituted with C ₁ -C ₃ alkyl, methoxy; and said dialkylformamidino protecting group is dimethylformamidino (DMF).

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₈ , wherein each R ₁₈ is independently selected from methyl, isopropyl, phenyl, benzyl or phenyloxymethylene (CH ₂ -OC ₆ H ₅ ), wherein C ₆ H ₅ is optionally substituted with C ₁ -C ₃ alkyl, methoxy; and said dialkylformamidino protecting group is dimethylformamidino (DMF).

In a further particularly preferred embodiment, said acyl protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv) is -C(O)-R ₁₉ , wherein each R ₁₉ is independently selected from methyl, isopropyl, phenyl, benzyl or phenyloxymethylene (CH ₂ -OC ₆ H ₅ ), wherein C ₆ H ₅ is optionally substituted with methyl, isopropyl; and said dialkylformamidino protecting group is dimethylformamidino (DMF).

The term "dialkylformamidino" as used herein refers to =C(H) _{-NR12R13 ,} wherein _R12 and _R13 are independently selected from _C1 - _C4 alkyl. In preferred embodiments, said dialkylformamidino is a protecting group of said exocyclic amino group in said nitrogenous base derivative (i), (ii), (iii) or (iv). The resulting compounds may have the (E)- or (Z)-configuration or both, and mixtures thereof in any ratio are intended to be included within the scope of the present invention. In a preferred embodiment, the disclosed compounds comprise dialkylformamidino, preferably dimethylformamidino (DMF), in the (Z)-configuration.

According to one embodiment, Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine and guanine. Preferably, Bx is selected from thymine, 5-methylcytosine, adenine and guanine. According to one embodiment, Bx is an aromatic heterocyclic fragment that can form base pairs when incorporated into DNA or RNA oligomers in place of the bases uracil, thymine, cytosine, 5-methylcytosine, adenine and guanine.

The term "nucleoside linking group" as used herein refers to any linking group known in the art that can, preferably does, link a disclosed compound of formula (IV), (V), or (VI) to an additional compound, preferably a nucleoside compound, including an additional disclosed compound of formula (IV), (V), or (VI), in the oligomers of the present invention. Exemplary patents that describe such possible linking groups include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,677,439; 5,646,269; and 5,792,608. Said additional compound is selected from a nucleoside compound or a non-nucleoside compound. Said nucleoside compound includes, but is not limited to, and is generally preferably selected from at least one of (i) a nucleoside, (ii) a nucleotide, (iii) an oligonucleotide, or (iv) modifications of (i), (ii), or (iii). Said non-nucleoside compound comprises and is generally preferably selected from a peptide, a protein, silicate compounds or even a solid support. The solid support includes, but is not limited to, surfaces, beads, glass supports, polymers or resins. In a preferred embodiment, the glass is glass with a given pore size, preferably with pores of 500 Å, 1000 Å or 2000 Å. The beads include, but are not limited to, glass beads, preferably glass with a given pore size or magnetic beads. The polymer includes, but is not limited to, polystyrenes, including, for example, divinyl benzene, styrene and chloromethyl styrene. In a preferred embodiment, the solid support is polystyrene beads with a high degree of crosslinking.

The term "nucleoside linking group" includes phosphorus-containing linking groups and non-phosphorus-containing linking groups. Non-phosphorus-containing linking groups do not contain a phosphorus atom, and examples of non-phosphorus-containing linking groups include and are generally preferably selected from alkyl, aryl, preferably phenyl, benzyl or benzoyl, cycloalkyl, alkylene aryl, alkylene diaryl, alkoxy, alkoxyalkylene, alkyl sulfonyl, alkyne, ether, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen; carboxyl, amide, amine, amino, imine, thiol, sulfide, sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane, or mixtures thereof. In a preferred embodiment, the non-phosphorus-containing linking group is aminopropyl, long chain alkylamino, ynyl, acetylamide, aminomethyl, formacetal, thioformacetal, thioformacetyl, riboacetyl, methyleneimino, methylenehydrazino, or a neutral nonionic nucleoside linking group such as amide-3 (3'- _CH2 -C(=O)-N(H)-5') or amide-4 (3'- _CH2 -N(H)-C(=O)-5'). In a preferred embodiment, the phosphorus-free linker group comprises a compound selected from alkyl, aryl, preferably phenyl, benzyl or benzoyl, cycloalkyl, alkylene aryl, alkylene diaryl, alkoxy, alkoxyalkylene, alkyl sulfonyl, alkyne or ether, wherein the compound comprises C ₁ -C ₉ , C ₁ -C ₆ or C ₁ -C ₄ .

In a preferred embodiment, said nucleoside linking group is a phosphorus-containing linking group, and said phosphorus-containing linking group refers to a moiety containing a phosphorus atom in the P ^III or P ^V valence state, represented by formula (XI):

where W is O, S, Se or an electron pair; preferably W is O or S;

R ₁₀ is H, halogen, OH, OR ₅ , NR ₆ R ₇ , SH, SR ₈ , C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ aminoalkyl; where R ₅ is C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; acetyl; a hydroxyl protecting group; wherein _R6 and _R7 , independently of one another, represent hydrogen, _C1 - _C9 alkyl optionally substituted with cyano nitro, halogen, _C2 - _C6 alkenyl, _C3 - _C6 cycloalkyl, _C1 - _C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein preferably said heterocyclic ring is selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being optionally substituted with _C1 - _C3 alkyl; and _R8 is a thiol protecting group; and each of the wavy lines represents the point of attachment of said phosphorus-containing linker group of formula (XI) to an additional compound, preferably a nucleoside compound, including an additional proposed compound of formula (IV), (V) or (VI), in the oligomers according to the present invention. If W is O, S or Se, then said P atom in said phosphorus-containing moiety is in the valence state P ^V . If W is an electron pair, then said P atom in said phosphorus-containing moiety has the valence P ^III . The moiety of formula (XI) includes any possible stereoisomers. Furthermore, said moieties represented by formula (XI) include salts thereof, wherein, as a rule and preferably, said salts are formed as a result of treatment with inorganic bases or amines and, as a rule and preferably, are salts obtained as a result of the reaction, wherein the OH or SH groups (independently of each other) are said R ₁₀ . Preferred inorganic bases or amines that form salts with OH or SH groups are well known in the art and, as a rule and preferably, are trimethylamine, diethylamine, methylamine or ammonium hydroxide. Said phosphorus-containing moieties included in the present invention, if appropriate, are also abbreviated as "O ^- HB ⁺ ", wherein said HB ⁺ refers to the resulting cation.

In a preferred embodiment, in the phosphorus-containing linker group of formula (XI), R ₆ and R ₇ independently of one another are hydrogen, C ₁ -C ₆ alkyl optionally substituted with cyano, nitro, halogen, C ₂ -C ₆ alkenyl; aryl optionally substituted with cyano, nitro, halogen, C ₁ -C ₃ alkyl; or an amino protecting group.

In a preferred embodiment, in the phosphorus-containing linker group of formula (XI), W is O or S; R ₁₀ is H, OH, OR ₅ , NR ₆ R ₇ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, C ₁ -C ₆ aminoalkyl; wherein R ₅ is C ₁ -C ₉ alkyl, C ₁ -C ₆ alkoxy, each of which, independently from the others, is optionally substituted with cyano, nitro, halogen, -NHC(O)C ₁ -C ₃ alkyl, -NHC(O)C ₁ -C ₃ haloalkyl, C ₁ -C ₃ alkylsulfonyl; aryl, _C1 - _C6 alkylenearyl, _C1 - _C6 alkylenediaryl, each of which, independently of the others, is optionally substituted with cyano, nitro, halogen, _C1 - _C4 alkoxy, _C1 - _C4 haloalkyl, _C1 - _C4 haloalkoxy, -NHC(O) _C1 - _C3 alkyl, NHC(O) _C1 - _C3 haloalkyl, _C1 - _C3 alkylsulfonyl; acetyl; or a hydroxyl protecting group; wherein _R6 and _R7 , independently of one another, are hydrogen, _C1 - _C6 alkyl optionally substituted with cyano, nitro, halogen, _C2 - _C6 alkenyl, _C3 - _C6 cycloalkyl, _C1 - _C3 alkoxy; aryl optionally substituted with cyano, nitro, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy; an amino protecting group; and _R8 is a thiol protecting group.

In a further preferred embodiment, said nucleoside linking group is a phosphorus-containing linking group, and said phosphorus-containing linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, preferably an H-phosphonate linking group or a methylphosphonate linking group; a phosphonothioate linking group, preferably an H-phosphonothioate linking group, a methylphosphonothioate linking group; a phosphinate linking group, a phosphorothioamidate linking group, a phosphoramidate linking group or a phosphite linking group. In another particularly preferred embodiment, said nucleoside linking group is a phosphorus-containing linking group, and said phosphorus-containing linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, or a phosphonate linking group, wherein the phosphonate is preferably an H-phosphonate linking group or a methylphosphonate linking group.

In another particularly preferred embodiment, said nucleoside linker group is a phosphorus-containing linker group, and said phosphorus-containing linker group is a phosphodiester linker group. In another particularly preferred embodiment, said nucleoside linker group is a phosphorus-containing linker group, and said phosphorus-containing linker group is a phosphorothioate linker group.

In a preferred embodiment, the phosphorus-containing linking group is selected from an alkyl phosphodiester linking group, an alkylene phosphodiester linking group, a thionoalkyl phosphodiester linking group or an aminoalkyl phosphodiester linking group, an alkyl phosphotriester linking group, an alkylene phosphotriester linking group, a thionoalkyl phosphotriester linking group or an aminoalkyl phosphotriester linking group, an alkyl phosphonate linking group, an alkylene phosphonate linking group, an aminoalkyl phosphonate linking group, a thionoalkyl phosphonate linking group or a chiral phosphonate linking group. More preferably, said nucleoside linking group is a phosphorus-containing linking group, and said phosphorus-containing linking group is a phosphodiester linking group -O-P(=O)(OH)O- or -O-P(=O)(O ^- )O-, where [HB ⁺ ] is a counterion, phosphorothioate -OP(=S)(OH)O- or -OP(=S)(O ^- )O-, where [HB ⁺ ] is a counterion, methylphosphonate -O-P(=O)(CH ₃ )O-. Included are various salts, mixed salts and free acid forms of the phosphorus-containing linking group.

In a further embodiment, said nucleoside linker group links a nucleoside, nucleotide or oligonucleotide to an additional nucleoside, nucleotide or oligonucleotide.

The wavy line in formulas (I) and (IV) representing the bond between Bx and the bicyclic core of the proposed compounds indicates that any spatial orientations of the nitrogenous base Bx are encompassed by formula (I) or (IV). This means that formulas (I) and (IV) include the alpha or beta conformation or any mixture of the alpha and beta anomers of the proposed compounds.

As used herein, the term "nucleoside" refers to a compound comprising a nitrogenous base and a sugar covalently linked to said nitrogenous base. The term "nucleotide" as used herein refers to a nucleoside further comprising a nucleoside linker group or a phosphorus-containing moiety, wherein said nucleoside linker group or said phosphorus-containing moiety is covalently linked to a sugar in said nucleoside.

As used herein, the term "nucleoside" or "nucleotide" is intended to include all natural or modified nucleosides or nucleoside mimetics, or natural or modified nucleotides or nucleotide mimetics, respectively, that can be incorporated into an oligomer by natural or chemical oligomer synthesis methods. Generally, preferably, the term "nucleoside" as used herein refers to a natural nucleoside, a modified nucleoside, or a nucleoside mimetic. Generally, preferably, the term "nucleotide" as used herein refers to a natural nucleotide, a modified nucleotide, or a nucleotide mimetic.

The term "modified nucleosides" is intended to include modifications of the sugar and/or nitrogenous base in a nucleoside known to those skilled in the art and described herein. The term "modified nucleotides" is intended to include modifications of the sugar and/or nitrogenous base and/or nucleoside linker group or phosphorus-containing moiety in a nucleotide known to those skilled in the art and described herein.

The term "nucleoside mimetic" is intended to include structures used to replace the sugar and the nucleobase. Examples of nucleoside mimetics include nucleosides in which the nucleobase is replaced with a phenoxazine moiety (e.g., a 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one group) and the sugar moiety is replaced with a cyclohexenyl or bicyclo[3.1.0]hexyl moiety. The term "nucleotide mimetic" as used herein is intended to include nucleotides used to replace the sugar and the nucleoside linker group. Examples of nucleotide mimetics include peptide nucleic acids (PNA) or morpholinos.

The term "nucleoside" or "nucleotide" also includes combinations of modifications, such as more than one nitrogenous base modification, more than one sugar modification, or at least one nitrogenous base modification and at least one sugar modification.

The sugar in the nucleoside or nucleotide includes, but is not limited to, a monocyclic, bicyclic or tricyclic ring system, preferably a tricyclic or bicyclic system, or a monocyclic ribose or de(s)oxyribose. Modifications of the sugar further include, but are not limited to, modified stereochemical configurations, at least one group substitution, or at least one group removal. The modified sugar is generally preferably a modified version of the ribosyl moiety that is commonly present in RNA and DNA (i.e., the furanosyl moiety), such as bicyclic sugars, tetrahydropyrans, 2'-modified sugars, 3'-modified sugars, 4'-modified sugars, 5'-modified sugars, or 4'-substituted sugars. Examples of suitable sugar modifications are known in the art and include, but are not limited to, 2'-, 3'- and/or 4'-substituted nucleosides (e.g., 4'-S-modified nucleosides); 2'-O-modified RNA nucleotide residues such as 2'-O-alkyl or 2'-O-(substituted)alkyl, e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl; 2'-O-(haloalkoxy)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DMCE), in particular the 2'-O-methyl or 2'-O-methoxyethyl (2'-O-MOE) modification; or other modified sugar moieties such as morpholino (PMO), cationic morpholino (PMO-plus) or a modified morpholino group such as PMO-X. The term "PMO-X" refers to a modified morpholino moiety having at least one modification at the 3' or 5' end, such as a 3' fluorescent label, a 3' quencher (e.g., 3'-carboxyfluorescein, 3'-Gene Tools Blue, 3'-lissamine, 3'-dabcyl), a 3' affinity label, and functional groups to form chemical linkers (e.g., 3'-biotin, 3'-primary amine, 3'-disulfidamine, 3'-dithiopyridyl), 5'-end modifications (5'-primary amine, 5'-dabcyl), 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne, or as described in WO 2011/150408 and US patent application US 2012/0065169.

"Bicyclic sugar moieties" contain two interconnected ring systems, such as bicyclic nucleosides where the sugar moiety contains a 2'-O-CH(alkyl)-4' or 2'-O- _CH2-4 ' group, locked nucleic acid (LNA), xylo-LNA, alpha-L-LNA, beta-D-LNA, cEt (2'-O,4'-C ethyl hindered) LNA, cMOEt (2'-O,4'-C methoxyethyl hindered) LNA, ethylene-bridged nucleic acid (ENA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl RNA (pRNA), or 3'-deoxypyranosyl DNA (pDNA). Alternatively, the sugar in the nucleoside or nucleotide comprises a tricyclic sugar moiety, such as, for example, described in WO 2013/135900 and WO 2014/140348.

The term "oligomer" as used herein refers to a compound comprising two or more monomeric units linked by nucleoside linker groups, wherein at least one of said two or more monomeric units is a compound of formula (IV), preferably a compound of formula (V) or a compound of formula (VI). In a preferred embodiment, the oligomer comprises at least one compound of formula (IV), (V) or (VI) and at least one ribonucleotide or deoxyribonucleotide. More preferably, the oligomer comprises at least one compound of formula (IV), (V) or (VI) and at least one deoxyribonucleotide.

The term "monomer unit" as used herein is intended to include all monomer units available for oligomer synthesis, which include, and generally preferably refer to, monomer units such as α-D-ribonucleosides, β-D-ribonucleosides, α-D-2'-deoxyribonucleosides, β-D-2'-deoxyribonucleosides, natural nucleosides, natural nucleotides, modified nucleosides, modified nucleotides, nucleoside mimetics, nucleotide mimetics, and compounds of the present invention, including any compounds of any of formulae (I)-(VI).

In a preferred embodiment, the oligomer is an oligonucleotide. The term "oligonucleotide" as used herein refers to a compound comprising at least two nucleosides linked to each other by a nucleoside linker group. Thus, the term "oligonucleotide" as used herein includes, and generally preferably refers to, compounds comprising at least two nucleosides linked by nucleoside linker groups, wherein said at least two nucleosides are independently selected from naturally occurring nucleosides, modified nucleosides, or nucleoside mimetics. Thus, the term "oligonucleotide" as used herein includes compounds comprising naturally occurring nucleotides, modified nucleotides, or nucleotide mimetics, and thus the term "oligonucleotide" as used herein includes oligonucleotides having sugar and/or nucleoside base and/or nucleoside linker group modifications known to those skilled in the art and described herein.

The oligomer may be single-stranded or double-stranded. In one embodiment, the oligomer is double-stranded (i.e., a duplex). In a preferred embodiment, the oligomer is single-stranded.

In a preferred embodiment, the oligomer is linked to a non-nucleoside compound, preferably to a solid support. The solid support is preferably selected from granules, polymers or resins. In certain embodiments, the oligomer has a length of up to 40 monomer units, preferably up to 30 monomer units, more preferably up to 30 monomer units, even more preferably up to 20 monomer units or up to 15 monomer units. In a further embodiment, said oligomer comprises from 5 to 40 monomer units, preferably from 8 to 30 monomer units, more preferably from 8 to 25 monomer units, even more preferably from 8 to 20 monomer units.

In certain embodiments, an oligomer such as provided herein is modified with one or more end groups covalently attached to the 5' or 7' end of the oligomer. The end group may also be attached to either end of the oligomer.

The term "'-end" refers to the end or tail of an oligomer, nucleic acid sequence, or compound of formula (IV), (V), or (VI), wherein the number (3', 5', or 7', etc.) denotes a carbon atom in a sugar incorporated into a nucleoside in the oligomer, nucleic acid sequence, or compound of formula (IV), (V), or (VI). The term "5'-terminal group" or "7'-terminal group" as used herein refers to a group located at the 5'-terminal or 7'-terminal, respectively, of a sugar incorporated into a compound of formula (IV), (V), or (VI). Examples of the "5'-terminal group" or "7'-terminal group" include, but are not limited to, a cap group, a diphosphate, a triphosphate, a label such as a fluorescent label (e.g., fluorescein or rhodamine), a dye, a reporter group suitable for tracking an oligomer, a solid support, a non-nucleoside group, an antibody, or a conjugating group. Preferably, the "5'-terminal group" or "7'-terminal group" is selected from a diphosphate, a triphosphate, a fluorescent label, a dye, a reporter group by which an oligomer can be tracked, a solid support, a non-nucleoside group, an antibody, or a conjugating group.

In certain embodiments, an oligomer as provided herein or a compound of formula (IV), (V), or (VI) is modified by covalently attaching one or more conjugating groups. In general, conjugating groups modify one or more properties of the compounds to which they are attached. These properties include, but are not limited to, nuclease stability, binding affinity, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, delivery, charge, and clearance. Conjugating groups, which are commonly used in the chemical arts, are linked directly or through an optional linker group to a parent compound, such as an oligomer. The term "conjugating group" includes, without limitation, and refers preferably to intercalators, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, lipophilic moieties or coumarins.

The terms "nucleic acid" or "nucleic acid sequence" are used interchangeably herein to mean an oligomeric or polymeric molecule comprising at least two interconnected nucleotides or at least two nucleosides linked by a nucleoside linker group. In the context of the present invention, nucleic acid includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) and is preferably selected from natural RNA, natural DNA, modified DNA, modified RNA, mixtures thereof, such as RNA-DNA hybrids. The modification may include modification of the backbone, such as the nucleoside linker group and/or the nucleoside and/or sugar, such as further described herein. Nucleic acids may be synthesized by chemical methods or by the action of polymerase enzymes.

The terms "natural" or "naturally occurring", used interchangeably herein, refer to compounds of natural origin.

The term "stereoisomers" refers to compounds that have the same chemical composition but different arrangements of atoms or groups in space.

"Diastereomer" refers to stereoisomers containing two or more chiral centers, where the compounds are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and chemical and biological reactivity. Mixtures of diastereomers can be separated by high-resolution analytical techniques such as electrophoresis and chromatography.

"Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

The stereochemical definitions and conventions used in this document generally follow S.P. Parker, ed., McRaw-Hiff Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

In this document, " _Tm " (melting temperature) is the temperature at which the two strands of a nucleic acid duplex separate. _Tm is often used as a measure of the stability of the antisense compound and complementary nucleic acid duplex.

According to a first aspect, the present invention provides a compound of formula (I):

where one of T ₁ and T ₂ is OR ₁ or OR ₂ ;

and the other of T ₁ and T ₂ is OR ₁ or OR ₂ ; where

R ₁ is H or a hydroxyl protecting group, and

R ₂ is a phosphorus-containing moiety; and

Bx is a nitrogenous base.

In a preferred embodiment, said compound of formula (I) according to the invention is a compound of formula (II)

Where

(i) T ₁ is OR ₁ , and T ₂ is OR ₁ or OR ₂ ; or

(ii) T ₁ is OR ₁ or OR ₂ , T ₂ is OR ₁ :

where preferably T ₁ is OR ₁ or OR ₂ , T ₂ is OR ₁ .

The compound of formula (II) is an alpha-anomer or an alpha-anomeric monomer, which differs from the beta-anomer by the spatial configuration Bx at the chiral first carbon atom at the 1'-end.

In another preferred embodiment, said compound of formula (I) is a compound of formula (III)

Where

(i) T ₁ is OR ₁ , and T ₂ is OR ₁ or OR ₂ ; or

(ii) T ₁ is OR ₁ or OR ₂ , T ₂ is OR ₁ :

where preferably T ₁ is OR ₁ , and T ₂ is OR ₁ or OR ₂ .

The compound of formula (III) is a beta-anomer or a beta-anomeric monomer, which differs from the alpha-anomer by the spatial configuration Bx at the chiral first carbon atom at the 1'-end.

In another preferred embodiment, in the compound of formula (I), said phosphorus-containing moiety R ₂ is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety. In another preferred embodiment, in the compound of formula (II), said phosphorus-containing moiety R ₂ is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety. In another preferred embodiment, in the compound of formula (III), said phosphorus-containing moiety R ₂ is selected from a phosphate moiety, a phosphoramidate moiety, and a phosphoramidite moiety.

In another preferred embodiment, in the compound of formula (I), (II) or (III), said Bx is selected from a purine base or a pyrimidine base, wherein preferably Bx is selected from (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U) or (vi) 5-methyluracil (MeU) or a derivative of (i), (ii), (iii), (iv), (v) or (vi), and further preferably Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. Even more preferably, in the compound of formula (I), (II) or (III), Bx is selected from thymine, 5-methylcytosine, adenine or guanine.

In another preferred embodiment, the compound of formula (I), (II) or (III) is linked to a non-nucleoside compound, preferably to a solid support.

In a preferred embodiment, the compound of formula (I) is selected from:

According to a second aspect, the invention provides an oligomer comprising at least one compound of formula (IV)

wherein, independently, in each said at least one compound of formula (IV), one of _T3 or _T4 is a nucleoside linking group; the other of _T3 and _T4 is _OR1 , _OR2 , a 5'-terminal group, a 7'-terminal group, or a nucleoside linking group, wherein _R1 is H or a hydroxyl protecting group, and _R2 is a phosphorus-containing moiety; and Bx is a nitrogenous base.

In a preferred embodiment, the oligomer according to the invention comprises at least one compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V):

Where

(i) _T3 is a nucleoside linker group and _T4 is a 7'-terminal group, _OR1 or _OR2 , preferably _T4 is a 7'-terminal group or _OR1 ; or

(ii) _T3 is a 5'-terminal group, _OR1 or _OR2 , preferably _T3 is a 5'-terminal group or _OR2 ; and _T4 is a nucleoside linker group; or

(iii) _T3 and _T4 independently of each other represent a nucleoside linker group.

In another preferred embodiment, the oligomer according to the invention comprises at least one compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (VI):

Where

(i) _T3 is a nucleoside linker group and _T4 is a 7'-terminal group, _OR1 or _OR2 , preferably _T4 is a 7'-terminal group or _OR2 ; or

(ii) _T3 is a 5'-terminal group, _OR1 or _OR2 , preferably _T3 is a 5'-terminal group or _OR1 ; and _T4 is a nucleoside linker group; or

(iii) _T3 and _T4 independently of each other represent a nucleoside linker group.

In a preferred embodiment, said oligomer is an oligonucleotide. In a further preferred embodiment, said oligomer is an oligonucleotide, wherein said compound of formula (IV) is a compound of formula (V). In another preferred embodiment, said oligomer is an oligonucleotide, wherein said compound of formula (IV) is a compound of formula (VI). In a more preferred embodiment, the oligomer comprising said at least one compound of formula (IV), (V) or (VI) is DNA.

In another embodiment, the proposed oligomer comprising said at least one compound of formula (IV), (V) or (VI) further comprises at least one nucleotide different from any compound of formula (IV), (V) or (VI), wherein preferably the at least one different nucleotide is (i) a nucleotide comprising a monocyclic sugar, i.e. a monocyclic nucleotide, or (ii) a nucleotide comprising a bicyclic sugar, i.e. a bicyclic nucleotide, or (iii) a nucleotide comprising a tricyclic sugar, i.e. a tricyclic nucleotide. Preferably, said at least one nucleotide different from the compound of formula (IV), (V) or (VI) is a nucleotide comprising a bicyclic sugar. Preferably, said at least one nucleotide different from the compound of formula (IV), (V) or (VI) is a nucleotide comprising a tricyclic sugar. More preferably, said at least one nucleotide other than the compound of formula (IV), (V) or (VI) is a nucleotide containing a monocyclic sugar.

In another preferred embodiment of the proposed oligomer, said compound of formula (IV) is a compound of formula (V), and said oligomer further comprises at least one nucleotide different from the compound of formula (V), wherein preferably the at least one different nucleotide is (i) a nucleotide containing a monocyclic sugar, i.e. a monocyclic nucleotide, or (ii) a nucleotide containing a bicyclic sugar, i.e. a bicyclic nucleotide, or (iii) a nucleotide containing a tricyclic sugar, i.e. a tricyclic nucleotide. Preferably, said at least one nucleotide different from the compound of formula (V) is a nucleotide containing a bicyclic sugar. Preferably, said at least one nucleotide different from the compound of formula (V) is a nucleotide containing a tricyclic sugar. More preferably, said at least one nucleotide different from the compound of formula (V) is a nucleotide containing a monocyclic sugar.

In another preferred embodiment of the proposed oligomer, said compound of formula (IV) is a compound of formula (VI), and said oligomer further comprises at least one nucleotide different from the compound of formula (VI), wherein preferably the at least one different nucleotide is (i) a nucleotide containing a monocyclic sugar, i.e. a monocyclic nucleotide, or (ii) a nucleotide containing a bicyclic sugar, i.e. a bicyclic nucleotide, or (iii) a nucleotide containing a tricyclic sugar, i.e. a tricyclic nucleotide. Preferably, said at least one nucleotide different from the compound of formula (VI) is a nucleotide containing a bicyclic sugar. Preferably, said at least one nucleotide different from the compound of formula (VI) is a nucleotide containing a tricyclic sugar. More preferably, said at least one nucleotide different from the compound of formula (VI) is a nucleotide containing a monocyclic sugar.

In another embodiment, the oligomer comprising a compound of formula (IV), (V) or (VI) further comprises at least two nucleotides different from the compound of formula (IV), (V) or (VI), wherein said at least two different nucleotides are linked to each other via a nucleoside linking group, wherein each nucleoside linking group is independently selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, a phosphinate linking group, a phosphorothioate linking group, a phosphinate linking group, a phosphorothioamidate linking group or a phosphoramidate linking group, and preferably each nucleoside linking group is independently a phosphodiester linking group or a phosphorothioate linking group. group, and even more preferably each nucleoside linking group is a phosphorothioate linking group.

In another preferred embodiment of the proposed oligomer, said compound of formula (IV) is a compound of formula (V), and said oligomer further comprises at least two nucleotides different from the compound of formula (V), wherein said at least two different nucleotides are linked to each other via a nucleoside linker group, wherein each nucleoside linker group, independently of the others, is selected from a phosphodiester linker group, a phosphotriester linker group, a phosphorothioate linker group, a phosphorodithioate linker group, a phosphonate linker group, a phosphinate linker group, a phosphorothioate linker group, a phosphinate linker group, a phosphorothioamide linker group, or a phosphoramidate linker group, and preferably each nucleoside linker group, independently of the others, is a phosphodiester linker group or a phosphorothioate linking group, and even more preferably each nucleoside linking group is a phosphorothioate linking group.

In another preferred embodiment of the proposed oligomer, said compound of formula (IV) is a compound of formula (VI), and said oligomer further comprises at least two nucleotides different from the compound of formula (VI), wherein said at least two different nucleotides are linked to each other via a nucleoside linker group, wherein each nucleoside linker group, independently of the others, is selected from a phosphodiester linker group, a phosphotriester linker group, a phosphorothioate linker group, a phosphorodithioate linker group, a phosphonate linker group, a phosphinate linker group, a phosphorothioate linker group, a phosphinate linker group, a phosphorothioamide linker group, or a phosphoramidate linker group, and preferably each nucleoside linker group, independently of the others, is a phosphodiester linker group or a phosphorothioate linking group, and even more preferably each nucleoside linking group is a phosphorothioate linking group.

In another preferred embodiment, in the oligomer according to the invention, Bx is selected from a purine base or a pyrimidine base, wherein preferably Bx is selected from (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U) or (vi) 5-methyluracil (MeU) or a derivative of (i), (ii), (iii), (iv), (v) or (vi), and even more preferably Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. More preferably, in the oligomer according to the invention, Bx is selected from thymine, 5-methylcytosine, adenine or guanine.

In another preferred embodiment, in the oligomer according to the invention, each nucleoside linking group, independently of the others, is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, a phosphonothioate linking group, a phosphinate linking group, a phosphorothioamidate linking group or a phosphoramidate linking group, and preferably each nucleoside linking group, independently of the others, is a phosphodiester linking group or a phosphorothioate linking group, and even more preferably each nucleoside linking group is a phosphorothioate linking group.

In another embodiment, the oligomer according to the invention comprises from 1 to 5, preferably from 1 to 4, more preferably from 1 to 2, even more preferably from 1 to 2, even more preferably exactly one compound of formula (IV), (V) or (VI). In a preferred embodiment of the proposed oligomer, said compound of formula (IV) is a compound of formula (VI), wherein said oligomer comprises from 1 to 5, preferably from 1 to 4, more preferably from 1 to 2, even more preferably from 1 to 2, even more preferably exactly one compound of formula (VI). It has been found that the incorporation of one compound of formula (VI) into an oligonucleotide and preferably the incorporation of one compound of formula (VI), wherein Bx is methylcytosine, into DNA duplexes has a significant stabilizing effect. Thus, in a particularly preferred embodiment of the proposed oligomer, said oligomer contains exactly one compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (VI), and said Bx is methylcytosine, wherein said oligomer is an oligonucleotide and further contains at least one nucleotide different from the compound of formula (VI), wherein preferably at least one different nucleotide is a nucleotide containing a monocyclic sugar.

In another preferred embodiment of the proposed oligomer, said oligomer comprises at least two compounds of formula (IV) and further comprises at least one nucleotide different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V), and EACH said compound of formula (V) is linked via the 5'-end to (i) the 5'-end of said at least one nucleotide different from the compound of formula (IV), or (ii) the 7'-end of another compound of formula (V); wherein said compound of formula (V) is linked via the 7'-end to (i) the 3'-end of said at least one nucleotide different from the compound of formula (IV), or (ii) the 5'-end of another compound of formula (V).

In another embodiment of the proposed oligomer, said oligomer comprises at least two compounds of formula (IV) and further comprises at least one nucleotide different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (VI), and EACH said compound of formula (VI) is linked via a 5'-end to (i) the 3'-end of said at least one nucleotide different from the compound of formula (IV), or (ii) the 3'-end of another compound of formula (VI); and EACH said compound of formula (VI) is linked via a 3'-end to (i) the 5'-end of said at least one nucleotide different from the compound of formula (IV), or (ii) the 5'-end of another compound of formula (VI).

In a preferred embodiment of the proposed oligomer, said oligomer further comprises at least two nucleotides different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V), and EACH said nucleotide different from the compound of formula (IV) is linked via a 3'-end to (i) the 7'-end of said compound of formula (V), or (ii) the 5'-end of another nucleotide different from the compound of formula (IV), and EACH said nucleotide different from the compound of formula (IV) is linked via a 5'-end to (i) the 5'-end of said compound of formula (V), or (ii) the 3'-end of another nucleotide different from the compound of formula (IV).

In a preferred embodiment of the proposed oligomer, said oligomer further comprises at least two nucleotides different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V), and EACH said nucleotide different from the compound of formula (IV) is linked via a 3'-end to (i) the 7'-end of said compound of formula (V), or (ii) the 5'-end of another nucleotide different from the compound of formula (IV), and EACH said nucleotide different from the compound of formula (IV) is linked via a 5'-end to (i) the 5'-end of said compound of formula (V), or (ii) the 3'-end of another nucleotide different from the compound of formula (IV).

In a preferred embodiment of the proposed oligomer, said oligomer further comprises at least two nucleotides different from the compound of formula (IV), where said compound of formula (IV) is a compound of formula (V), and each of at least one compound of formula (V) is linked via a 5'-end and a 7'-end to said nucleotide different from the compound of formula (IV).

In a preferred embodiment of the proposed oligomer, said oligomer further comprises at least two nucleotides different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V), and EACH said nucleotide different from the compound of formula (IV) is linked via a 3'-end to (i) the 7'-end of said compound of formula (V), or (ii) the 5'-end of another nucleotide different from the compound of formula (IV), and EACH said nucleotide different from the compound of formula (IV) is linked via a 5'-end to (i) the 5'-end of said compound of formula (V), or (ii) the 3'-end of another nucleotide different from the compound of formula (IV).

In a preferred embodiment of the proposed oligomer, said oligomer further comprises at least two nucleotides different from the compound of formula (IV), where said compound of formula (IV) is a compound of formula (VI), and each of at least one compound of formula (VI) is linked via a 5'-end and a 7'-end to said nucleotide different from the compound of formula (IV).

In another preferred embodiment of the proposed oligomer, said oligomer contains at least two compounds of formula (IV) and further contains at least two nucleotides different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (V), and each of said at least one compound of formula (V) is linked via the 7'-end to the 3'-end of a nucleotide different from the compound of formula (IV); and via the 5'-end to the 5'-end of a nucleotide different from the compound of formula (IV).

In another preferred embodiment of the proposed oligomer, said oligomer contains at least two compounds of formula (IV) and further contains at least two nucleotides different from the compound of formula (IV), wherein said compound of formula (IV) is a compound of formula (VI), and each of the at least one compound of formula (VI) is linked via the 5'-end to the 3'-end of a nucleotide different from the compound of formula (IV); and via the 7'-end to the 5'-end of a nucleotide different from the compound of formula (IV).

In a preferred embodiment of the proposed oligomer, said oligomer comprises at least one compound of formula (IV), wherein said compound is a compound of formula (VI), wherein each of the at least one compound of formula (VI) is linked via the 5'-end and via the 7'-end to a nucleotide different from the compound of formula (IV), and Bx is cytosine or 5-methylcytosine, preferably 5-methylcytosine.

In a further preferred embodiment of the proposed oligomer, said oligomer contains exactly one compound of formula (IV), wherein said compound is a compound of formula (VI), wherein said compound of formula (VI) is linked via the 5'-end and via the 7'-end to a nucleotide different from the compound of formula (IV), and Bx is cytosine or 5-methylcytosine, preferably 5-methylcytosine.

In another preferred embodiment, the oligomer according to the invention comprises from 1 to 5, preferably from 1 to 4, more preferably from 1 to 2, even more preferably from 1 to 2, even more preferably exactly one compound of formula (IV), (V) or (VI), preferably of formula (VI), wherein Bx is a pyrimidine base, more preferably cytosine or 5-methylcytosine, even more preferably 5-methylcytosine. In a more preferred embodiment, the oligomer according to the invention comprises exactly one compound of formula (VI), wherein Bx is cytosine or 5-methylcytosine, preferably 5-methylcytosine. The incorporation of exactly one or more compounds of formula (IV), (V) or (VI), preferably of formula (VI), wherein Bx is a pyrimidine base, leads to only slight destabilization or even stabilizes the duplex formation of the oligomers according to the invention. In particular, if the nitrogenous base is cytosine or a derivative of cytosine, preferably 5-methylcytosine, then a significant stabilization of the duplexes of the oligomer according to the invention containing the compounds of formula (IV), (V) or (VI), preferably of formula (VI), with complementary DNA occurs. Such a stabilizing effect is more pronounced for nucleosides containing 5-methylcytosine. In another embodiment, the oligomer according to the invention contains from 1 to 5, preferably from 1 to 4, more preferably from 1 to 2, even more preferably from 1 to 2, even more preferably exactly one compound of formula (IV), (V) or (VI), preferably of formula (VI), wherein Bx is a purine base. The purine nitrogenous base stabilizes the duplexes of the oligomers according to the invention containing the compounds of formula (IV), (V) or (VI), preferably of formula (VI), with complementary RNA.

In another preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least two adjacent compounds of formula (IV), wherein each of the adjacent compounds of formula (IV) is independently linked to an adjacent adjacent compound of formula (IV) by a nucleoside linking group, wherein the nucleoside linking group links the 5'-end and the 7'-end of the two adjacent compounds of formula (IV). In another preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least two adjacent compounds of formula (V), wherein each of the adjacent compounds of formula (V) is independently linked to an adjacent adjacent compound of formula (V) by a nucleoside linking group, wherein the nucleoside linking group links the 5'-end and the 7'-end of the two adjacent compounds of formula (V). In another preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least two adjacent compounds of formula (VI), wherein each of the adjacent compounds of formula (VI) is independently linked to an adjacent adjacent compound of formula (VI) by a nucleoside linker group, wherein the nucleoside linker group links the 5'-end and the 7'-end of the two adjacent compounds of formula (VI).

In a further preferred embodiment, the oligomer according to the invention comprises or preferably consists of 10-40 adjacent compounds of formula (IV), preferably 10-30 adjacent compounds of formula (IV), more preferably 10-25 adjacent compounds of formula (IV), even more preferably 10-20 adjacent compounds of formula (IV) or 10-15 adjacent compounds of formula (IV). In a further preferred embodiment of the proposed oligomer, said at least one compound of formula (IV) is a compound of formula (V), wherein said oligomer comprises or preferably consists of 10-40 adjacent compounds of formula (V), preferably 10-30 adjacent compounds of formula (V), more preferably 10-25 adjacent compounds of formula (V), even more preferably 10-20 adjacent compounds of formula (V) and even more preferably 10-15 adjacent compounds of formula (V). In a further preferred embodiment of the proposed oligomer, said at least one compound of formula (IV) is a compound of formula (VI), wherein said oligomer comprises or preferably consists of 10-40 contiguous compounds of formula (VI), preferably 10-30 contiguous compounds of formula (VI), more preferably 10-25 contiguous compounds of formula (VI), even more preferably 10-20 contiguous compounds of formula (VI), and even more preferably 10-15 contiguous compounds of formula (VI).

In a further preferred embodiment of the proposed oligomer, said at least one compound of formula (IV) is a compound of formula (V), wherein said oligomer comprises or preferably consists of 10-40 contiguous compounds of formula (V), preferably 10-30 contiguous compounds of formula (V), more preferably 10-25 contiguous compounds of formula (V), even more preferably 10-20 contiguous compounds of formula (V) and even more preferably 10-15 contiguous compounds of formula (V), and each of the contiguous compounds of formula (V) is independently linked to an adjacent contiguous compound of formula (V) by a nucleoside linking group, wherein the nucleoside linking group links the 5'-end and the 7'-end of two contiguous compounds of formula (V), and said nucleoside linking group is a phosphorus-containing linking group, wherein said phosphorus-containing linking group is selected from a phosphodiester linking group, a phosphotriester linker group and a phosphorothioate linker group, preferably said phosphorus-containing linker group is a phosphodiester linker group or a phosphorothioate linker group.

In a further preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least one nucleic acid sequence, wherein said nucleic acid sequence comprises said at least one compound of formula (IV), and said nucleic acid sequence is selected from SEQ ID NO: 1-24, preferably SEQ ID NO: 24. In a further preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least one nucleic acid sequence, wherein said nucleic acid sequence comprises said at least one compound of formula (V), and said nucleic acid sequence is selected from SEQ ID NO: 16-24, preferably SEQ ID NO: 21-24, more preferably SEQ ID NO: 24. In a further preferred embodiment, the oligomer according to the invention comprises or preferably consists of at least one nucleic acid sequence, wherein said nucleic acid sequence comprises said at least one compound of formula (VI), and each said the nucleic acid sequence is selected from SEQ ID NO: 1-15, preferably SEQ ID NO: 13-15. In a more preferred embodiment, the oligomer according to the invention consists of a nucleic acid sequence selected from SEQ ID NO: 13-15 or 21-24, preferably SEQ ID NO: 24. In another preferred embodiment, said oligomer is a nucleic acid sequence selected from SEQ ID NO: 13-15 or 21-24, and preferably said oligomer is SEQ ID NO: 24.

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (IV), wherein said nucleic acid sequence comprises at least one nucleotide or nucleoside at the 5'-end or 7'-end, which is different from the compound of formula (IV). In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (V), wherein said nucleic acid sequence comprises at least one nucleotide or nucleoside at the 5'-end or 7'-end, which is different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV). In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (VI), wherein said nucleic acid sequence comprises at the 5'-end or 7'-end at least one nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably from the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (IV), wherein said nucleic acid sequence comprises at least one nucleotide or nucleoside at the 5'-end and 7'-end, which is different from the compound of formula (IV). In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (V), wherein said nucleic acid sequence comprises at least one nucleotide or nucleoside at the 5'-end and 7'-end, which is different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV). In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (VI), wherein said nucleic acid sequence comprises at the 5'-end and 7'-end at least one nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (V), wherein said nucleic acid sequence comprises at the 5'-end or 7'-end at least one nucleotide or nucleoside different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV), wherein the 5'-end of said nucleic acid sequence is linked to the 5'-end of a nucleotide different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV); or wherein the 7'-end of said nucleic acid sequence is linked to the 3'-end of a nucleotide or nucleoside other than the compound of formula (V), preferably other than the compound of formula (V) or (VI), and more preferably other than the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (V), wherein said nucleic acid sequence comprises at the 5'-end and 7'-end at least one nucleotide or nucleoside different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV), wherein the 5'-end of said nucleic acid sequence is linked to the 5'-end of a nucleotide or nucleoside different from the compound of formula (V), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV); and wherein the 7'-end of said nucleic acid sequence is linked to the 3'-end of a nucleotide or nucleoside other than the compound of formula (V), preferably other than the compound of formula (V) or (VI), and more preferably other than the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (VI), wherein said nucleic acid sequence comprises at the 5'-end or 7'-end at least one nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV), wherein the 5'-end of said nucleic acid sequence is linked to the 3'-end of a nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV); or wherein the 7'-end of said nucleic acid sequence is linked to the 5'-end of a nucleotide or nucleoside other than the compound of formula (VI), preferably other than the compound of formula (V) or (VI), and more preferably other than the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said oligomer comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent compounds of formula (VI), wherein said nucleic acid sequence comprises at the 5'-end and the 7'-end each at least one nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV), wherein the 5'-end of said nucleic acid sequence is linked to the 3'-end of a nucleotide or nucleoside different from the compound of formula (VI), preferably different from the compound of formula (V) or (VI), and more preferably different from the compound of formula (IV); and wherein the 7'-end of said nucleic acid sequence is linked to the 5'-end of a nucleotide or nucleoside other than the compound of formula (VI), preferably other than the compound of formula (V) or (VI), and more preferably other than the compound of formula (IV).

In a further preferred embodiment of the proposed oligomer, said compound of formula (IV) is selected from

In a further preferred embodiment, the oligomer according to the invention is double-stranded. In a particular embodiment, exactly one or both chains in said double-stranded oligomer comprise at least one compound of formula (IV), (V) or (VI).

According to a third aspect, the present invention provides a compound of formula (I), (II) or (III) or an oligomer according to the invention for use as a medicament for the prevention, treatment or diagnosis of a disease.

In a particular embodiment, the compound of formula (I), (II) or (III) is used as a medicament for preventing or treating a disease. The invention provides a method for preventing a disease in a patient or treating a patient suffering from a disease by administering a therapeutically effective amount of a compound of formula (I), (II) or (III) to the patient. In another embodiment, the compound of formula (I), (II) or (III) is used to prepare a medicament for preventing or treating a disease.

In a further embodiment, the oligomer according to the invention is used as a medicine for preventing or treating a disease. The invention provides a method for preventing a disease in a patient or treating a patient suffering from a disease by administering a therapeutically effective amount of the oligomer according to the invention to the patient. In another preferred embodiment, the oligomer according to the invention is used to produce a medicine for preventing or treating a disease.

The term "patient" as used herein refers to a human or animal, wherein the animal is preferably a mammal. The term "patient" is not limited to subjects exhibiting symptoms of a disease or disorder, but includes healthy subjects (i.e., asymptomatic) or subjects at risk of developing a symptom. A "therapeutically effective amount" refers to an amount administered to a subject, in a single dose or as part of a multi-dose regimen, that is effective to provide a desired physiological response or therapeutic effect in the subject. Examples of desired therapeutic effects include, but are not limited to, amelioration of symptoms or pathology, slowing the progression of symptoms or pathology, and slowing the onset of symptoms or pathology in a disease. A therapeutically effective amount may vary depending on the nature of the formulation employed and the type and condition of the user. Determining appropriate amounts of any given composition is within the general skill in the art and includes conducting a standard series of tests designed to evaluate the appropriate therapeutic level. Typical and preferred therapeutically effective amounts of the antisense oligonucleotide are in the range of from about 0.05 to 1000 mg/kg body weight, and in particular from about 5 to 500 mg/kg body weight.

In a further embodiment, the oligomer of the invention is an antisense oligonucleotide. In a preferred embodiment, the antisense oligonucleotide of the invention is used to prevent, treat or diagnose a disease. As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide that can hybridize to a target nucleic acid sequence. In a preferred embodiment, the antisense oligonucleotide is complementary to the target nucleic acid sequence. An oligonucleotide is complementary to a target nucleic acid sequence if a sufficient number of complementary positions in the oligonucleotide and the target nucleic acid are occupied by complementary nitrogenous bases that can form hydrogen bonds with each other, resulting in specific binding between the oligonucleotide and the target nucleic acid. For example, adenine and thymine are complementary nitrogenous bases that pair by hydrogen bonding. Hybridization can occur under a variety of conditions. It is understood in the art that the sequence of an antisense oligonucleotide does not have to include nucleotides that are 100% complementary to the nucleotides of the target nucleic acid in order for them to hybridize. In an antisense oligonucleotide, one or more nucleotides can hybridize without the embedded or adjacent nucleotides participating in the hybridization. The oligonucleotide fragment in the antisense oligonucleotide according to the present invention should preferably have a sequence that is at least 70% complementary to a target region in a target nucleic acid, more preferably they should have 85% or 90% complementary sequence, and even more preferably have a sequence that is 95% complementary to a target region in a target nucleic acid sequence.

In a particular embodiment, the oligomer of the invention is used to prevent or treat a disease where the oligomer can affect replication, translation, transcription, translocation, catalytic activity, complex formation, splicing, or integrity of a target nucleic acid. In a particular embodiment, the oligomer of the invention is used to prevent or treat a disease where the oligomer can bind to a target nucleic acid, downregulate expression of a target nucleic acid, sterically block a target nucleic acid sequence, or trigger nucleic acid interference, gene silencing, or target nucleic acid disruption, or exon skipping. In a preferred embodiment, the oligomer of the invention is used to prevent or treat a disease where the oligomer can bind to a target nucleic acid and downregulate expression of said target nucleic acid. In another preferred embodiment, the oligomer according to the invention is used for preventing or treating a disease, wherein the oligomer can bind to a target nucleic acid, sterically block said target nucleic acid and trigger exon skipping in said target nucleic acid. In a preferred embodiment, said target nucleic acid is DNA or RNA. The RNA is preferably pre-mRNA (preprocessed messenger RNA or messenger RNA precursor) or mature RNA. The RNA can be mRNA or a functional form of non-coding RNA, such as long non-coding RNA, micro RNA, small interfering RNA, small nucleolar RNA, Piwi-interacting RNA, small RNA derived from tRNA, RNA derived from small rDNA, rRNA or tRNA. In a particular embodiment, the oligomer of the invention is used to prevent or treat a disease, wherein the oligomer can alter the splicing process in a target nucleic acid, wherein preferably the target nucleic acid is a pre-mRNA. Preferably, said oligomer can trigger exon skipping in the target pre-mRNA. "Exon" refers to a specific fragment of nucleic acid that codes for a protein, or to a nucleic acid sequence that is present in the mature form of an RNA molecule after pre-mRNA fragments have been removed by splicing.

In a further embodiment, the oligomer according to the invention is used as a medicine for preventing or treating a disease, wherein said disease is a genetic disease. In a preferred embodiment, the oligomer according to the invention is used as a medicine for preventing or treating a disease, wherein said disease is muscular dystrophy, preferably Duchenne muscular dystrophy. In another preferred embodiment, the oligomer for use as a medicine for preventing, treating or diagnosing a disease is the nucleic acid sequence of SEQ ID NO: 21 or the nucleic acid sequence of SEQ ID NO: 24, preferably the nucleic acid sequence of SEQ ID NO: 24, and wherein the disease is muscular dystrophy, preferably Duchenne muscular dystrophy. The oligomers according to the invention and in particular SEQ ID NO: 24 retain good affinity for RNA, and oligomers consisting of compounds of formula (V) are likely to provide significantly improved biological stability compared to the corresponding natural DNA. In addition, the nucleic acid of SEQ ID NO: 24 does not significantly activate complement, and complement activation is an important toxic response often associated with the in vivo use of antisense oligonucleotides. Thus, the oligomers according to the invention, preferably oligomers comprising or preferably consisting of compounds of formula (V), are promising antisense agents. Finally, oligomers consisting of compounds of formula (V), preferably nucleic acid with SEQ ID NO: 24, can trigger exon 23 skipping, double skipping of exons 22 and 23 with high activity. These promising results indicate that the oligomers according to the invention, in particular oligomers consisting of compounds of formula (V), meet the conditions for inducing a highly active therapeutic effect in patients suffering from muscular dystrophy, such as Duchenne dystrophy.

According to another aspect, the present invention provides an oligomer according to the invention for use as a medicament for the prevention or treatment of a disease.

According to a further aspect, the oligomer according to the invention is used to diagnose a disease. According to a further aspect, the oligomer according to the invention is used as a drug for diagnosing a disease. In another preferred embodiment, the oligomer according to the invention is used to produce a drug for diagnosing a disease. The invention provides a method for diagnosing a disease in a patient. Said diagnosis or said diagnosis comprises

(i) administering an effective amount of an oligomer according to the invention to a patient, wherein the oligomer is labeled, and

(ii) non-invasive or invasive, preferably non-invasive, in vivo imaging of the labeled oligomer, or

(i') obtaining a sample from the patient,

(ii') adding an oligomer according to the invention to a sample, wherein the oligomer is labeled, and

(iii') analyzing the sample to determine the binding of the labeled oligomer to nucleic acids contained in the sample. In a preferred embodiment, the oligomer of the invention used in diagnosing a disease is an oligonucleotide, more preferably an antisense oligonucleotide. The sample obtained from a patient is blood, serum, cerebrospinal fluid or a tissue sample. The term "labeled oligomer" as used herein refers to an oligomer comprising a label. Preferably, the label is selected from a fluorescent label, a dye, a reporter group or a radioactive label.

According to a further aspect, the invention provides a pharmaceutical composition comprising at least one compound selected from formula (I), (II) or (III). According to a further aspect, the invention provides a pharmaceutical composition comprising at least one oligomer according to the invention. In a preferred embodiment, said pharmaceutical composition comprises one or more oligomers according to the invention, wherein at least one of said one or more oligomers is an oligonucleotide, more preferably an antisense oligonucleotide. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of at least one compound of formula (I), (II) or (III) or at least one oligomer according to the invention, preferably together with one or more pharmaceutically acceptable carriers. In one embodiment, a unit dose of the pharmaceutical composition according to the invention comprises from about 1 microgram to 20,000 micrograms of the oligomer or compound of formula (I), (II) or (III) per dose, and preferably from about 10 to 1000 micrograms. For intravenous delivery, a unit dose of the pharmaceutical composition preferably contains from 0.5 to 500 micrograms per kg of body weight, more preferably from 5 to 300 micrograms of the oligomer according to the invention per kg of body weight. In the pharmaceutical compositions according to the invention, the oligomer or compound of formula (I), (II) or (III) is typically present in an amount of about 0.5-95% by weight, based on the total weight of the composition.

In a preferred embodiment, a pharmaceutical composition comprising at least one compound of formula (I), (II) or (III) or at least one oligomer according to the invention further comprises at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, processing aid (e.g., a lubricant, talc, magnesium, calcium or zinc stearate or stearic acid) or a solvent encapsulating material. A pharmaceutically acceptable carrier can be used to carry or transport a compound of the invention from one organ or body part to another. Methods for delivering nucleic acids are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, eds. Akhtar; Sullivan et al., PCT WO 94/02595. These and other protocols can be used to deliver virtually any nucleotide or nucleic acid molecule, including a compound of formula (I), (II), or (III) and the oligomers of the present invention. The invention also provides a pharmaceutical composition of the invention further comprising P-glycoprotein inhibitors (such as Pluronic P85) that can promote drug delivery to various tissues; biodegradable polymers such as poly(DL-lactide-glycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al., 1999, Cell Transplant, 8, 47-58, Alkermes, Inc. Cambridge, Mass); nanoparticles such as those made from polybutyl cyanoacrylate, which can deliver drugs across the blood-brain barrier and alter cellular uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999); or liposomes containing polyethyleneglycol lipids.

The administration of the pharmaceutical composition, the compound of formula (I), (II) or (III) or the oligomer according to the invention can be carried out using various mechanisms known in the art. In a preferred embodiment, the pharmaceutical composition, the compound of formula (I), (II) or (III) or the oligomer according to the invention is administered locally or systemically. In a preferred embodiment, the pharmaceutical composition, compound of formula (I), (II) or (III) or oligomer according to the invention is administered orally (e.g. as an aqueous or non-aqueous solution or suspension, tablet, bolus, powder, granule, paste), parenterally (e.g. by subcutaneous, intramuscular, intravenous, intraperitoneal or epidural injection, e.g. as a sterile solution or suspension or a sustained release formulation), topically (e.g. as a cream, ointment or patch with sustained release or spray formulation), intravaginally or intrarectally (e.g. as a pessary, cream or foam), sublingually, intraocularly, transdermally, intranasally, intracellularly or by direct local injection into a tumor. In a preferred embodiment, a pharmaceutical composition comprising an oligomer according to the invention is used to down-regulate the expression of a target nucleic acid, sterically block the sequence of a target nucleic acid, or trigger nucleic acid interference, gene silencing, disruption, or exon skipping in a target nucleic acid.

According to a further aspect, the oligomer according to the invention is used in vitro to bind to a target nucleic acid sequence. In a preferred embodiment, the oligomer according to the invention is used in vitro to downregulate the expression of a target nucleic acid, sterically block a target nucleic acid sequence, or trigger nucleic acid interference, gene silencing, disruption or exon skipping in a target nucleic acid. In a particular embodiment, the oligomer according to the invention is used in vitro to alter replication, translation, transcription, translocation, catalytic activity, complex formation, splicing or integrity of a target nucleic acid. In a preferred embodiment, the oligomer according to the invention is used in vitro to bind to a target nucleic acid, wherein exon skipping is triggered in said target nucleic acid. In a preferred embodiment, the present invention provides a method for in vitro down-regulation of expression of a target gene in the cytosol of a cell by delivering an oligomer according to the invention through a cell membrane. In a preferred embodiment, the target nucleic acid is DNA, pre-mRNA or mature mRNA.

According to a further aspect, the invention provides a method for the solid-phase synthesis of an oligomer according to the invention, comprising using any of the compounds of formula (I) to (VI).

EXAMPLES

General methods

All reactions were carried out in dried laboratory glassware under an inert atmosphere of argon. Anhydrous reaction solvents were obtained by filtration through activated alumina or by storing over molecular sieves (4 Ǻ). Column chromatography (CC) was carried out on silica gel (SiliaFlash P60, 40-63 μm, 60 Ǻ). Methanol used in CC was of HPLC grade, all other solvents used in CC were of technical grade and were distilled before use. Thin-layer chromatography was carried out on silica gel plates (Macherey-Nagel, TLC plates with pre-coated Sil G-25 UV254). Compounds were visualized under a UV lamp or by immersing the plates in a staining solution with p-anisaldehyde [p-anisaldehyde (3.7 mL), glacial acetic acid (3.7 mL), concentrated sulfuric acid (5 mL), ethanol (135 mL)] and subsequent heating with a hair dryer. NMR spectra were obtained at 300 or 400 MHz ⁽¹ H), 75 or 101 MHz ( ¹³ C), and 122 MHz ( ³¹ P) in _CDCl3 , _CD3OD , or _CD3CN . Chemical shifts (δ) are reported relative to the peak of residual unmodified solvent [ _CDCl3 : 7.26 ppm ( ¹ H), 77.16 ppm ( ¹³ C); _CD3OD : 3.31 ppm ( ¹ H), 49.00 ppm ( ¹³ C)]. Signals were assigned based on APT and DEPT and ^1H , ^1H and ^1H , ^13C correlation experiments (COSY, HSQC, HMBC). High-resolution mass spectrometry was performed using electron spray ionization in positive ion detection mode (ion trap, ESI ⁺ ).

In the examples section, for simplicity, the nucleotides or nucleosides referred to are beta-anomers unless specifically designated as alpha-anomers. In addition, in accordance with the above, the oligomers or oligonucleotides referred to in the examples section contain beta-anomers unless specifically designated as containing alpha-anomers.

Melting point

UV melting experiments were performed on a Varian Cary Bio 100 UV/Vis spectrophotometer _. Experiments were performed at 2 μM duplex, 10 mM _NaH2PO4 , 0 M–150 mM NaCl (alpha anomer) or 0.05 M–1.00 M NaCl (beta anomer), and pH adjusted to 7.0. Samples were protected from evaporation using a dimethylpolysiloxane protective layer. Absorbance at 260 nm was monitored. Each experiment included three cooling-heating cycles with a temperature gradient of 0.5°C/min. The maximum values for the first derivative of the curve were determined using Varian WinUV software, and the _Tm values were reported as the average of six temperature changes.

Circular dichroism spectroscopy

CD spectra were obtained on a Jasco J-715 spectropolarimeter equipped with a Jasco PFO-350S temperature controller. The conditions for the samples were the same as in the UV melting experiments. Spectra were acquired in the range from 210 to 320 nm at a rate of 50 nm/min, and the temperature was measured directly in the sample. In each experiment, a blank sample was analyzed, which had the same salt concentrations as the sample under study. These spectra were obtained using a smoothed average spectrum for three scans, from which the corresponding spectrum of the blank sample was subtracted.

EXAMPLE 1

Synthesis of the proposed compounds

(General overview)

Bicyclic scaffolds 7 and 10, intended for subsequent nucleoside synthesis, can be obtained from the previously described intermediate compound 1 ( M.; Bolli, M.; Schweizer, B.; Leumann, C. Helv. Chim. Acta 1993, 76, 481) (Scheme 1). The epoxide ring in 1 was efficiently opened by LiHMDS-mediated intramolecular elimination at -78°C to afford the unsaturated ester 2 in good yield. Subsequent nickel-catalyzed reduction of 2 using NaBH ₄ , which occurred specifically from the convex side of the bicyclic core structure, afforded ester 3 as the only detectable diastereomer. The hydroxyl functionality in 3 was then protected using TBDPS to afford 4 in quantitative yield. Intermediate 4 was then reduced using DIBAL at -78 °C to give aldehyde 5. The acetonide protecting group in 5 was then hydrolyzed under mild conditions using In(OTf) ₃ as a catalyst (Golden, K.C; Gregg, B.T.; Quinn, J.F. Tetrahedron Lett. 2010, 51, 4010) in a mixture of MeCN and H ₂ O, and the resulting bicyclic hemiacetal was converted to methyl glycoside 6 by a simple solvent exchange with MeOH. Compound 6 was then acetylated to give the protected precursor 7, which was used to synthesize the corresponding purine nucleosides using the Voorbruggen method.

Scheme 1: (a) LiHMDS, THF, -78°C, 2 h, 74%; (b) NaBH ₄ , NiCl ₂ , EtOH, 0°C → room. temp., 2 hours, 90%; (c) TBDPSCl, I ₂ , N-methylimidazole, THF, room. temp., 3 h, quantity; (d) DiBAL-H, CH ₂ Cl ₂ , -78°C, 90 min, 89%; (e) i) In(OTf) ₃ , MeCN/H ₂ O, room. temp., 48 h, ii) MeOH, 6 h, 81%; (f) Ac ₂ O, DMAP, DCM, room. temp., 2 hours, 96%; (g) i) TMSOTf, 2,6-lutidine, DXM, room. temp., 60 min, ii) TBAF, THF, 0°C, 20 min, 92%; (h) DMTr-Cl, AgOTf, DCM/lutidine, room. temp., 4 hours, 93%; (i) TBAF, THF, room. temp., 20 h, quantity The synthesis of the preferred pyrimidine nucleosides of the present invention involved the well-characterized β-stereoselective NIH-induced addition of nitrogenous bases to the corresponding bicyclic glycal (Medvecky, M.; Istrate, A.; Leumann, C. J J Org. Chem. 2015, 80, 3556; Dugovic, B.; Leumann, C.J. Journal of Organic Chemistry 2014, 79, 1271; Lietard, J.; Leumann, C.J. J. Org. Chem. 2012, 77, 4566). First, N-iodosuccinimide-induced synthesis was performed to incorporate the nitrogenous base thymine ( NIS) nucleosidation of the immediate glycal precursor 8, where R ₁ = TMS, which was readily prepared from 6 by treatment with TMSOTf alone. This approach resulted in stereoselective formation of the corresponding β-nucleoside, which nevertheless contained a significant 7% amount of impure α-anomer, which could not be separated by standard chromatographic methods. It was suggested that β-selectivity could be increased by increasing the steric volume of R ₁ and a decrease in the size of R ₂ , as in glycal 10. This may facilitate the initial α-addition of electrophilic iodine at the C(4) position. For this, compound 6 was converted to glycal 8 using TMSOTf, followed by short-term treatment with TBAF to selectively remove newly incorporated TMS group. Intermediate 8 was then converted to dimethoxytrityl compound 9, in which the TBDPS protecting group was removed to yield the target sugar moiety 10.

In situ NIS nucleosidation of TMS-protected glycal 10 and subsequent radical reduction of the iodide intermediate using Bu ₃ SnH afforded the DMTr-protected thymidine derivative 11 in good yield, containing only trace amounts (<2% by ¹ H NMR) of the α-anomer (Scheme 2). Final phosphorylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite afforded the thymidine phosphoramidite building block 12. The synthesis of the 5-methylcytosine-containing nucleoside was accomplished by conversion of the core thymine. For this purpose, nucleoside 11 was protected with TMS and converted to the corresponding triazolide by treatment with 1,2,4-triazole and POCl ₃ . Subsequent treatment of the resulting triazolide in a mixture of ammonia and 1,4-dioxane afforded the corresponding nucleoside containing 5-methylcytosine, which was directly protected with _Bz2O to afford 13 in 88% yield in three steps. Phosphoramidite 14 was prepared by phosphitylation as described above.

Scheme 2: (a) i) thymine, BSA, NIS, DXM, room. temp., 7 h; ii) Bu ₃ SnH, AIBN, toluene, 70°C, 30 min, 73%; (b) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, ETT, DCM, room. temp., 30 min, 70% for 12, 75% for 14; (c) i) BSA, triazole, POCl ₃ , Et ₃ N, CH ₃ CN, room. temp., 5 h, ii) 1,4-dioxane/NH ₄ OH, room. temp., 2 h, iii) Bz ₂ O, Et ₃ N, DMF, room. temp., 20 h, 88%.

Classical Vorbruggen nucleosidation, which was performed to incorporate purine nucleobases, generally yielded predominantly α-nucleosides. Conversion of precursor 7 using N ⁶ -benzoyladenine or 2-amino-6-chloropurine afforded an inseparable mixture of anomers 15 and 20, respectively, with α/β ratios of 4:1 and 7:3 (Scheme 3). Separation of the anomers was possible after deacetylation to afford pure β-anomers 16 and 21. Using these compounds, the adenine building block 19 could be obtained by standard dimethoxytritylation (→17), subsequent TBAF-mediated cleavage of the silyl protecting group (→19), and phosphitylation. Synthesis of the guanine building block required conversion of the 2-amino-6-chloropurine nitrogen base. This was accomplished by treatment of 21 with 3-hydroxypropanenitrile and TBD, followed by protection of the 2-amino group with DMF, yielding protected guanosine derivatives 22. Using the chemical sequence described above, synthesis of the guanine building block 25 was accomplished by dimethoxytritylation (→23), subsequent removal of the silyl protecting group (→24), and phosphitylation.

Scheme 3: (a) N ⁶ -benzoyladenine, BSA, TMSOTf, MeCN, 70°C, 20 min, 64%; (b) NaOH, THF/MeOH/H ₂ O, 0°C, 20 min, 69%; (c) DMTr-Cl, pyridine, room temp., 24 h, 87%; (d) TBAF, THF, room temp., 48 h, 87%; (e) CEP-Cl, DIPEA, THF, room temp., 2 h, 71%; (f) 2-amino-6-chloropurine, BSA, TMSOTf, MeCN, 55°C, 50 min, 77%; (g) NaOH, THF/MeOH/H ₂ O, 0°C, 20 min, 85%; (i) i) TBD, 3-hydroxypropanenitrile, DCM, 48 h, ii) N,N-dimethylformamide dimethyl acetal, DMF, 55°C, 2 h, 73%; (j) DMTr-Cl, pyridine, room. temp., 18 h, 70%; (j) TBAF, THF, room. temp., 7 hours, 87%; (l) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, ETT, DCM, room. temp., 50 min, 69%.

A scheme for the synthesis of four preferred phosphoramidite building blocks of the present invention was developed using protected sugar 7 as a starting material. Treatment of a mixture of sugar 7 and in situ silylated thymine with TMSOTf resulted in the efficient formation of nucleotide 35, which has a preferred α/β anomer ratio of approximately 85:15 (as determined by ¹ H NMR) (Scheme 4). The chemical reaction sequence that leads to the thymidine phosphoramidite containing a DMTr group at position 5' does not allow the anomers to be separated by standard chromatographic methods. Thus, to introduce a polarity-reversed modification in the DNA strand, a DMTr group was introduced at position 7' by removing the silyl group in 35 by brief treatment with TBAF (→36), followed by standard dimethoxytritylation (→37). Separation of the two anomers was possible after standard deacetylation, yielding pure a-anomer 38. The thymidine building block 39 was finally prepared by phosphitylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the presence of 5-(ethylthio)-1H-tetrazole. Intermediate 38 also provides a short route to the 5-methylcytosine-containing nucleoside by in situ conversion of TMS-protected nucleoside 38 to the corresponding triazolide using POCl ₃ and 1,2,4-triazole followed by treatment with ammonia and 1,4-dioxane. Direct protection with Bz ₂ O in DMF afforded the efficient formation of nucleoside 40, at which time the labile silyl group was cleaved. Final phosphitylation under conditions such as described above afforded 5-methylcytidine phosphoramidite 41.

Scheme 4: (a) thymine, BSA, TMSOTf, MeCN, room. temp., 18 h, 82%; (b) TBAF, THF, 2 h, 75%; (c) DMTr-Cl, pyridine, room. temp., 24 h, 96%; (d) K ₂ CO ₃ , MeOH, 3 hours, 86%; (e) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, ETT, DCM, room. temp., 1 hour, 81% for 39, 30 min, 80% for 41; f) i) BSA, 1,2,4-triazole, POCl ₃ , Et ₃ N, MeCN, room. temp., 7 h, ii) 1,4-dioxane/NH ₄ OH, room temp., 3 h, iii) Bz ₂ O, Et ₃ N, DMF, room temp., 18 h, 83%.

In the case of purine nitrogenous bases, the introduction of purines was accomplished by brief nucleosidation at slightly elevated temperatures using N ⁶ -benzoyladenine or 2-amino-6-chloropurine to afford nucleosides 15 and 20, respectively, with α/β ratios of 4:1 and 7:3 (Scheme 5). To resolve the anomers, the acetyl groups were removed under mild conditions to afford pure α-anomers 42 and 48. To obtain the adenosine building block, the acetyl protecting group was reintroduced (→ 43), the TPDPS protecting group was removed using TBAF (→ 44), and then standard dimethoxytritylation was performed (→ 45). Selective removal of the acetyl protecting group (→ 46) and subsequent phosphitylation under the conditions described above afforded the adenine building block 47.

In the case of the guanine building block, after separation of the two anomers, 6-chloropurine was converted to the nitrogenous base guanine by treatment with TBD and 3-hydroxypropanenitrile, yielding the guanosine nucleoside 49. During acetylation for 48 h, concomitant protection of the 5'-hydroxy and 2-amino groups occurred, yielding the protected nucleoside 50. Similar to the above description, the DMTr group was introduced by removal of the silyl protecting group using TBAF (→ 51), followed by dimethoxytritylation (→ 52). The two acetyl groups were removed by treatment with K ₂ CO ₃ and the resulting polar product was directly protected with DMF to yield the guanosine nucleoside 53. Final phosphitylation afforded the building block 54.

Scheme 5: (a) N ⁶ -benzoyladenine, BSA, TMSOTf, MeCN, 70°C, 20 min, 64%; (b) NaOH, THF/MeOH/H ₂ O, 0°C, 20 min, 51% α-anomer, 18% β-anomer; (c) Ac ₂ O, DMAP, DCM, room temp., 18 h, 90%; (d) TBAF, THF, room temp., 3.5 h, 90%; (e) DMTr-Cl, pyridine, room temp., 24 h, 89%; (f) NaOH, THF/MeOH/H ₂ O, 0°C, 30 min, 94% (g) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, ETT, DCM, room. temp., 1 hour, 77% for 47, 50 min, 67% for 54; (h) 2-amino-6-chloropurine, BSA, TMSOTf, MeCN, 55°C, 50 min, 77%; (i) NaOH, THF/MeOH/H ₂ O, 0°C, 20 min, 85%; (j) TBD, 3-hydroxypropanenitrile, DCM, 48 h, 87%; (k) Ac ₂ O, DMAP, DXM, room. temp., 48 h, 76%; (1) TBAF, THF, room. temp., 4 hours, 87%; (m) DMTr-Cl, pyridine, room. temp., 48 h, 99%; (n) i) K ₂ CO ₃ , MeOH, room. temp., 7 hours, ii) N,N-dimethylformamide dimethyl acetal, DMF, 55°C, 2 hours, 77%.

EXAMPLE 2

Ethyl-(E and Z,1'R,5'S,7'R)-(7'-hydroxy-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-ylidene )acetate (2a/b)

A solution of epoxide 1 (4.46 g, 18.4 mmol) in dry THF (100 mL) was cooled to -78 °C. LiHMDS (1 M in THF, 22.1 mL, 22.1 mmol) was then slowly added. The solution was stirred for 2 h at -78 °C, then allowed to warm to rt and neutralized by adding 1 M aqueous HCl (22.1 mL). The mixture was then diluted with EtOAc (100 mL) and the THF was removed under reduced pressure. The mixture was then washed with 0.5 M NaH ₂ PO ₄ (50 mL) and the aqueous phase was extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (EtOAc/hexane 3:1) to give two isomers 2a/b (3.30 g, 74%) as light yellow solids.

Data for 2a: R _f = 0.37 (EtOAc/hexane 1:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 6.07-5.98 (m, 1H, H-C(2)), 5.59 (d, J=6.0 Hz, 1H, H-C( 5')), 4.94-4.81 (m, 1H, H-C(1')), 4.65 (t, J=5.6 Hz, 1H, H-C(7')), 4.18 (q, J=7.1 Hz, 2H, CH ₃ CH ₂ ), 2.67 (br., 1H, OH), 2.37 (dd, J=13.5, 7.5 Hz, 1H, H-C(8')), 1.55-1.42 (m, 1H, H-C(8')), 1.40, 1.33 (2s, 6H, (CH ₃ ) ₂ C), 1.26 (t, J=7.1 Hz, 3H, CH ₂ CH ₃ ).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 165.75 (C(1)), 161.61 (C(6')), 116.53 (C(2)), 110.69 (C(3') )), 76.55 (C(5')), 75.52 (C(1')), 71.63 (C(7')), 60.51 (CH ₂ CH ₃ ), 37.46 ( C(8')), 26.44, 24.11 ((CH ₃ ) ₂ C), 14.27 (CH ₂ CH ₃ ).

ESI ⁺ -MSHR m/z, calculation for C ₁₂ H ₁₉ O ₅ ([M+H] ⁺ ) 243.1227, experiment 243.1231.

Data for 2b: R _f = 0.52 (EtOAc/hexane 1:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 6.15-6.05 (m, 1H, H-C(2)), 5.37-5.02 (m, 2H, H-C(5') , OH), 4.87 (d, J=3.4 Hz, 1H, H-C(1')), 4.67 (t, J=4.9 Hz, 1H, H-C(7') ), 4.20 (qd, J=7.1, 0.9 Hz, 2H, CH ₃ CH ₂ ), 2.55 (dd, J=14.6, 8.1 Hz, 1H, H-C( 8')), 1.94-1.77 (m, 1H, H-C(8')), 1.39-1.25 (m, 9H, (CH ₃ ) ₂ C, _{CH2CH3 )} .

¹³ C NMR (75 MHz, CDCl ₃ ) δ 167.91 (C(1)), 167.43 (C(6')), 120.13 (C(2)), 111.75 (C(3') )), 81.62 (C(5')), 78.08 (C(1')), 70.85 (C(7')), 61.25 (CH ₂ CH ₃ ), 36.53 ( C(8')), 27.38, 25.45 ((CH ₃ ) ₂ C), 14.19 (CH ₂ CH ₃ ).

ESI ⁺ -MSHR m/z, calculation for C ₁₂ H ₁₉ O ₅ ([M+H] ⁺ ) 243.1227, experiment 243.1227.

EXAMPLE 3

Ethyl (1'R,5'S,6'S,7'R)-(7'-hydroxy-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-yl)acetate (3)

To a solution of alcohols 2a/b (12.65 g, 52.2 mmol) and nickel chloride hexahydrate (2.48 g, 10.4 mmol) in EtOH (300 mL) was added sodium borohydride (9.88 g, 261 mmol) portionwise at 0 °C. The resulting dark solution was stirred for 30 min at 0 °C and 90 min at rt. It was then carefully concentrated to remove EtOH under reduced pressure, the resulting solid was diluted with EtOAc (200 mL), and the excess NaBH ₄ was quenched by adding water (100 mL) at 0 °C, then stirred at rt for 30 min. The two phases were then separated. The organic phase was washed with water (100 mL). The aqueous phases were then combined, filtered and extracted with EtOAc (2×100 mL). The combined organic phases were dried over _MgSO4 , filtered and concentrated. The crude product was purified by CX (EtOAc/hexane 2:1) to give 3 (11.4 g, 90%) as a white solid.

Data for 3: R _f = 0.40 (EtOAc/hexane 1:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 4.65-4.52 (m, 2H, HC(1'), H-C(5')), 4.15 (qd, J=7.1, 1.4 Hz, 2H, CH ₃ CH ₂ ), 4.05 (ddd, J=10.0, 9.99, 6.2 Hz, 1H, H-C(7')), 2.86 (shir . s, 1H, OH), 2.65 (qd, J=16.9, 7.1 Hz, 2H, H-C(2)), 2.24 (dd, J=13.7, 6.2 Hz, 1H, H-C(8')), 1.93 (dt, J=12.7, 7.1 Hz, 1H, H-C(6')), 1.56 (ddd, J=13.9, 10.2, 5.5 Hz, 1H, H-C(8')), 1.38 (s, 3H, (CH ₃ ) ₂ C), 1, 30-1.21 (m, 6H, (CH ₃ ) ₂ C, CH ₂ CH ₃ ).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 174.38 (C(1)), 109.06 (C(3')), 79.65 (C(5')), 77.19 (C(1 '), 74.32 (C(7'), 60.80 (CH ₂ CH ₃ ), 46.66 (C(6')), 40.38 (C(8')), 32.43 (C (2)), 26.00, 23.69 ((CH ₃ ) ₂ C), 14.17 (CH ₂ CH ₃ ).

ESI ⁺ -MSHR m/z, calculation for C ₁₂ H ₂₁ O ₅ ([M+H] ⁺ ) 245.1384, experiment 245.1388.

EXAMPLE 4

Ethyl (1'R,5'S,6'S,7'R)-(7'-(tert-butyldiphenylsilyl)oxy)-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6'-yl)acetate (4)

To a solution of alcohol 3 (2.50 g, 10.2 mmol), N-methylimidazole (12.6 g, 153 mmol) and iodine (7.80 g, 30.6 mmol) in dry THF (60 mL) was added dropwise tert-butyl(chloro)diphenylsilane (3.0 mL, 11.2 mmol) at room temp. The solution was stirred for ₃ h at room temp. Then the THF was evaporated, the mixture was diluted with EtOAc (50 mL) and washed with 10% aqueous _Na2O3S2 ( ₂ ×40 mL). Then the aqueous phases were combined and extracted with EtOAc (50 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (EtOAc/hexane 1:10) to give 4 (5.01 g, quantitative yield) as a white solid.

Data for 4: R _f = 0.87 (DCM/MeOH 10:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 7.77-7.59 (m, 4H, H-arom.), 7.51-7.32 (m, 6H, H-arom.), 4.61 (t, J=5.7 Hz, 1H, H-C(5')), 4.49 (t, J=5.7 Hz, 1H, H-C(1')), 4.15 (q , J=6.9 Hz, 2H, CH ₃ CH ₂ ), 3.96 (dd, J=15.5, 9.5 Hz, 1H, H-C(7')), 2.64-2, 32 (m, 2H, H-C(2)), 2.15 (tt, J=9.0, 4.3 Hz, 1H, H-C(6')), 1.83 (dd, J=12.7, 5.2 Hz, 1H, H-C(8')), 1.61-1.45 (m, 1H, H-C(8')), 1.27 ( td, J=7.1, 1.9 Hz, 3H, CH ₂ CH ₃ ), 1.18 (s, 6H, (CH ₃ ) ₂ C), 1.09, 1.08 (2s, 9H, ( CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 173.07 (C(1)), 135.87, 135.85(CH-arom.), 134.08, 133.73 (C-arom.), 129.80, 129.75, 127.67, 127.58 (CH-arom.), 108.82 (C(3')), 77.92 (C(5')), 76.96 (C(1')), 74.93 (C(7')), 60.24 (CH ₂ CH ₃ ), 47.27 (C(6')), 40.27 (C(8')), 31.10 (C(2)), 27.04 (CH ₃ ) ₃ -C-Si), 25.86 ((CH ₃ ) ₂ C), 23.83 ((CH ₃ ) ₂ C), 19.23 (CH ₃ ) ₃ -C-Si), 14.24 (CH ₂ -CH ₃ ).

ESI ⁺ -MSHR m/z, calculation for C ₂₈ H ₃₉ O ₅ Si ([M+H] ⁺ ) 483.2561, experiment 483.2562.

EXAMPLE 5

(1'R,5'S,6'S,7'R)-(7'-(tert-butyldiphenylsilyl)oxy)-3',3'-dimethyl-2',4'-dioxabicyclo[3.3.0]oct-6' -yl)acetaldehyde (5)

A solution of ester 4 (8.56 g, 16.3 mmol) in dry DCM (120 mL) was cooled to -78 °C and then DiBAL-H (1 M in cyclohexane, 18 mL, 18 mmol) was slowly added. The solution was stirred at -78 °C for an additional 90 min and then allowed to warm to rt. The reaction was quenched by adding 0.5 M aqueous NaH ₂ PO ₄ (100 mL). The organic phase was separated and the aqueous phase was further extracted with DCM (2 x 100 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (EtOAc/hexane, 2:10 to 2:1) to give aldehyde 5 (6.36 g, 89%) and alcohol 34 (0.637 g, 9%).

Data for 5: R _f = 0.65 (EtOAc/hexane 2:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.72 (s, 1H, H-C(1)), 7.65 (td, J=8.0, 1.6 Hz, 4H, H-arom. ), 7.47-7.33 (m, 6H, H-arom.), 4.57 (t, J=5.7 Hz, 1H, H-C(5')), 4.51 (t, J=5.7 Hz, 1H, H-C(1')), 3.99 (td, J=10.0, 5.9 Hz, 1H, H-C(7')), 2.58- 2.43 (m, 2H, H-C(2)), 2.20-2.08 (m, 1H, H-C(6')), 1.87 (dd, J=13.5, 5.9 Hz, 1H, H-C(8')), 1.53 (ddd, J=13.5, 10.1, 5.5 Hz, 1H, H-C(8 ')), 1.16 (d, J=3.5 Hz, 6H, ((CH ₃ ) ₂ C), 1.05 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 201.87 (C(1)), 135.93, 135.90 (CH-arom.), 133.96, 133.73 (C-arom.), 129.96, 129.89, 127.79, 127.68 (CH-arom.), 108.89 (C(3')), 77.76 (C(5')), 77.17 (C(1')), 74.96 (C(7'), 45.44 (C(6')), 41.31 (C(2)), 40.16 (C(8')), 27.08 (CH ₃ ) ₃ -C-Si), 25.87 ((CH ₃ ) ₂ C), 23.79 ((CH ₃ ) ₂ C), 19.25 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₆ H ₃₅ O ₄ Si ([M+H] ⁺ ) 439.2299, experiment 439.2297.

EXAMPLE 6

(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methylhexahydro-2H-cyclopenta[b]furan-6-ol (6)

Indium(III) trifluoromethanesulfonate (703 mg, 1.25 mmol) was added to a solution of aldehyde 5 (13.73 g, 31.31 mmol) in MeCN (170 mL) and H ₂ O (19 mL). The solution was stirred for an additional 48 h, then the solvents were removed under reduced pressure and co-evaporated with toluene. The residue was dissolved in dry MeOH and stirred for 6 h. After evaporation of the solvent, the crude product was purified by CHX (EtOAc/hexane 3:10) to give a mixture of 6 (10.50 g, 81%) with an anomer ratio of α/β ≈ 4:1 as a colorless oily substance.

Data for 6: R _f = 0.53 (EtOAc/hexane 1:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.63 (dd, J=7.1, 0.6 Hz, 4H, H-arom.), 7.46-7.34 (m, 6H, H- arom.), 4.98 (d, J=4.8 Hz, 0.8H, H-C(2)), 4.91 (dd, J=5.9, 1.3 Hz, 0.2 N , H-C(2)), 4.63-4.54 (m, 1H, H-C(6a)), 4.53-4.37 (m, 1H, H-C(6)), 4 .09 (m, 0.2Н, Н-С(4)), 3.92 (lat., 0.8Н, Н-С(4)), 3.29, 3.27 (2s, 3Н, MeО), 2.79 (dd, J=17.0, 8.2 Hz, 0.8Н, Н-С(3а)), 2.64-2.51 (m, 0.2 Н, Н -C(3a)), 2.29 (d, J=8.1 Hz, 1H, OH), 2.10-1.80 (m, 2.4 H, H-C(3), H-C (5)), 1.65 (ddd, J=13.2, 9.1, 4.4 Hz, 0.8 N, N-C(5)), 1.44-1.34 (m, 0 ,2 N, N-S(3)), 1.22 (ddd, J=13.2, 8,1,4,9 Hz, 0.8 N, N-S(3)), 1.05 ( s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 135.78, 135.74 (CH-arom.), 133.96, 133.84 (C-arom.), 129.78, 127.72 (CH-arom.), 107.21, 106.50 (C(2)), 85.37, 81.76 (C(6a)), 78.11, 77.19 (C(4)), 73.03, 72.44 (C(6)), 55.30, 54.46 (MeO), 50.91, 49.67 (C(3a)), 41.13, 40.29 (C(3)), 38.16, 37.98 (C(5)), 26.96, 26.92 (CH ₃ ) ₃ -C-Si), 19.07 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₆ H ₃₅ O ₄ Si ([M+H] ⁺ ) 435.1962, experiment 435.1950.

EXAMPLE 7

(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsil)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-yl-acetate (7)

To a solution of sugar 6 (3.35 g, 8.12 mmol) and 4-dimethylaminopyridine (1.29 g, 10.6 mmol) in dry DCM (100 mL) was added acetic anhydride (3.8 mL, 41 mmol) at room temp. After stirring for 2 h, the reaction was quenched by slow addition of sat. NaHCO ₃ (10 mL). The mixture was then diluted with sat. NaHCO ₃ (50 mL) and extracted with DCM (3×50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CHX (EtOAc/hexane 1:2) to give a mixture of 7 (3.53 g, 96%) with an anomer ratio of α/β ≈ 4:1 as a colorless oily substance.

Data for 7: R _f = 0.42 (EtOAc/hexane 1:2);

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.70-7.59 (m, 4H, H-arom.), 7.48-7.34 (m, 6H, H-arom.), 5.41 (dt, J=11.0, 5.6 Hz, 0.8Н, Н-С(6)), 5.28 (ddd, J=11.7, 6.6, 5.2 Hz, 0.2Н , Н-С(6)), 4.99 (d, J=4.8 Hz, 0.8Н, Н-С(2)), 4.89 4.81 (m, 0.4Н, Н-С (2), H-C(6a)), 4.76-4.69 (m, 0.8H, H-C(6a)), 4.11 (d, J=5.1 Hz, 0.2H, H-C(4)), 3.90 (d, J=4.0 Hz, 0.8H, H-C(4)), 3.27, 3.24 (2s, 3H , MeO), 2.81 (dd, J=16.6, 7.6 Hz, 0.8H, H-C(3a)), 2.60 (dd, J=10.1, 7.0 Hz, 0.2Н, Н-С(3а)), 2.30-2.18 (m, 0.2 Н, Н-С(5)), 2.12, 2.10 (2s, J=4.7 Hz, 3H, MeCO ₂ ), 2.07-1.82 (m, 2.8H, H-C(5), H-C(3)), 1.24 (ddd, J=12.9, 7 ,6, 3.7 Hz, 1H, H-C(3)), 1.07 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.75, 170.66 (MeCO ₂ ), 135.77, 135.73, 135.72 (CH-arom.), 133.75, 133.65 (C-arom.), 129.82, 129.74, 127.76, 127.75, 127.71 (CH-arom.), 106.19, 106.15 (C(2)), 83.17, 79.80 (C(6a)), 77.49, 76.46 (C(4)), 75.64, 74.41 (C(6)), 54.34, 54.25 (MeO), 51.48, 50.17 (C(3a)), 38.05, 37.98 (C(3)), 36.96, 36.21 (C(5)), 26.95, 26.90 ( _CH3 ) ₃ -C-Si), 21.09, 21.04 ( _MeCO2 ), 19.04 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₆ H ₃₄ O ₅ NaSi ([M+Na] ⁺ ) 477.2068, experiment 477.2063.

EXAMPLE 8

(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsil)oxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-6-ol (8)

To a solution of sugar 6 (2.08 g, 5.04 mmol) in dry DCM (35 mL) was added 2,6-lutidine (2.95 mL, 25.2 mmol) at 0 °C. After stirring for 20 min at 0 °C, TMSOTf (2.73 mL, 15.1 mmol) was added dropwise and then the solution was allowed to warm to room temp and stirred for an additional 60 min. The reaction was then quenched by adding saturated NaHCO ₃ (40 mL). The organic phase was separated and the aqueous phase was further extracted with DCM (3 x 30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The resulting product was dissolved in dry THF (35 mL), cooled to 0 °C, and TBAF (1 M in THF, 5.6 mL, 5.6 mmol) was added. The solution was stirred for 10 min and then diluted with sat. NaHCO ₃ (30 mL) and extracted with DCM (4 x 40 mL). The combined organic phases were dried over MgSO ₄ , filtered, and evaporated. The crude product was purified by chromatography (EtOAc/hexane 1:4) to give glycal 8 (1.76 g, 92%).

Data for 8: R _f = 0.49 (EtOAc/hexane 1:2);

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.66 (m, 4H, H-arom.), 7.42 (m, 6H, H-arom.), 6.22 (t, J=2.1 Hz, 1H, H-C(2)), 4.91 (dd, J=8.2, 5.3 Hz, 1H, H-C(3)), 4.70 (dt, J=11.1, 5 .6 Hz, 1H, Н-С(6)), 4.56 (t, J=2.8 Hz, 1H, Н-С(6а)), 3.97 (d, J=4.0 Hz, 1H, H-C(4)), 3.24 (d, J=8.2 Hz, 1H, H-C(3a)), 2.30 (br, 1H, OH), 2.03 (dd, J=12.6, 5.4 Hz, 1H, H-C(5)), 1.51 (ddd, J=12.7, 11.2, 4.2 Hz, 1H, H- C(5)), 1.08 (s, 9H, ( _CH3 ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 146.24 (C(2)), 135.72, 135.69 (CH-arom.), 134.03, 133.74 (C-arom.), 129.80, 129.78, 127.73 (CH-arom.), 101.84 (C(3)), 84.59 (C(6a)), 76.79(C(4)), 74.10 (C(6)), 55.56 (C(3a)), 39.38 (C(5)), 26.93 (CH ₃ ) ₃ -C-Si), 19.08 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₃ H ₂₉ O ₃ Si ([M+H] ⁺ ) 381.1880, experiment 381.1893.

EXAMPLE 9

(3aR,4R,6R,6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-yl)oxy)( tert-butyl)diphenylsilane (9)

To a solution of glycal 8 (1.34 g, 3.52 mmol) and DMTr-Cl (1.43 g, 4.23 mmol) in a mixture of dry DCM (15 mL) and dry 2,6-lutidine (15 mL) was added silver triflate (1.13 g, 4.40 mmol) portionwise to give a dark red suspension. After stirring for 2 h at room temperature, an additional portion of DMTr-Cl (239 mg, 0.705 mmol) was added. The suspension was stirred for an additional 2 h and then filtered. The organic phase was washed with saturated NaHCO ₃ (100 mL) and the aqueous phase was extracted with DCM (3×30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (EtOAc/hexane 1:7, +0.5% _Et3N ) to afford the protected glycal 9 (2.24 g, 93%) as a white foam.

Data for 9: R _f = 0.59 (EtOAc/hexane 1:2);

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.76 (d, J=7.4 Hz, 2H, H-arom.), 7.69-7.60 (m, J=9.3, 5, 9, 4.6 Hz, 8H, H-arom.), 7.56-7.39 (m, 8H, H-arom.), 7.33 (t, J=7.3 Hz, 1H, H- arom.), 7.00-6.93 (m, 4H, H-arom.), 6.47-6.37 (m, 1H, H-C(2)), 4.67-4.58 ( m, 1H, H-C(6)), 4.58-4.50 (m, 2H, H-C(3), H-C(6a)), 3.86, 3.85 (2s, 6H, MeO), 3.82 (d, J=4.0 Hz, 1H, H-C(4)), 3.08 (d, J=8.1 Hz, 1H, H -C(3a)), 1.67 (td, J=12.4, 4.2 Hz, 1H, H-C(5)), 1.28 (dd, J=12.7, 5.4 Hz , 1H, H-C(5)), 1.11 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 158.67 (MeO-C-arom.), 147.61 (C(2)), 146.26, 137.36, 137.21 (C-arom.), 135.81, 135.78 (CH-arom.), 134.17, 134.04 (C-arom.), 130.48, 129.83, 129.81, 128.37, 127.98, 127.76, 127.73, 126.79, 113.32, 113.28 (CH-arom.), 100.29 (C(3)), 86.96 (C(Ph) ₃ ), 84.95 (C(6a)), 76.17 (C(6)), 76.07(C(4)), 55.26 (MeO-DMTr), 55.11 (C(3a)), 37.32 (C(5)), 27.04 ( _CH3 ) ₃ -C-Si), 19.21 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₄₄ H ₄₆ O ₅ NaSi ([M+Na] ⁺ ) 705.3007, experiment 705.3021.

EXAMPLE 10

(3aS,4R, 6R, 6aS)-6-(bis(4-methoxyphenyl)(phenyl)methoxy)-3a,5,6,6a-tetrahydro-4H-cyclopenta[b]furan-4-ol (10)

To a solution of glycal 9 (2.23 g, 3.27 mmol) in dry THF (20 mL) was added TBAF (1 M in THF, 20 mL, 20 mmol) at rt. The solution was stirred for 20 h and then diluted with saturated NaHCO ₃ (100 mL) and extracted with DCM (3 x 80 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (0.5% MeOH in DMC, +0.5% Et ₃ N) to give 10 (1.45 g, quant.) as a white foam.

Data for 10: R _f = 0.44 (EtOAc/hexane 1:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 7.53-7.46 (m, 2H, H-arom.), 7.43-7.35 (m, 4H, H-arom.), 7.21 (dd, J=10.7, 5.3 Hz, 2 H, H-arom.), 7.16-7.08 (m, 1H, H-arom.), 6.80-6.71 (m , 4H, H-arom.), 6.30 (t, J=2.1 Hz, 1H, H-C(2)), 4.68 (t, J=2.8 Hz, 1H, H-C (3)), 4.29-4.14 (m, 2H, H-C(6), H-C(6a)), 3.71 (s, 6H, MeO), 3.65 (d, J=3.5 Hz, 1H, H-C(4)), 2.87 (d, J=7.9 Hz, 1H, H-C(3a)), 1.59 ( ddd, J=13.2, 11.6, 4.3 Hz, 1H, H-C(5)), 1.05-0.95 (m, 2H, H-C(5), OH).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 158.54 (MeO-C-arom.), 147.64 (C(2)), 145.82, 137.12, 137.08 (C-arom.) , 130.26, 128.29, 127.81, 126.71, 113.13 (CH-arom), 100.17 (C(3)), 86.75 (C(Ph) ₃ ), 84, 42 С(6а)), 75.54 (С(6)), 74.59 (С(4)), 55.22 (MeO-DMTr), 54.25 (С(3а)), 37.56 ( C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₀ H ₂₇ O ₅ ([M+H] ⁺ ) 467.1853, experiment 467.1844.

EXAMPLE 11

(3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (11)

To a solution of glycal 10 (1.45 g, 3.27 mmol) in dry DCM (45 mL) at 0 °C was added BSA (2.0 mL, 8.18 mmol) dropwise and then the solution was allowed to warm to rt. After stirring for 45 min, thymine (595 mg, 4.91 mmol) was added and the reaction mixture was stirred for an additional 60 min at rt. The mixture was then cooled to 0 °C and N-iodosuccinimide (875 mg, 3.92 mmol) was added. After stirring for 3 h at 0 °C and 4 h at rt, the reaction mixture was diluted with _EtOAc (100 mL) and then washed with 10% aq. _Na2S2O3 ( ₁₀₀ mL) and sat. NaHCO ₃ (100 ml). The aqueous phases were combined and extracted with DCM (3×50 ml). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was dissolved in dry toluene (45 mL) and then _Bu3SnH (1.32 mL, 4.91 mmol) and azoisobutyronitrile (AIBN, 53 mg, 0.33 mmol) were added at rt. After heating at 70 °C for 30 min, the mixture was cooled to rt and TBAF (1 M in THF, 6.5 mL, 6.5 mmol) was added. The solution was stirred for an additional 25 min and diluted with sat. _NaHCO3 (100 mL) and extracted with DCM (4×70 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM, +0.5% _Et3N ) to give 11 (1.45 g, 73% over two steps) as a white foam.

Data for 11: R _f = 0.29 (6% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.37 (br, 1H, HN(3)), 7.83 (d, J=1.1 Hz, 1H, H-C(6)), 7.58-7.52 (m, 2H, H-arom.), 7.48-7.41 (m, 4H, H-arom.), 7.28 (t, J=7.7 Hz, 2H, H-arom.), 7.21 (t, J=7.2 Hz, 1H, H-arom.), 6.84 (dd, J=8.9, 1.2 Hz, 4H, H-arom.), 5.91 (dd, J=8.0, 5.5 Hz, 1H, H-C(1')), 4.25 (dt, J=10.8, 6.0 Hz, 1H, H-C(5')), 4.13-4.08 (m, 1H, H-C(4')), 3.86 (d,J=3.4 Hz, 1H, H-C(7'), 3.79 (s, 6H, MeO), 2.70 (ddd, J=12.8, 10.2, 5.5 Hz, 1H-C(2') ), 2.61 (dd, J=16.9, 8.2 Hz, 1H, H-C(3')), 1.84 (d, J=0.8 Hz, 3H, Me-C(5)), 1.80 (lat, 1H, OH), 1.60 (ddd, J=14.2, 10.5, 4.2 Hz, 1H, H-C(6')), 1.3 3(dt, J=12.9, 8.0 Hz, 1H, H-C(2')), 1.14 (dd, J=13.7, 6.1 Hz, 1H, H-C(6')).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.17 (C(4)), 158.64 (MeO-C-arom.), 150.47 (C(2)), 145.65, 136.85, 136.71 (C-arom.), 135.52 (C(6)), 130.20, 128.12, 127.91, 126.90, 113.22, 113.21 (CH-arom.), 110.69 (C(5)), 87.21 (C(Ph) ₃ ), 86.57 (C(1')), 82.02 (C(4')), 74.19 (C(5')), 74.13 (C(7')), 55.25 (MeO-DMTr), 49.40 (C(3')), 38.51 (C(6')), 37.64 (C(2')), 12.58 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₃ H ₃₄ O ₇ N ₂ Na ([M+Na] ⁺ ) 593.2258, experiment 593.2250.

EXAMPLE 12

(3'S,5'R,7'R)-1-{7'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4,-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}thymine (12)

To a solution of nucleoside 11 (232 mg, 0.406 mmol) and 5-(ethylthio)-1H-tetrazole (90 mg, 0.69 mmol) in dry DCM (10 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.26 mL, 0.81 mmol) at room temp. After stirring for 30 min, the reaction mixture was diluted with DCM (50 mL) and washed with sat. NaHCO ₃ (2×30 mL) and sat. NaCl (30 mL). The aqueous phases were combined and extracted with DCM (50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (1.8% MeOH in DCM, +0.5% _Et3N ) to give 12 (219 mg, mixture of two isomers, 70%) as a white foam.

Data for 11: R _f = 0.68 (6% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.93 (br, 1H, HN(3)), 7.85 (d, J=1.2 Hz, 1H, H-C(6)), 7 ,65-7.52 (m, 2H, H-arom.), 7.52-7.40 (m, 4H, H-arom.), 7.40-7.21 (m, 3H, H-arom. .), 6.96-6.81 (m, 4H, H-arom.), 6.00, 5.94 (2dd, J=8.3, 5.2 Hz, 1H, H-C(1' )), 4.29-4.17 (m, 1H, H-C(5')), 4.12-3.89 (m, 2H, H-C(4'), H-C(7')), 3.85, 3.84 (2s, 6H, MeO), 3.81-3.63 (m, 2H, OCH ₂ CH ₂ CN) , 3.56-3.41 (m, 2H, (Me ₂ CH) ₂ N), 2.88-2.69 (m, 2H, H-C(3'), H-C(2')) , 2.61, 2.56 (dt, J=12.9 6.3 Hz, 2H, OCH ₂ CH ₂ CN), 1.92, 1.82 (2d, J=0.8 Hz, 3H, Me -C(5)), 1.75-1.56 (m, 1H, H-C(6')), 1.52-1.36 (m, 2H, H-C(6'), H-C(2')), 1.22-1.01 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 163.86 (C(4)), 158.66, 158.64 (MeO-C-arom), 150.29, 150.27 (C(2)) , 145.58, 145.52, 136.76, 136.71, 136.69, 136.60 (C-arom.), 135.49, 135.35 (C(6)), 130.21, 130 ,16, 128.17, 128.13, 127.88, 126.91, 126.89 (CH-aromatic), 117.49 (OCH ₂ CH ₂ CN), 113.18 (CH-aromatic), 110.74 (C(5)), 87.27, 87.25 (C(Ph) ₃ ), 86.58, 86.45 (C(1')), 81.79 , 81.68 (C(4')), 76.02, 75.50 (J _C,P =16.5, 15.7 Hz, C(7')), 74.22 (C(5') ), 58.26, 58.06, 57.87 (OCH ₂ CH ₂ CN), 55.26, 55.22 (MeO-DMTr), 48.85, 48.62 (Jc,p=2.6, 5.0 Hz, C(3')), 43.10, 43.04 (Jc,p=12.3, 12.4 Hz (Me2Gtf)2N), 37.78 (J _C,P =5.3 Hz C(6')), 37.62, 37.48 (C(2')), 37.41 (J _C,P =3.6 Hz C(6')), 24.57, 24.53, 24.50, 24.46, 24.44, 24.39, 24.37 (Me ₂ CH) ₂ N) , 20.35, 20.25 (J _C,P = 7.1, 7.0 Hz, OCH ₂ CH ₂ CN, 12.58, 12.41 (7s, Me-C(5)).

³¹ P NMR (122 MHz, CDCl ₃ ) δ 147.32, 146.98.

ESI ⁺ -MSHR m/z, calculation for C ₄₂ H ₅₂ O ₈ N ₄ P ([M+H] ⁺ ) 771.3517, experiment 771.3512.

EXAMPLE 13

(3'S,5'R,7'R)-N4-benzoyl-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine (13)

To a solution of nucleoside 11 (302 mg, 0.530 mmol) in dry MeCN (5 mL) was added BSA (0.31 mL, 1.27 mmol) dropwise at 0 °C, and then the solution was stirred overnight at rt. In another flask, a suspension of 1,2,4-triazole (1.28 g, 18.55 mmol) in dry MeCN (50 mL) was cooled to 0 °C and POCl ₃ (0.40 mL, 4.2 mmol) and Et ₃ N (2.96 mL, 21.2 mmol) were added. The suspension was stirred for 30 min at 0 °C, and then the previously prepared solution of silylated compound 11 was added to the suspension and the mixture was stirred for an additional 5 h at rt. The reaction was quenched by adding sat. NaHCO ₃ (10 ml), MeCN was removed under reduced pressure and the resulting mixture was diluted with sat. NaHCO ₃ (35 ml) and extracted with DCM (3×40 ml). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (10 mL) and conc. NH ₄ OH (10 mL). After stirring for 2 h at room temperature, the mixture was concentrated to half volume in vacuo, diluted with sat. NaHCO ₃ (30 mL) and extracted with DCM (4×30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was then dissolved in dry DMF (13 mL), _Et3N (90 µL, 0.64 mmol) was added followed by _Bz2O (300 mg, 1.33 mmol) at rt and the solution was stirred overnight. The resulting brown solution was quenched by careful addition of sat. _NaHCO3 (50 mL) and extracted with DCM (4×50 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CC (hexane/EtOAc 1:2, +0.5% _Et3N ) to give 13 (315 mg, 88%) as a white foam.

Data for 13: R _f = 0.57 (4% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 13.39 (br, 1H, NH), 8.46-8.26 (m, 2H, H-arom), 8.13 (d, J=0 .5 Hz, 1H, C(6)), 7.61 (d, J=7.3 Hz, 2H, H-arom.), 7.58-7.43 (m, 7H, H-arom.) , 7.34 (t, J=7.4 Hz, 2H, H-arom.), 7.30-7.23 (m, 1H, H-arom.), 6.89 (d, J=8, 8 Hz, 4H, H-arom.), 5.96 (dd, J=7.5, 5.8 Hz, 1H, H-C(1')), 4.38-4.25 (m, 1H, H-C(5')), 4.22-4.12 (m, 1H, H-C(4')), 3.90 (d, J= 3.6 Hz, 1H, H-C(7')), 3.83 (s, 6H, MeO), 2.82 (ddd, J=13.3, 10.2, 5.7 Hz, 1H, H-C(2')), 2.66 (dd, J=17.0, 8.1 Hz, 1H, H-C(3')), 2.08 (s, 3H, Me-C(5 )), 1.77 (br., 1H, OH), 1.71-1.57 (m, 1H, H-C(6')), 1.49-1.36 (m, 1H, H-C(2')), 1.21 (dd, J=13.7, 6.2 Hz, 1H, H-C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 179.56 (CONH), 160.01 (C(4)), 158.70 (MeO-C-arom.), 147.96 (C(2)), 145.65 (C-arom.), 137.26 (C(6)), 136.99, 136.83, 136.71 (C-arom.), 132.41, 130.22, 129.89, 128.16, 128.14, 127.95, 126.94, 113.25 (CH-arom.), 111.57 (C(5)), 87.34 (C(Ph) ₃ ), 87.32 (C(1')), 82.57 (C(4')), 74.30 (C(5')), 74.16 (C(7')), 55.27 (MeO-DMTr), 49.56 (C(3')), 38.52 (C(6')), 38.00 (C(2')), 13.63 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₄₀ H ₄₀ O ₇ N ₃ ([M+H] ⁺ ) 674.2861, experiment 674.2862.

EXAMPLE 14

(3'R,5'R,7'R)-N4-benzoyl-1-{7'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}-5-methylcytosine (14)

To a solution of nucleoside 13 (276 mg, 0.409 mmol) and 5-(ethylthio)-1H-tetrazole (69 mg, 0.53 mmol) in dry DCM (10 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.20 mL, 0.61 mmol) at room temp. After stirring for 60 min, the reaction mixture was diluted with DCM (50 mL) and washed with sat. NaHCO ₃ (2×30 mL) and sat. NaCl (30 mL). The aqueous phases were combined and extracted with DCM (50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (EtOAc/hexane 2:3, +0.5% _Et3N ) to give 14 (268 mg, mixture of two isomers, 75%) as a white foam.

Data for 14: R _f = 0.77 (5% MeOH in DCM);

^1H NMR (400 MHz, CDCl ₃ ) δ 13.32 (s, 1H, NH), 8.41-8.28 (m, 2H, H-arom), 8.13-8.04 (m, 1H, C(6)), 7.61-7.51 (m, 3H, H-arom.), 7.51-7.40 (m, 6H, H-arom.), 7.37-7, 29 (m, 2H, H-arom), 7.29-7.20 (m, 1H, H-arom), 6.92-6.82 (m, 4H, H-arom), 6, 07-5.87 (m, 1H, Н-С(1')), 4.24 (dq, J=11.7, 5.8 Hz, 1H, H-C(5')), 4.13-4.00 (m, 1H, H-C(4')), 3.94 (ddd, J=14.5, 10.5, 2, 8 Hz, 1H, H-C(7')), 3.83, 3.82 (2s, 6H, MeO), 3.69 (m, 2H, OCH ₂ CH ₂ CN), 3.53 3.40 (m, 2H, (Me ₂ CH) ₂ N), 2.91-2.70 (m, 2H, H-C(2'), H-C(3')), 2.57, 2.53 (2t, J=6.3 Hz, 2H, OCH ₂ CH ₂ CN), 2.08, 1.99 (2d, J=0.6 Hz, 3H, Me-C(5)), 1.72-1.56 (m, 1H, H-C(6')), 1.54-1.36 (m, 2H, H-C(2'), H -C(6')), 1.10 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 179.54 (CONH), 159.98 (C(4)), 158.69 (MeO-C-arom.), 147.90 (C(2)), 145.58, 145.54 (C-arom.), 137.30, 136.93 (C(6)), 136.81, 136.80, 136.73, 136.70, 136.67, 136.60 (C-arom.), 132.37, 132.35, 130.22, 130.17, 129.89, 128.17, 128.15, 128.11, 127.93, 126.94 (CH-arom.), 117.49 (OCH ₂ CH ₂ CN), 113.23 (CH-arom.), 111.60 (C(5)), 87.36, 87.35 (C(Ph) ₃ ), 87.33, 87.25 (C(1')), 82.33, 82.2 5 (C(4')), 76.05, 75.52 (J _C,P =16.4, 15.6 Hz, C(7')), 74.32 (C(5')), 58.18, 57.98 (J _C,P =19.5 Hz OCH ₂ CH ₂ CN), 55.28, 55.24 (MeO-DMTr), 48.93, 48.72 (J _C,P =2.7, 4.9 Hz, C(3')), 43.11, 43.05 (J _C,P =12.4 Hz (Me ₂ CH) ₂ N), 38.02, 37.88 (C(2')), 37.74, 37.40 (J _C,P =5.3, 3.4 Hz, C(6')), 24 ,58, 24.54, 24.50, 24.47, 24.40, 24.38 (6s, Me ₂ CH) ₂ N), 20.36, 20.26 (J _C,P = 7.1 Hz, OCH ₂ CH ₂ CN),), 13.66, 13.49 (Me-C (5)).

³¹ P NMR (122 MHz, CDCl ₃ ) δ 147.37, 147.07.

ESI ⁺ -MSHR m/z, calculation for C ₄₉ H ₅₇ O ₈ N ₅ P ([M+H] ⁺ ) 874.3939, experiment 874.3937.

EXAMPLE 15

(3'R,5'R,7'R)-N6-benzoyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}adenine (15)

To a suspension of sugar 7 (1.86 g, 4.10 mmol) and N ⁶ -benzoyladenine (1.96 g, 8.20 mmol) in dry MeCN (40 mL) was added BSA (4.00 mL, 16.4 mmol) at rt. After stirring for 25 min, the suspension turned into a clear solution, and then it was heated to 70 °C. TMSOTf (1.48 mL, 8.20 mmol) was added dropwise and the solution was stirred for an additional 20 min at 70 °C. Then the solution was cooled to rt, quenched by addition of saturated NaHCO ₃ (100 mL) and extracted with EtOAc (4×50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (2% MeOH in DCM) to give mixture 15 (1.74 g, 64%) with an anomer ratio of α/β≈4:1 as a white foam.

Data for 15: R _f = 0.33 (EtOAc/hexane 4:1);

^1H NMR (400 MHz, CDCl ₃ ) δ 9.33 (br, 1H, NH), 8.68 (d, J=5.4 Hz, 0.8H, H-C(2)), 8, 64 (d, J=5.6 Hz, 0.2Н, Н-С(2)), 8.10 (d, J=1.5 Hz, 0.2Н, Н-С(8)), 7, 99 (d, J=7.3 Hz, 2H, H-arom.), 7.95 (s, 0.8H, H-C(8)), 7.63 (t, J=8.7 Hz, 4H, H-arom.), 7.55 (dd, J=13.0, 6.4 Hz, 1H, H-arom.), 7.50-7.34 (m, 8H, H-arom.), 6.20 (dd, J=6.3, 2.5 Hz, 0.8H, H-C(1')), 6.05 (t, J=6.5 Hz, 0 ,2Н, Н-С(1')), 5.43-5.32 (m, 1H, Н-С(5')), 5.03-4.97 (m, 0.8Н, Н-С (4')), 4.83 (t, J=6.0 Hz, 0.2H, H-C(4')), 4.14 (lat, 0.2H, H-C(7') ), 4.08 (d, J=3.7 Hz, 0.8Н, Н-С(7')), 3.02 (dd, J=16.1, 6.6 Hz, 0.8Н, Н -C(3')), 2.83 (dd, J=16.9, 7.7 Hz, 0.2H, H-C(3')), 2.59-2.39 (m, 1H, H-C(2')), 2.18-2 .11 (m, 1H, H-C(6')), 2.07 (d, J=1.6 Hz, 2.4H, MeCO ₂ ), 2.02 (d, J=1.9 Hz, 0.6H, MeSO ₂ ), 2.01-1.92 (m, 1H, H-C(6')), 1.91-1.80 (m, 1H, H-C(3')), 1.07 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.57, 170.49 (MeSO ₂ ), 164.82 (CONH), 152.50 (C(2)), 151.27 (C(4)), 149.56 (C(6)), 141.37, 141.06 (C(8)), 135.72, 135.68, 135.66 (CH-aromatic), 133.67, 133.57, 133.24, 133.22 (C-arom.), 132.73, 130.03, 129.98, 128.80, 128.78, 127.92, 127.86, 127.85 (CH-arom.), 123.61 (C(5)), 87.19, 86.17 (C(1')), 83.22, 80.96 (C(4'), 76 ,50, 76.04 (C(7')), 74.38 (C(5')), 51.07 (C(3')), 37.29, 37.15, 36.80, 36, 60 (C(2'), C(6')), 26.89 (CH ₃ ) ₃ -C-Si), 20.97, 20.90 (MeCO ₂ ), 19.01 (CH ₃ ) ₃ - C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₃₇ H ₄₀ O ₅ N ₅ Si ([M+H] ⁺ ) 662.2793, experiment 662.2787.

EXAMPLE 16

(3'R,5'R,7'R)-N6-benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}adenine (16):

Nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH in THF/methanol/ _H2O (5:4:1, 80 mL) at 0 °C. The reaction mixture was stirred for 20 min and quenched by adding _NH4Cl (1.06 g). The solvents were then removed under reduced pressure and the product was purified by QC (5% isopropanol in DCM) to afford 16 (287 mg, 18%) and the corresponding α-anomer (836 mg, 51%) as a white foam.

Data for 16: R _f = 0.44 (6% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 8.70 (s, 1H, H-C(2)), 8.09-7.98 (m, 2H, H-arom), 7.97 (s , 1H, H-C(8)), 7.63 (ddd, J=7.4, 5.7, 1.5 Hz, 4H, H-arom.), 7.59-7.55 (m, 1H, H-arom), 7.51 (m, 2H, H-arom), 7.44-7.33 (m, 6H, H-arom), 6.02 (dd, J=9, 4, 5.5 Hz, 1H, Н-С(1')), 4.57 (dd, J=8.1, 5.0 Hz, 1H, Н-С(4')), 4.43 (dd, J=11.8, 5.3 Hz, 1H, Н-С(5')), 4.26 (br., 1H, Н-С( 7')), 2.78 (q, J=8.9 Hz, 1H, H-C(3')), 2.32-1.80 (m, 5H, H-C(2'), H -C(6'), OH), 1.06 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.85 (CONH), 152.56 (C(2)), 151.17 (C(4)), 149.86 (C(6)), 141.25 (C(8)), 135.68 (CH-arom.), 133.87, 133.39 (C-arom.), 132.78, 129.92, 128.78, 128.01, 127.78 (CH-arom.), 123.51 (C(5)), 87.65 (C(1')), 82.91 (C(4')), 76.66 (C(7')), 72.54 (C(5')), 50.44 (C(3')), 41.42 (C(6')), 36.17 (C(2')), 26.89 (CH ₃ ) ₃ -C-Si), 19.03 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₃₅ H ₃₈ O ₄ N ₅ Si ([M+H] ⁺ ) 620.2688, experiment 620.2671.

EXAMPLE 17

(3'R,5'R,7'R)-N6-benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (17)

To a solution of nucleoside 16 (307 mg, 0.495 mmol) in dry pyridine (6 mL) was added DMTr-Cl (503 mg, 1.49 mmol) at room temp. The solution was stirred for 1 day and then diluted with saturated NaHCO ₃ (50 mL) and extracted with DCM (3×70 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (1.5% MeOH in DCM, +0.5% Et ₃ N) to give 17 (395 mg, 87%) as a yellow foam.

Data for 17: R _f = 0.65 (5% MeOH in DCM);

¹ NMR (300 MHz, MeOD) δ 8.64 (s, 1H, H-C(2)), 8.61 (s, 1H, H-C(8)), 8.08 (d, J=1 ,2 Hz, 2Н, Н-aromatic), 7.68-7.17 (m, 22Н, Н-aromatic), 6.86-6.75 (m, 4Н, Н-aromatic), 6, 14 (dd, J=7.4, 6.3 Hz, 1H, H-C(1')), 4.48-4.31 (m, 1H, H-C(5')), 4.28 -4.15 (m, 1Н, Н-С(4')), 3.88 (d, J=3.8 Hz, 1Н, Н-С(7')), 3.75, 3.74 (2s, 6H, MeO), 2.67 (dd, J=16.6, 6.7 Hz, 1H, H-C(3')), 2.47 (ddd, J=13.3 , 10.2, 6.1 Hz, 1H, Н-С(2')), 2.15-1.94 (m, 1Н, Н-С(6')), 1.71 (ddd, J= 13.0, 11.3, 4.4 Hz, 1H, Н-С(2')), 1.11 (dd, J=12.2, 4.9 Hz, 1H, Н-С(6') ), 0.95 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.69 (CONH), 158.61, 158.60 (MeO-C-arom.), 152.42 (C(2)), 151.27 (C(4)), 149.41 (C(6)), 145.81 (C-arom.), 141.25 (C(8)), 137.00, 136.85 (C-arom.), 135.60, 135.57 (CH-arom.), 133.80, 133.69, 133.43 (C-arom.), 132.70, 130.28, 130.25, 129.85, 129.81, 128.84, 128.18, 127.89, 127.71, 127.65, 126.78 (CH-arom.), 123.52 (C(5)), 113.22, 113.19 (CH-arom.), 87.09 (C(Ph) ₃ ), 86.41 (C(1')), 83.52 (C(4')), 76.05 (C(7')), 74.78 (C(5')), 55.20 (MeO-DMTr), 50.43 (C(3')), 38.10 (C(2'), C(6')), 26.84 ( _CH3 ) ₃ -C-Si), 19.00 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₅₆ H ₅₆ O ₆ N ₅ Si ([M+H] ⁺ ) 922.3994, experiment 922.3953.

EXAMPLE 18

(3'S,5'R,7'R)-N6-benzoyl-9-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (18)

To a solution of nucleoside 17 (376 mg, 0.408 mmol) in dry THF (9 mL) was added TBAF (1 M in THF, 1.22 mL, 1.22 mmol) at rt. The solution was stirred for 2 days and then diluted with saturated NaHCO ₃ (25 mL) and extracted with DCM (4 × 25 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (4% MeOH in DCM, + 0.5% Et ₃ N) to give 18 (242 mg, 87%) as a white foam.

Data for 18: R _f = 0.33 (5% MeOH in DCM);

¹ H NMR (300 MHz, CD ₃ CN) δ 9.35 (br, 1H, NH), 8.67 (s, 1H, C(2)), 8.46 (s, 1H, C(8) ), 8.01 (d, J=7.4 Hz, 2H, H-arom.), 7.54 (m, 5H, N-arom.), 7.35 (m, 4H, H-arom.) , 7.30-7.17 (m, 3H, H-arom.), 6.84 (d, J=8.9 Hz, 4H, H-arom.), 6.09 (dd, J=7, 8, 6.2 Hz, 1H, Н-С(1')), 4.12 (dt, J=11.2, 5.8 Hz, 1H, С(5')), 3.87-3.79 (m, 2H, C(4'), C(7')), 3.75 (s, 6H,MeO), 2.83-2.64 (m, 2H, C( 2'), OH), 2.58 2.46 (m, 1H, C(3')), 2.21 (dd, J=13.9, 7.1 Hz, 1H, C(2')) , 1.92 1.82 (m, 1H, C(6')), 1.29-1.17(m, 1H, C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 165.03 (CONH), 158.57 (MeO-C-arom.), 152.40 (C(2)), 151.23 (C(4)), 149.52 (C(6)), 145.68 (C-arom.), 141.49 (C(8)), 136.86, 136.84, 133.77 (C-arom.), 132.77, 130.22, 128.81, 128.16, 128.02, 127.89, 126.84 (CH-arom.), 123.40 (C(5)), 113.19 (CH-arom.), 87.06 (C(Ph) ₃ ), 86.74 (C(1')), 83.58 (C(4')), 74.62 (C(5')), 74.38 (C(8')), 55.25 (MeO-DMTr), 49.77 (C(3')), 38.55, 38.32 (C(6'), C(2')).

ESI ⁺ -MSHR m/z, calculation for C ₄₀ H ₃₈ O ₆ N ₅ ([M+H] ⁺ ) 684.2817, experiment 684.2830.

EXAMPLE 19

(3'R,5'R,7,R)-N6-benzoyl-9-{7'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}adenine (19)

To a solution of nucleoside 18 (173 mg, 0.253 mmol) and N,N-diisopropylethylamine (0.18 mL, 1.0 mmol) in dry THF (8 mL) was added N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.50 mmol) at room temp. The solution was stirred for 2 h and then diluted with sat. NaHCO ₃ (40 mL) and extracted with DCM (4 X 40 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (EtOAc, +0.5% Et ₃ N) to give 19 (177 mg, mixture of two isomers, 71%) as a white foam.

Data for 19: R _f = 0.38, 0.44 (EtOAc);

^1H NMR (400 MHz, CDCl ₃ ) δ 9.05 (br, 1H, NH), 8.70, 8.70 (2s, 1H, H-C(2)), 8.47, 8.46 (2s, 1H, H-C(8)), 7.97 (d, J=7.5 Hz, 2H, H-arom.), 7.57-7.50 (m, 1H, H-arom. ), 7.49-7.41 (m, 4H, N-arom.), 7.39-7.31 (m, 4H, N-arom.), 7.24-7.17 (m, 5. 4 Hz, 2H, H-arom.), 7.13 (dt, J=12.5, 6.2 Hz, 1H, H-arom.), 6.83-6.70 (m, 4H, H-arom.), 6.14-5.97 (m, 1H, H-C(1')), 4.14 (ddd, J=11.1 , 7.8, 3.4 Hz, 1H, Н-С(5')), 3.91 -3.74 (m, 2Н, Н-(4'), Н-С(7')), 3 .71, 3.70 (2s, 6H, MeO), 3.65-3.50 (m, 2H, OCH ₂ CH ₂ CN), 3.37 (ddq, J=13.9, 10.2, 6 ,8 Hz, 2H, (Me ₂ CH) ₂ N), 2.90-2.76 (m, 1H, H-C(2')), 2.75-2.60 (m, 1H, H-C(3')), 2.47, 2.42 (2t, J=6.3 Hz, 2H, OCH ₂ CH ₂ CN 2.11 (dt, J=12.7, 6.1 Hz, 1H, Н-С(2')), 1.73 (ddt, J=13.6, 10.4, 5.1 Hz, 1Н, Н-С(6')), 1, 39 (ddd, J=50.2, 13.4, 6.2 Hz, 1H, H-C(6')), 1.10-0.89 (t, 12H, (Me ₂ CH) ₂ N) .

¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.66 (CONH), 158.57 (MeO-C-arom.), 152.46 (C(2)), 151.32, 151.26 (C(4)), 149.45, 149.43 (C(6)), 145.60, 145.59 (C-arom.), 141.52, 141.47 (C(8)), 136.88, 136.83, 136.81, 133.78 (C-arom.), 132.75, 132.73, 130.22, 130.21, 130.19, 130.17, 128.87, 128.17, 127.87, 126.82, 126.80 (CH-arom.), 123.59 (C(5)), 117.53, 117.50 (OCH ₂ CH ₂ CN) _, 113.17 (CH-arom.), 87.10, 87.07 (C(Ph) ₃ ) _, 86.72, OCH _{2CH 2} CN), 55,25, 55,21 (MeO-DMTr), 49,29, 49,08 (J _C,P =2,6, 4,7 Гц, C(3')), 43,12, 43,00 (J _C,P =2,4, 2,3 Гц (Me ₂ CH) ₂ N), 38,27, 38,23 (C(2')), 37,41, 37,22 (J _C,P =5,3, 3,5 Гц, C(6')) 24,56, 24,53, 24,49, 24,47, 24,43, 24,41, 24,36, 24,33 (8s, Me ₂ CH) ₂ N), 20,36, 20,25 (J _C,P =7,2, 7.0 Hz, OCH ₂ CH ₂ CN).

³¹ P NMR (122 MHz, CDCl ₃ ) δ 147.64, 146.87.

ESI ⁺ -MSHR m/z, calculation for C ₄₉ H ₅₅ O ₇ N ₇ ([M+H] ⁺ ) 884.3895, experiment 884.3898.

EXAMPLE 20

(3'R,5'R,7'R)-2-amino-6-chloro-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}purine (20)

To a suspension of sugar 7 (1.75 g, 3.85 mmol) and 2-amino-6-chloropurine (1.05 g, 6.17 mmol) in dry MeCN (20 mL) was added BSA (3.80 mL, 15.4 mmol) at rt. The suspension was heated to 55 °C and stirred for 30 min. TMSOTf (1.05 mL, 5.78 mmol) was then added dropwise and the solution was stirred for an additional 50 min at 55 °C. The solution was cooled to rt, quenched by adding saturated NaHCO ₃ (10 mL), diluted with EtOAc (50 mL), and filtered through a small pad of SiO ₂ . The SiO ₂ was washed with additional EtOAc. The mixture was then washed with sat. NaHCO ₃ (2 × 80 mL), the aqueous phases were combined and extracted with EtOAc (3 × 50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (2.5% MeOH in DCM) to give a mixture of 20 (1.77 g, 77%) with an anomer ratio α/β ≈ 7:3 as a white foam.

Data for 20: R _f = 0.54 (EtOAc/hexane 5:1);

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.86 (s, 0.3H, H-C(8)), 7.69 (s, 0.7H, H-C(8)), 7.68 -7.60 (m, 4H, H-arom.), 7.47-7.34 (m, 6H, H-arom.), 6.04 (dd, J=6.9, 3.0 Hz, 0.7Н, Н-С(Г)), 5.87 (dd, J=8.0, 6.2 Hz, 0.3Н, Н-С(1')), 5.37 (dt, J= 14.2, 4.6 Hz, 1H, H-C(5')), 5.16 (lat, 2H, 1H2), 4.91 (dd, J=6.5, 5.1 Hz, 0.7Н, Н-С(4')), 4.79 (dd, J=6.9, 5.2 Hz, 0.3Н, Н-С(4')), 4, 13 (broad, 0.3Н, Н-С(7')), 4.06 (d, J=4.0 Hz, 0.7Н, Н-С(7')), 2.95 (dd, J=16.3, 6.6 Hz, 0.7H, N-C(3')), 2.81 (dd, J=17.0, 7.4 Hz, 0.3H, N-C(3 ')), 2.49-2.30 (m, 1H, H-C(2')), 2.14 (dd, J=13.1, 6.7 Hz, 1H, H-C(6' )), 2.08 (s, 2.1H,MeCO ₂ ), 2.02 (s, 0.9H, MeC02), 2.02-1.91 (m, 1H, H-C(6')), 1.80 (td, J= 13.4, 6.8 Hz, 1H, H-C(2')), 1.07, 1.06 (2s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.55, 170.44 (MeCO ₂ ), 158.98, 158.91 (C(2)), 153.18, 152.95 (C(4)), 151.40, 151.34 (C(6)), 140.38, 140.14 (C(8)), 135.73, 135.70 (CH-arom.), 133.78, 133.62, 133.24, 133.17 (C-arom.), 130.03, 130.00, 127.88, 127.86 (CH-arom.), 125.65, 125.57 (C(5)), 86.59, 85.74 (C(G)), 82.93, 80.99 (C(4')), 76.57, 76.14 (C(7')), 74.34, 74.32 (C(5')), 51.15, 51.10 (C(3')), 37.19, 36.99 (C(6')), 36.70, 36.25 (C(2')), 26.87 ( _CH3 ) ₃ -C-Si), 20.95, 20.86 ( _MeCO2 ), 19.00 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₃₀ H ₃₅ O ₄ N ₅ ClSi ([M+H] ⁺ ) 592.2141, experiment 592.2158.

EXAMPLE 21

(3'R,5'R,7'R)-2-amino-6-chloro-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2,,3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}purine (22b)

Nucleoside 20 (1.78 g, 3.01 mmol) was dissolved in 0.5 M NaOH in THF/methanol/ _H2O (5:4:1, 15 mL) at 0 °C. The reaction mixture was stirred for 20 min at 0 °C and quenched by adding _NH4Cl (484 mg). The suspension was then diluted with saturated _NaHCO3 (100 mL) and extracted with DCM (4×75 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM) to give 21 (428 mg, 25%) and the corresponding a-anomer (992 mg, 60%) as a white foam.

Data for 21: R _f = 0.43 (5% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 7.71 (s, 1H, H-C(8)), 7.68-7.60 (m, 4H, H-arom), 7.44 -7 .33 (t, 6H, H-arom.), 5.85 (dd, J=9.3, 5.8 Hz, 1H, H-C(1')), 5.33 (br., 2H, ~NH2), 4.62 (dd, J=8.4, 4.9 Hz, 1H, H-C(4')), 4.44 (dd, J=10.7, 5.3 Hz, 1H , H-C(5')), 4.40-4.15 (m, 2H, H-C(7'), OH), 2.79 (q, J=8.7 Hz, 1H, H-C(3')), 2.22 (dd, J=15.2, 9.3 Hz, 1H, H-C(6')), 2.11-2.02 (m, 1H , H-C(6')), 2.02-1.85 (m, 2H, H-C(2')), 1.06 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 158.73 (C(2)), 152.78 (C(4)), 151.94 (C(6)), 140.70 (C(8)), 135.70 (CH-arom.), 133.91, 133.48 (C-arom.), 129.90, 127.78 (CH-arom.), 125.97 (C(5)), 87.96 (C(T)), 82.88 (C(5')), 76.85 (C(7')), 72.36 (C(5')), 50.41 (C(3')), 41.96 (C(6')), 35.73 (C(2')), 26.90 (CH ₃ ) ₃ -C-Si), 19.02 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₈ H ₃₃ O ₃ N ₅ ClSi ([M+H] ⁺ ) 550.2036, experiment 550.2015.

EXAMPLE 22

(3'R,5'R,7'R)-N2-(N,N-dimethylformamidino)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-D-ribofuranosyl}guanine (22)

To a solution of 21 (380 mg, 0.645 mmol) and 3-hydroxypropanenitrile (0.22 mL, 3.23 mmol) in dry DCM (15 mL) was added 1,5,7-triazabicyclo[4.4.0]dec-5-ene (400 mg, 2.87 mmol) at 0 °C. The solution was stirred for 3 h at 0 °C and then for 2 days at room temperature. The reaction was quenched by adding silica. After evaporation of the solvent, the SiO ₂ powder was filtered off, washed with MeOH, and the solvent was evaporated to give a brown foam.

The crude product was dissolved in dry DMF (5 mL) and N,N-dimethylformamide dimethyl acetal (0.43 mL, 3.2 mmol) was added. The solution was stirred for 2 h at 55 °C and then the solvents were removed under reduced pressure. The crude product was purified by CX (6% MeOH in DCM) to give 23 (274 mg, 73%) as a yellowish foam.

Data for 22: R _f = 0.45 (12% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.52 (s, 1H, NH), 8.46 (s, 1H, NC#N(CH3)2), 7.63 (dd, J=7.7 , 1.5 Hz, 4H, H-arom.), 7.50 (s, 1H, HC(8)), 7.44-7.30 (m, 6H, H-arom.), 5.83 ( dd, J=9.3, 6.0 Hz, 1H, Н-С(Г)), 4.61 (dd, J=8.7, 5.0 Hz, 1H, Н-С(4')) , 4.43-4.32 (m, 1H, H-C(5')), 4.29 (dd, J=7.0, 4.8 Hz, 1H, H-C(7')), 3.95 (d, J=5.1 Hz, 1H, OH), 2.98 (s, 6H, NCHN(CH ₃ ) ₂ ), 2.79 (dd, J =18.0, 7.0 Hz, 1H, Н-С(3')), 2.20 (dt,J=12.8, 5.4 Hz, 1H, Н-С(6')), 2 .09-1.88 (m, 3H, H-C(b'), H-C(2')), 1.05 (s, 9H, (CH ₃ ) ₃ -C-Si)).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 158.73 (C(2)), 157.79 (C(6)), 156.91 (NCHN(CH ₃ ) ₂ ), 149.84 (C(4)), 137.00 (C(8)), 135.70, 135.67 (CH-arom.), 133.78, 133.60 (C-arom.), 129.93, 129.86, 127.78, 127.72 (CH-arom.), 121.61 (C(5)), 88.04 (C(H)), 82.21 (C(4')), 77.49 (C(7')), 71.94 (C(5')), 50.13 (C(3')), 42.23 (C(6')), 41.20 (NCHN( _CH3 ) ₂ ), 35.50 (C(2')), 34.97 (NCHN( _CH3 ) ₂ ), 26.87 ( _CH3 ) ₃ -C-Si), 19.02 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₃₁ H ₃₈ O ₄ N ₆ Si ([M+H] ⁺ ) 586.2718, experiment 586.2703.

EXAMPLE 23

(3'R,5'R,7'R)-M2-(N,N-dimethylformamidino)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (23)

To a solution of 22 (139 mg, 0.237 mmol) in dry pyridine (2 mL) was added DMTr-Cl (240 mg, 0.708 mmol) in six portions over 3 h at room temp. After stirring overnight, the orange solution was diluted with saturated NaHCO ₃ (20 mL) and extracted with DCM (3 X 20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CHX (4% MeOH in DCM, + 0.5% Et ₃ N) to give 23 (148 mg, 70%) as a yellowish foam.

Data for 23: R _f = 0.52 (10% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.49 (s, 1H, NH), 8.38 (s, 1H, NCHN(CH ₃ ) ₂ ), 7.80 (s, 1H, C(8) ), 7.50-7.43 (m, 2H, H-arom.), 7.42-7.27 (m, 10H, H-arom.), 7.26-7.15 (m, 6H, H-arom), 7.14-7.08 (m, 1H, H-arom), 6.77-6.68 (m, 4H, H-arom), 5.78 (dd, J= 8.2, 5.9 Hz, 1H, H-C(1')), 4.25 (dt, J=11.0, 5.6 Hz, 1H, HC(5')), 4.14-4.03 (m, 1H, HC(4')), 3.70-3.64 (m, 7H, MeO, HC(7 ')), 3.00 (s, 3H, NCHN(CH ₃ ) ₂ ), 2.97 (s, 3H, NCHN(CH ₃ ) ₂ ), 2.43 (dd, J=16.7, 7, 5 Hz, 1H, HC(3')), 2.24 (ddd, J=13.3, 10.1, 5.8 Hz, 1H, HC(2')), 1.62 (td, J= 13.1, 4.3 Hz, 1H, HC(6')), 1.43 (dt, J=13.5, 8.0 Hz, 1H, HC(2')), 0.99 (dd, J=13.3, 6.2 Hz, 1H), 0.86 (s, 9H, (CH ₃ ) ₃ -C-Si)).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 158.51, 158.49 (MeO-C-aromatic), 158.04 (C(2)), 157.91 (C(6)), 156.60 (NCHN(CH ₃ ) ₂ ), 149.76 (C(4)), 145.83, 137.12, 136.94 (C-aromatic), 136.01 (C(8)), 135.60 , 135.59 (CH-aromatic), 133.81, 133.47 (C-aromatic), 130.32, 130.26, 129.77, 128.24, 127.82, 127.65, 127.62, 126.67 (CH-arom.), 120.65 (C(5)), 113.13, 113.09 (CH-arom.), 86.82 (C(Ph) ₃ ), 85 ,01 (C(1')), 82.26 (C(4')), 76.14 (C(7')), 74.61 (C(5')), 55.19 (MeO-DMTr ), 50.18 (C(3')), 41.29 (NCHN(CH ₃ ) ₂ ), 38.01 (C(6')), 37.76 (C(2')), 35.14 (NCHN(CH ₃ ) ₂ ) 26.81 87 (CH ₃ ) ₃ -C-Si), 19.01 ( _CH3 ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₅₂ H ₅₇ O ₆ N ₆ Si ([M+H] ⁺ ) 889.4103, experiment 889.4128.

EXAMPLE 24

(3'S,5'R,7'R)-M2-(N,N-dimethylformamidino)-9-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (24)

To a solution of 23 (243 mg, 0.273 mmol) in dry THF (2 mL) was added TBAF (1 M in THF, 1.65 mL, 1.63 mmol) at rt. The solution was stirred for 7 h and then diluted with sat. NaHCO ₃ (30 mL) and extracted with DCM (4 × 30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (7% MeOH in DCM, + 0.5% Et ₃ N) to give 24 (155 mg, 87%) as a white foam retaining traces of TBAF.

Data for 24: R _f = 0.44 (10% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.55 (s, 1H, NH), 8.45 (s, 1H, NCHN(CH ₃ ) ₂ ), 8.00 (s, 1H, H-C( 8)), 7.60-7.50 (m, 2H, N-arom.), 7.49-7.39 (m, 4H, N-arom.), 7.31-7.23 (m, 2H, H-arom.), 7.21-7.12 (m, 1H, H-arom.), 6.81 (d,J=8.5 Hz, 4H, H-arom.), 5.93 (dd, J=7.5, 6.1 Hz, 1H, H-C(1')), 4.26 (dt, J=11.1, 5.8 Hz, 1H, H-C(5')), 4.07-3.98 (m, 1H, H-C(4')), 3.91 ( d, J=4.3 Hz, 1H, H-C(7')), 3.77 (s, 6H, MeO), 3.14 (s, 3H, NCHN(CH ₃ ) ₂ ), 3.04 (s, 3H, NCHN(CH ₃ ) ₂ ), 2.73 (ddd, J=13.3, 10.1, 6.0 Hz, 1H, H-C(2')), 2.63-2 ,48 (m, 1H, H-C(3')), 2.12 (br., 1H, OH), 1.95- 1.82 (m, 2H, Н-С(b'), Н-С(2')), l,14(dd,J=13.4, 6.1 Hz, 1H, Н-С(6')).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 158.52 (MeO-C-arom.), 158.12 (C(2)), 157.88 (C(6)), 156.65 (NCHN(CH ₃ ) ₂ ), 149.78 (C(4)), 145.69, 137.02, 136.99 (C-arom.), 136.07 (C(8)), 130.26, 128.26, 127.82, 126.74 (CH-arom.), 120.53 (C(5)), 113.12 (CH-arom.), 86.81 (C(Pb)3), 85.35 (C(1')), 82.64 (С(4')), 74,61 (С(7')), 74,48 (С(5')), 55,23 (MeO-DMTr), 49,63 (С(3')), 41,37 (NCHN(CH ₃ ) ₂ ), 38,55 (С(6')), 38,23 (С(2')), 35,14 (NCHN(CH ₃ ) ₂ ).

ESI ⁺ -MSHR m/z, calculation for C ₃₆ H ₃₉ O ₆ N ₆ ([M+H] ⁺ ) 651.2926, experiment 651.2912.

EXAMPLE 25

(3'R,5'R,7'R)-N2-(N,N-dimethylformamidino)-9-{7'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}guanine (25)

To a solution of nucleoside 24 (143 mg, 0.220 mmol) and 5-(ethylthio)-1H-tetrazole (43 mg, 0.33 mmol) in dry DCM (10 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.12 mL, 0.38 mmol) at room temp. After stirring for 50 min, the reaction mixture was diluted with saturated NaHCO ₃ (20 mL) and extracted with DCM (3 × 20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (3.5% MeOH in DCM, +0.5% _Et3N ) to give 25 (130 mg, mixture of two isomers, 69%) as a white foam.

Data for 25: R _f = 0.60 (10% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.54, 9.47 (2s, 1H, NH), 8.54, 8.52 (2s, 1H, NC#N(CH3)2), 8.02 , 8.00 (2s, 1H, HC(8)), 7.58-7.49 (m, 2H, H-arom), 7.46-7.36 (m, 4H, H-arom) , 7.25 (dd, J=11.0, 3.5 Hz, 2H, H-arom.), 7.21-7.13 (m, 1H, H-arom.), 6.80 (dd, J=8.8, 2.2 Hz, 4H, H-aromatic), 6.00-5.82 (m, 1H, HC(1')), 4.16 (dd, J=10.7, 5.4 Hz, 1H, HC(5')), 4.00-3.82 (m, 2H, HC(4') , HC(7')), 3.77, 3.77 (2s, 6H,MeO), 3.62 (dt, J=12.2, 6.1 Hz, 2H, OCH ₂ CH ₂ CN), 3 .51-3.33 (m, 2H, (Me ₂ CH) ₂ N), 3.15, 3.14 (2s, 3H, NCHN(CH ₃ ) ₂ ), 3.07 (s, 3H, NCHN( CH ₃ ) ₂ ), 2.85-2.61 (m, 2H, C(2'), C(3')), 2.59-2.44 (m, 2H, OCH ₂ CH ₂ CN), 2.00-1.79 (m, 2H, H-(C2'), HC(6')), 1.53-1 .26 (m, 1H, HC(6')), 1.10, 1.01 (2t, J=6.4 Hz, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 158.50 (MeO-C-aromatic), 158.04, 158.00 (C(2)), 157.93 (C(6)), 156.61 , 156.60 (NCHN(CH ₃ ) ₂ ), 149.73, 149.72 (C(4)), 145.62, 145.62, 136.97, 136.94 (C-aromatic), 136 ,14(C(8)), 130.27, 130.24,130.22, 128.26,127.81, 126.73 (CH-aromatic), 120.81, 120.76 (C(5)), 117.67, 117.56 (OCH ₂ CH ₂ CN), 113.10 (CH-arom), 86.88, 86.85 (C(Pb)z), 85.58 , 85.37 (C(1')), 82.41, 82.07 (C(4')), 77.08, 76.01 (J _C,P =37.0, 15.1 Hz, C (7')), 74.52, 74.46 (C(5')), 58.19, 57.74 (J _C,P = 18.9, 19.0 Hz OCH ₂ CH ₂ CN), 55 ,25, 55.21 (MeO-DMTr), 49.10, 48.83 (J _C,P =2.2, 4.8 Hz, C(3')), 43.12, 43.00 ((Me ₂ CH) ₂ N), 41.34, 41.33 (NCHN(CH ₃ ) ₂ ), 38.48, 38.41 (C(2'))s 37.23, 36.92 (J _C,P =5.7, 3.3 Hz C(6')), 35.17 ((Me ₂ CH) ₂ N), 24.56, 24.53, 24.48, 24.47, 24.43, 25.36, 24.35 (7s, Me ₂ CH) ₂ N), 20.39, 20, 28 (J _C,P =7.1, 6.9 Hz, OCH ₂ CH ₂ CN).

³¹ P NMR (122 MHz, CDCl ₃ ) δ 147.69, 146.37.

ESI ⁺ -MSHR m/z, calculation for C ₄₅ H ₅₆ O ₇ N ₈ P ([M+H]+) 851.4004, experiment 851.4018.

EXAMPLE 26

(3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-β-B-ribofuranosyl}uracil (26)

To a solution of sugar 6 (669 mg, 1.62 mmol) in dry DCM (13 mL) was added 2,6-lutidine (0.94 mL, 8.10 mmol) at 0 °C. After stirring for 20 min at 0 °C, TMSOTf (0.89 mL, 4.86 mmol) was added dropwise and then the solution was allowed to warm to rt and stirred for an additional 3 h. The reaction was then quenched by adding saturated NaHCO ₃ (20 mL). The organic phase was separated and the aqueous phase was further extracted with DCM (2 × 20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was dissolved in dry DCM (12 mL) and then uracil (545 mg, 4.86 mmol) and BSA (1.8 mL, 7.29 mmol) were added at room temp. After stirring for 60 min at room temp, the resulting fine suspension was cooled to 0 °C and N-iodosuccinimide (578 mg, 2.52 mmol) was added. After stirring for 30 min at 0 °C and 4 h at room temp, the reaction mixture was diluted _{with EtOAc (50 mL) and then washed with 10% aq. Na2S2O3 ( 30} mL) and sat. _NaHCO3 (30 mL). The aqueous phases were combined and extracted with DCM (2 × 20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was dissolved in dry toluene (15 mL) and then _Bu3SnH (0.65 mL, 2.43 mmol) and azoisobutyronitrile (AIBN, 13 mg, 0.081 mmol) were added at rt. After heating at 95 °C for 2 h, the mixture was cooled to rt and MeOH (7 mL) and HCl (1 M in water, 1.6 mL, 1.6 mmol) were added. The solution was stirred for an additional 15 min and then diluted with sat. _NaHCO3 (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by chromatography (EtOAc/hexane 4:1) to give 26 (490 mg, 61% over three steps) as a white foam.

Data for 26: R _f = 0.15 (EtOAc/hexane 2:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.95 (br, 1H, HN(3)), 7.69 (d, J=6.4 Hz, 4H, H-arom), 7.54 -7.39 (m, 7H, H-C(6), H-arom), 5.98 (dd, J=9.3, 5.6 Hz, 1H, H-C(1')), 5.71 (d, J=8.1 Hz, 1H, H-C(5)), 4.51 (dd, J=13.7, 6.3 Hz, 2H, H-C(4'), H-C(5')), 4.14 (lat, 1H, H-C(7')), 3.25 (lat, 1H, OH), 2.74 (dd, J=17.1 , 8.7 Hz, 1H, H-C(3')), 2.26-1.87 (m, 3H, H-C(2'), H-C(6')), 1.49-1 .19 (m, 1H, H-C(2')), 1.12 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 163.65 (C(4)), 150.46 (C(2)), 139.85 (C(6)), 135.69, 135.66 (CH-arom.), 133.71, 133.42 (C-arom.), 129.98, 129.93, 127.85, 127.81 (CH-arom.), 102.84 (C(5)), 86.17 (C(1')X 81.83 (C(4')), 76.94 (C(7')), 72.45 (C(5')), 50.09 (C(3')), 40.93 (C(6')), 35.83 (C(2')), 26.91 (CH ₃ ) ₃ -C-Si), 19.03 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₂₇ H ₃₂ O ₅ N ₂ NaSi ([M+Na] ⁺ ) 515.1973, experiment 515.1963.

EXAMPLE 27

(3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4,-dimethyltriphenyl)methyl]-β-D)-ribofuranosyl}uracil (27)

To a solution of nucleoside 26 (438 mg, 0.889 mmol) in dry pyridine (7 mL) was added DMTr-Cl (1.20 g, 3.55 mmol) at rt. The solution was stirred for 1 day at rt and then diluted with saturated NaHCO ₃ (30 mL) and extracted with DCM (3 × 40 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (1.5% MeOH in DCM, + 0.5% Et ₃ N) to give 27 (601 mg, 80%) as a yellow foam.

Data for 27: R _f = 0.48 (EtOAc/hexane 2:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.26 (br, 1H, HN(3)), 7.84 (d, J=8.1 Hz, 1H, H-C(6)), 7 ,40-7.08 (m, 19Н, Н-aromatic), 6.69 (dd, J=8.8, 4.9 Hz, 4H, Н-aromatic), 5.70 (dd, J= 7.8, 5.8 Hz, 1H, HC(1')), 5.49 (dd, J=8.1, 1.5 Hz, 1H, HC(5)), 4.24-4.11 (m, 1H, HC(5')), 4.05-3.95 (m, 1H, HC(4')), 3.65 (d, J=1.7 Hz, 6H, MeO), 3.62 (d, J=3.0 Hz, 1H, HC(7')), 2.41 (dd, J=17.2, 8.5 Hz, 1H, HC(3' )), 2.24 (ddd, J=13.5, 10.2, 5.7 Hz, 1H, HC(2')), 1.39-1.24 (m, 1H, HC(6') ), 1.04 (dd, J=13.1, 5.7 Hz, 1H, HC(6')), 0.89 (dt, J=13.8, 8.3 Hz, 1H, HC(2 ')), 0.81 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 163.58 (C(4)), 158.66 (MeO-C-arom.), 150.38 (C(2)), 145.61 (C-arom.), 139.92 (C(6)), 136.71, 136.56(C-arom.), 135.61, 135.55 (CH-arom.), 133.55, 133.41 (C-arom.), 130.30, 129.92, 129.84, 128.16, 127.90, 127.74, 127.67, 126.90, 113.19, 113.15 (CH-arom.), 102.12 (C(5)), 87.41 (C(Ph) ₃ ), 86.80 (C(1')), 82.32 (C4')), 75.54 (C(7')), 74.41 (C(5')), 55.23 (MeO-DMTr), 50.05 (C(3')), 38.49 (C(b')), 37.5 3 (C(2')), 26.81 (CH ₃ ) ₃ -C-Si), 18.99 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₄₈ H ₅₀ O ₇ N ₂ NaSi ([M+Na] ⁺ ) 817.3279, experiment 817.3286.

EXAMPLE 28

(3'S,5'R,7'R)-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4,-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (28)

To a suspension of 1,2,4-triazole (1.83 g, 26.5 mmol) in dry MeCN (70 mL) at 0 °C was added POCl ₃ (0.57 mL, 6.05 mmol) followed by Et ₃ N (4.2 mL, 30.2 mmol). The suspension was stirred for 30 min at 0 °C and then a solution of nucleoside 27 (601 mg, 0.756 mmol) in dry MeCN (4 mL) was added at 0 °C. After stirring for 4 h at rt, the reaction was quenched by addition of sat. NaHCO ₃ (20 mL), MeCN was removed under reduced pressure and the resulting mixture was diluted with sat. NaHCO ₃ (30 mL) and extracted with DCM (3 × 60 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (18 mL) and conc. NH4OH (18 mL). After stirring for 3 h at room temp, the mixture was concentrated to half volume in vacuo, diluted with _{saturated NaHCO3} (30 mL) and extracted with DCM (3 x 30 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (5% MeOH in DCM, +0.5% _Et3N ) to give 28 (520 mg, 87%) as a white foam.

Data for 28: R _f = 0.41 (10% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.96 (d, J=7.4 Hz, 1H,H-C(6)), 7.45 (d, J=7.4 Hz, 2H, H -arom.), 7.38-7.08 (m, 17H, H-arom.), 6.73 (dd, J=8.7, 4.7 Hz, 4H, H-arom.), 5, 73 (t, J=8.6 Hz, 2H, H-C(5), H-C(1')), 4.32-4.16 (m, 1H, H-C(5')), 4.03 (t, J=5.6 Hz, 1H, H-C(4')), 3.66 (d, J=0.9 Hz, 6H,MeO), 3.61 (d, J=2.9 Hz, 1H, H-C(7')), 2.50-2.33 (m, 2H, H-C(2'), H-C(3')), 1.47 -1.28 (m, 1H, H-C(6')), 1.03 (dd, J=12.9, 5.6 Hz, 1H, H-C(6')), 0.92- 0.75 (m, 10H, H-C(2'), (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCb) 8 165.78 (C(4)), 158.59 (MeO-C-arom.), 155.94 (C(2)), 145.88 (C-arom.), 140.68 (C(6)), 136.93, 136.78 (C-arom.), 135.59, 135.53 (CH-arom.), 133.60, 133.54 (C-arom.), 130.31, 129.86, 129.77, 128.15, 127.88, 127.71, 127.64, 126.79, 113.18, 113.14 (CH-arom.), 94.53 (C(5)), 87.55 (C(Ph) ₃ ), 87.22 (C(1')), 82.23 (C(4')), 75.76 (C(7')), 74.68 (C(5')), 55.21 (MeO-DMTr), 50.18 (C(3')), 38.25 (C(b')), 38.0 8 (C(2')), 26.83 (CH ₃ ) ₃ -C-Si), 19.00 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₄₈ H ₅₂ O ₆ N ₃ Si ([M+H] ⁺ ) 794.3620, experiment 794.3649.

EXAMPLE 29

(3'S,5'R,7'R)-N4-benzoyl-1-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4,-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (29)

To a solution of nucleoside 28 (519 mg, 0.653 mmol) in dry DMF (15 mL) was added _Et3N (110 µL, 0.784 mmol) followed by _BZ2O (370 mg, 1633 mmol) at rt, and the solution was stirred overnight. The reaction in solution was then quenched by careful addition of sat. _NaHCO3 (60 mL) and extracted with DCM (3×70 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CC (hexane/EtOAc 2:3, +0.5% _Et3N ) to give 29 (580 mg, 99%) as a white foam.

Data for 29: R _f = 0.51 (EtOAc);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.61 (d, J=7.4 Hz, 1H, H-C(6)), 7.81 (d, J=7.5 Hz, 2H, H -arom.), 7.49-7.13 (m, 24H, H-arom., H-C(5)), 6.77 (dd, J=8.5, 4.4 Hz, 4H, H -arom.), 5.73 (t, J=6.4 Hz, 1H, HC(l')), 4.39-4.20 (m, 1H, HC(5')), 4.05 ( t, J=6.1 Hz, 1H, HC(4')), 3.70 (s, 6H,MeO), 3.63 (d, J=2.3 Hz, 1H, HC(7')), 2.72-2.55 (m, 1H, HC(2')), 2.48 (dd,J=16.0, 8.4 Hz, 1H, HC(3') ), 1.42-1.29 (m, 1H, HC(6')), 1.19-1.11 (m, 1H, HC(6')), 1.07-0.96 (m, 1H, HC(2')), 0.85 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 166.64 (CONH), 162.25 (C(4)), 158.70 (MeO-C-aromatic), 154.84 (C(2)), 145.71 (C-aromatic), 144.84 (C(6)), 136.74, 136.67 (C-aromatic), 135.59, 135.51 (CH-aromatic), 133, 52, 133.42, 133.24 (C-aromatic), 133.11, 130.30, 129.92, 129.85,129.02,128.12, 127.97, 127.76, 127.68, 127.61, 126.94, 113.25, 113.22(CH-arom), 96.22 (C(5)), 89.07 (C(Ph) ₃ ), 87.53 (C(1')), 83.46 (C(4')), 75.59 (C(7')), 74.71 (C(5')), 55.24 (MeO-DMTr), 50.35 (C(3')), 38.61 (C(b')), 38.15 (C(2')), 26.82 (CH ₃ ) ₃ -C-Si), 19.00 (CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₅₅ H ₅₆ O ₇ N ₃ Si ([M+H] ⁺ ) 898.3882, experiment 898.3898.

EXAMPLE 30

(3'S,5'R,7'R)-N4-benzoyl-1-{-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (30)

To a solution of 29 (580 mg, 0.648 mmol) in dry THF (14 mL) was added TBAF (1 M in THF, 3.25 mL, 3.25 mmol) at rt. The solution was stirred for 1 day and then diluted with saturated NaHCO ₃ (50 mL) and extracted with DCM (3 x 40 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM, + 0.5% Et ₃ N) to give 30 (366 mg, 85%) as a white foam.

Data for 30: R _f = 0.31 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.90 (br, 1H, NH), 8.73 (d, J=7.5 Hz, 1H, H-C(6)), 7.82 ( d, J=7.3 Hz, 2H, H-arom.), 7.55-7.31 (m, JH, H-arom., H-C(5)), 7.28-7.09 ( m, 3H, H-arom.), 6.76 (dd, J=8.8, 1.7 Hz, 4H, H-arom.), 5.73 (t, J=6.3 Hz, 1H, H-C(1')), 4.28-4.13 (m, 1H, H-C(5')), 3.83 (t, J=6.0 Hz, 1H, H-C(4')), 3.75 (d, J=3.6 Hz, 1H, H-C(7')), 3.70 (s, 6H, MeO), 2.86 (d, J=14.7 Hz, 1H, H-C-(2')), 2.54 (dd, J=17.4, 7.4 Hz, 1H, H-C(3')), 1.68 -1.55 (m, 1H, H-C(6')), 1.45-1.13 (m, 3H, H-C(2'), H-C(6'), OH).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 166.63 (CONH), 162.34 (C(4)), 158.65 (MeO-C-arom.), 155.00 (C(2)), 145.62 (C-arom.), 145.11 (C(6)), 136.72, 136.64, 133.16 (C-arom.), 130.25, 129.02, 128.12, 127.93, 127.61, 126.95, 113.20 (CH-arom.), 96.24 (C(5)), 89.20 (C(Ph) ₃ ), 87.48 (C(H)), 83.40 (C(4')), 74.50, (C(5')) 73.90 (C(7')), 55.25 (MeO-DMTr), 50.05 (C(3')), 38.90 (C(b')), 38.40 (C(2')).

ESI ⁺ -MSHR m/z, calculation for C ₃₉ H ₃₈ O ₇ N ₃ ([M+H] ⁺ ) 660.2704, experiment 660.2707.

EXAMPLE 31

(3'S,5'R,7'R)-N4-benzoyl-1-{7'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofuranosyl}cytosine (31)

To a solution of nucleoside 30 (67 mg, 0.101 mmol) and 5-(ethylthio)-1H-tetrazole (22 mg, 0.17 mmol) in dry DCM (3 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (65 μL, 0.20 mmol) at room temp. After stirring for 40 min, the reaction mixture was diluted with DCM (20 mL) and washed with sat. NaHCO ₃ (2 × 15 mL) and sat. NaCl (15 mL). The aqueous phases were combined and extracted with DCM (20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (EtOAc, +0.5% _Et3N ) to give 31 (75 mg, mixture of two isomers, 86%) as a white foam.

Data for 31: R _f = 0.67 (4% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.88 (s, 1H, NH), 8.79 (d, J=7.5 Hz, 1H, H-C(6)), 7.93 (d , J=7.5 Hz, 2H, H-arom.), 7.67-7.40 (m, 10H, H-arom., H-C(5)), 7.39-7.22 (m , 3H, H-arom.), 6.93-6.79 (m, 4H, H-arom.), 5.97-5.77 (m, 1H, H-C(1')), 4, 22 (dt, J=14.5, 5.6 Hz, 1Н, Н-(5')), 3.98-3.84 (m, 2Н, H-C(4'), H-C(7')), 3.82 (s, 6H,MeO), 3.66 (ddd, J=16.8, 13.5, 6.7 Hz, 2H , OCH ₂ CH ₂ CN), 3.53-3.37 (m, 2H, (Me ₂ CH) ₂ N), 3.14-2.93 (m, 1H, H-C(2')), 2.84-2.66 (m, 1H, H-C(3')), 2.53 (dt, J=12.4, 6.3 Hz, 2H, OCH ₂ CH ₂ CN), 1.83 -1.56 (m, 2H, H-C(6')), 1.46 (td, J=14.1, 7.0 Hz, 1H, H-C(2')), 1.18-0.97 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 166.70 (CONH), 162.32, 162.28 (C(4)), 158.68 (MeO-C-arom.), 154.93 (C( 2)), 145.53 (C-arom.), 144.95, 144.89 (C(6)), 136.69, 136.63, 136.56, 136.52, 133.24 (C- arom.), 133.10, 130.24, 130.20, 129.01, 128.10, 127.94, 127.60, 126.96 (CH-arom.), 117.53 (OCH ₂ CH ₂ CN), 113.20 (CH-arom.), 96.24 (C(5)), 89.15, 89.10 (C(Ph) ₃ ), 87.55, 87.54 (C (1')), 83.11, 83.04 (С(4')), 75.93, 75.37 (J _C,P =16.7, 15.5 Hz, С(7')), 74.48 (C(5')), 58.25, 57.99 (J _C,P = 17.9, 18.1 Hz OCH ₂ CH ₂ CN), 55.27, 55.24 (MeO-DMTr ), 49.27, 49.03 (J _C,P =3.1, 4.8 Hz, C(3')), 43.15, 42.98 ((Me ₂ CH) ₂ N), 38.89, 38.80 (C(2')), 37.44, 37.24 (J _C,P = 5.2, 3 ,2 Hz, С(6')), 24.58, 24.54, 24.48, 24.45, 24.35 (5s, Me ₂ CH) ₂ N 20.33, 20.24 (J _{C, P} =5.8, 5.7 Hz, OCH ₂ CH ₂ CN).

³¹ P NMR (121 MHz, CDCl ₃ ) δ 147.19, 146.94.

ESI ⁺ -MSHR m/z, calculation for C ₄₈ H ₅₅ O ₈ N ₅ P ([M+H] ⁺ ) 860.3783, experiment 860.3791.

EXAMPLE 32

(3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-O-(4-nitrobenzoate)-5'-O-[(4,4'-dimethoxytriphenyl)methyl]-β-D-ribofurantyl}thymine (32)

To a solution of nucleoside 11 (100 mg, 0.175 mmol) and 4-dimethylaminopyridine (26 mg, 0.21 mmol) in dry DCM (8 mL) was added 4-nitrobenzoyl chloride (59 mg, 0.315 mmol) at rt. After stirring for 6 h, the reaction was quenched by adding sat. NaHCO ₃ (5 mL). The mixture was then diluted with sat. NaHCO ₃ (15 mL) and extracted with DCM (3 X 15 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (2.5% MeOH in DCM, + 0.5% Et ₃ N) to give 32 (98 mg, 78%) as a white foam containing traces of Et ₃ N.

Data for 32: R _f = 0.42 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.26 (t, J=7.3 Hz, 3H, H-arom., HN(3)), 8.00 (d, J=8.9 Hz, 2H, H-arom.), 7.72 (d, J=1.0 Hz, 1H, H-C(6)), 7.55 (d, J=6.9 Hz, 2H, H-arom. ), 7.44 (dd, J=8.8, 6.6 Hz, 4H, H-arom.), 7.35-7.18 (m, 3H, H-arom.), 6.83 (dd , J=9.0, 2.6 Hz, 4H, H-arom.), 6.01 (dd,J=8.2, 5.2 Hz, 1H, H-C(G)), 4.96 (d,J=3.3 Hz, 1H, H-C(7')), 4.33 -4.24 (m, 1H, H-C(4')), 4.24-4 .13 (m, 1H, H-C(5')), 3.78 (d, J=0.9 Hz, 6H,MeO), 2.92-2.72 (m, 2H, H-C( 3'), H-C(2')), 1.81 (d, J=0.6 Hz, 3H, Me-C(5)), 1.79-1.62 (m, 2H, H- C(6')), 1.22 (d, J=5.9 Hz, 1H, H-C(2')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.05, 163.84 (C(4), C02R), 158.81 (MeO-C-arom.), 150.64, 150.52 (02N-C -apoM., С(2)), 145.29, 136.43, 136.34 (С-aromatic), 135.18 (С(6)), 130.62, 130.20, 130.17, 128.16, 128.01, 127.15, 123.58, 113.30, 113.27 (C-arom), 111.17 (C(5)), 87.53 (C(Pb)z) , 86.29 (C(G)), 81.59 (C(4')), 78.65 (C(7')), 74.16 (C(5')), 55.26 (MeO-DMTr ), 47.07 (C(3')), 37.35 (C(2')), 35.71 (C(b')), 12.51 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₄₀ H ₃₇ O ₁₀ N ₃ Na ([M+Na] ⁺ ) 742.2371, experiment 742.2375.

EXAMPLE 33

((3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-O-(4-nitrobenzoate)-β-D-ribofuranosyl}thymine (33)

To a solution of 32 (60 mg, 0.083 mmol) in a mixture of dry DCM (1 mL) and MeOH (0.4 mL) was added dichloroacetic acid (0.2 mL) dropwise at rt. After stirring for 3 h, the mixture was diluted with saturated NaHCO ₃ (15 mL) and extracted with DCM (3 × 10 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CXC (5% MeOH in DCM) to give 33 (29 mg, 84%) as a white foam. Crystals suitable for X-ray analysis were obtained by recrystallization in H ₂ O/MeOH.

Data for 33: R _f = 0.18 (5% MeOH in DCM);

^1H NMR (400 MHz, DMSO) δ 11.33 (s, 1H, HN(3)), 8.34 (d, J=8.8 Hz, 2H, H-arom), 8.27-8 .13 (m, 2H, H-arom), 7.78 (s, 1H, H-C(6)), 5.96 (dd, J=9.3, 5.6 Hz, 1H, H- C(1')), 5.18 (t, J=3.8 Hz, 1H, H-C(7')), 5.12 (d, J=6.0 Hz, 1H, OH), 4 .33 (dd, J=7.3, 4.7 Hz, 1H, Н-С(4')), 4.27 (td, J=10.5, 5.5 Hz, 1H, H-C(5')), 2.90 (dd, J=17.2, 8.5 Hz, 1H, H-C(3')), 2.58-2.46 (m, 1H, H -С(2')), 2.30 (ddd, J=13.8, 8.8, 5.3 Hz, 1Н, Н-С(6')), 2.03 (dd, J=9, 6, 4.2 Hz, 1H, H-C(6')), 1.92-1.76 (m, 4H, H-C(2'), Me-C(5)).

¹³ C NMR (101 MHz, DMSO) δ 164.33, 164.23 (C(4), C02R), 150.91, 150.75 (02N-C-apoM., C(2)), 136.79 (C-arom.), 135.69 (C(6)), 131.20, 124.32 (CH-arom.), 109.89 (C(5)), 85.31 (C(1') ), 81.48 (C(4')), 80.07 (C(7')), 71.72 (C(5')), 47.18 (C(3')), 37.77 ( С(6')), 35.48 (С(2')), 12.66 12.58 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₁₉ H ₂₀ O ₈ N ₃ ([M+H] ⁺ ) 418.1245, experiment 418.1242.

EXAMPLE 35

(3'R,5'R,7'R)-1-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α,β-D-ribofuranosyl}thymine (35)

To a solution of sugar 7 (933 mg, 2.05 mmol) and thymine (372 mg, 3.08 mmol) in dry MeCN (12 mL) was added dropwise BSA (1.5 mL, 6.15 mmol) at rt. After stirring for 50 min at rt, the solution was cooled to 0 °C and TMSOTf (0.45 mL, 2.5 mmol) was added dropwise. After additional stirring for 3 h at 0 °C and 15 h at rt, the reaction mixture was diluted with saturated NaHCO ₃ (100 mL) and extracted with DCM (4 × 40 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (2.5% isopropanol in DCM) to give mixture 35 (924 mg, 82%) with an anomer ratio α/β ≈ 85:15 as a white foam.

Data for 35: R _f = 0.56 (7% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.14 (br, 1H, HN(3)), 7.53 (dd, J=7.7, 1.6 Hz, 4H, H-arom) , 7.39-7.23 (m, 6H, H-arom.), 7.09 (d, J=1.0 Hz, 0.15 H, H-C(6)), 6.87 (d , J=1.0 Hz, 0.85 N, N-S(6)), 5.83 (t, J=6.2 Hz, 0.85 N, N-S(G)), 5.80 -5.70 (t, 0Д5Н, Н-С(1')), 5.36 -5.04 (m, 1Н, Н-С(5')), 4.89 (dd, J=6.3, 5.2 Hz, 1H, Н-С(4')), 4.62 (dd, J=7.1, 5.6 Hz, 0Д5Н, Н-С(4')), 4.01-3.85 (m, 1H, H-C(7')), 2.76-2.55 (m, 1H, H-C(3')), 2.09-1.91 ( m, 4H, H-C(6'), MeSO ₂ ), 1.90-1.58 (m, 6H, H-C(6'), H-C(2'), Me-C(5) ), 0.96 (s, 9H, (CH ₃ ) ₃ -C-Si).

13CNMR (75 MHz, _CDCl3 ) δ 170.70 ( _MeCO2 ), 163.87 (C(4)), 150.29 (C(2)), 135.69, 135.67 (CH-arom.), 134.99 (C(6)), 133.58, 133.18 (C-arom.), 130.03, 127.87 (CH-arom.), 111.05 (C(5)), 87.56 (C(1')) 82.85 (C(4')), 76.50 (C(7')), 74.76 (C(5')), 50.72 (C(3')), 37.79 (C(6')), 36.94 (C(2')), 26.88 ((CH ₃ ) ₃ -C-Si), 20.95 (MeCO ₂ ), 19.01 ((CH ₃ ) ₃ -C-Si), 12.63 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₀ H ₃₇ O ₆ N ₂ Si ([M+H] ⁺ ) 549.2415, experiment 549.2401.

EXAMPLE 36

(3'S,5'R,7'R)-1-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7,-hydroxy-α,β-D-ribofuranosyl}thymine (36)

To a solution of nucleoside 35 (924 mg, 1.68 mmol) in dry THF (10 mL) was added TBAF (1 M in THF, 3.4 mL, 3.4 mmol) at rt. After stirring for 2 h at rt, the reaction mixture was diluted with saturated NaHCO ₃ (80 mL) and extracted with EtOAc (3 X 80 mL) and DCM (2 X 80 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (5% MeOH in DCM) to give a mixture of anomers 36 (391 mg, 75%).

Data for 36: R _f = 0.24 (7% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.66 (br, 0.15H, HN(3)), 9.63 (br, 0.85H, HN(3)), 7.27 (d , J=1.0 Hz, 0.15Н, Н-С(6)), 7.06 (d, J=1.0 Hz, 0.85Н, Н-С(6)), 6.00 (t , J=6.1 Hz, 0.85Н, Н-С(1')), 5.91 (dd, J=8.8, 5.5 Hz, 0.15Н, Н-С(1')) , 5.26-5.10 (m, 1H, Н-С(5')), 4.92 (dd, J=6.5, 5.3 Hz, 0.85Н, H-C(4')), 4.65 (dd, J=6.9, 5.7 Hz, 0.15H, H-C(4')), 4.19-4.03 (m, 1H , H-C(7')), 2.91-2.72 (m, 2H, H-C(3'), OH), 2.64 (ddd, J=13.3, 9.8, 5 .5 Hz, 0.15Н, Н-С(2')), 2.25-2.15 (m, 1.70Н, Н-С(2')), 2.05 (s, 0.45Н, MeCO ₂ ), 2.04 (s, 2.55H, MeCO ₂ ), 2.03-1.89 (m, 2H, H-C(6')), 1.88 (d, J=0.7 Hz, 0.45H, Me-C(5)), 1.85 (d, J=0.6 Hz, 2.55H, Me-C(5)), 1.42-1.28 (m, 0 ,15Н, Н-С(2')).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.87 (MeCO ₂ ), 164.26 (C(4)), 150.66 (C(2)), 135.54 (C(6)), 111.22 (C(5)), 87.97 (C(1'), 82.97 (C(4')), 75.08 (C(7')), 74.52 (C(5')), 50.07 (C(3')), 37.81 (C(2')), 37.23 (C(6')), 21.02 (MeCO ₂ ), 12.67 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₁₄ H ₁₉ O ₆ N ₂ ([M+H] ⁺ ) 311.1238, experiment 311.1234.

EXAMPLE 37

(3'S,5'R,7'R)-1-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α,β-D-ribofuranosyl}thymine (37)

To a solution of nucleoside 36 (364 mg, 1.17 mmol) in dry pyridine (7 mL) was added DMTr-Cl (1.19 g, 3.51 mmol) at room temp. The solution was stirred for 1 day and then diluted with saturated NaHCO ₃ (50 mL) and extracted with DCM (3 X 50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CHX (EtOAc/hexane 2:1, +0.5% Et ₃ N) to give a mixture of anomers 37 (690 mg, 96%) as a yellow foam.

Data for 37: R _f = 0.70 (8% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.17 (br, 0.85H, HN(3)), 8.56 (br, 0.15H, HN(3)), 7.38-7 .32 (m, 2H, H-arom.), 7.29-7.15 (m, 7H, H-arom.), 6.82 (d, J=1.1 Hz, 1H, H-C( 6)), 6.76 (d, J=8.9 Hz, 4H, H-arom.), 5.86 (t, J=6.0 Hz, 0.85H, H-C(1')) , 5.71 (dd, J=8.9, 5.4 Hz, 0.15H, H-C(1')), 5.25 (dd, J=10.2, 5.6 Hz, 0.15Н, Н-С(5')), 5.21-5.11 (m, 0.85Н, Н-(С5')), 4.78 (dd, J=6, 7, 4.8 Hz, 0.85Н, Н-С(4')), 4.49 (dd, J=7.1, 5.3 Hz, 0.15Н, Н-С(4')), 3.84 (br., 1H, H-C(7')), 3.72, 3.71 (2s, 6H, MeO), 2.34-2.23 (m, 1H, H-C(3 ')), 2.01, 1.99 (2s, 3H, MeCO ₂ ), 1.82 (d, J=0.5 Hz, Me-C(5)), 1.80-1.56 (m, 4H, H-C(2'), H-C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.69 (MeCO ₂ ), 163.91 (C(4)), 158.82 (MeO-C-arom.), 150.33 (C(2)), 145.34, 136.64, 136.58 (C-arom.), 135.00 (C(6)), 130.25, 128.39, 128.07, 127.15, 113.41 (CH-arom.), 111.04 (C(5)), 87.70 (C(Ph) ₃ ), 87.31 (C(1')), 83.15 (C(4')), 77.16 (C(7')), 74.96 (C(5')), 55.37 (MeO-DMTr), 49.12 (C(3')), 37.55 (C(2')), 36.82 (C(6')), 21.07 (MeCO ₂ ), 12.66 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₅ H ₃₆ O ₈ N ₂ ([M+H] ⁺ ) 612.2466, experiment 612.2453.

EXAMPLE 38

(3'S,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7,-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}thymine (38)

To a solution of nucleoside ₃₇ (690 mg, 1.12 mmol) in dry MeOH (10 mL) was added _K2CO3 (467 mg, 3.36 mmol) at room temp. The solution was stirred for 3 h and then diluted with sat. NaCl (60 mL) and extracted with DCM (3 X 60 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% isopropanol in _Et2O , +0.5% _Et3N ) to give the α-anomer 38 (550 mg, 86%) as a white solid.

Data for 38: R _f = 0.39 (5% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.37 (br, s, 1H, HN(3)), 7.39-7.31 (m, 2H, H-arom), 7.25 ( d, J=8.3 Hz, 4H, H-arom.), 7.20 (t, J=1.1 Hz, 2H, H-arom.), 7.16-7.08 (m, 1H, H-arom), 6.78 (d, J=1.1 Hz, 1H, H-C(6)), 6.74 (d, J=8.8 Hz, 4H, H-arom), 5.91 (dd, J=6.5, 4.9 Hz, 1H, H-C(1')), 4.57 (dd, J=7.2, 4.4 Hz, 1H, H-C(4')), 4.35-4.18 (m, 1H, H-C(5')), 3.86 (d, J=4.7 Hz, 1H, H-C (7')), 3.69 (s, 6H, MeO), 2.53 (br, 1H, OH), 2.22 (dd, J=15.3, 6.3 Hz, 1H, H- C(3')), 1.85-1.69 (m, 5H, Me-C(5), H-C(2'), H-C(6')), 1.66-1.49 (m, 2H, H-C(2'), H-C(6')).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 163.98 (C(4)), 158.67 (MeO-C-arom.), 150.47 (C(2)), 145.48, 136.80, 136.75 (C-arom.), 134.94 (C(6)), 130.19, 130.18, 128.35, 127.97, 127.01, 113.31 (CH-arom.), 111.04 (C(5)), 87.82 (C(Ph) ₃ ), 87.05 (C(1')), 85.74 (C(4')), 78.26 (C(7')), 73.33 (C(5')), 55.31 (MeO-DMTr), 48.81 (C(3')), 40.21 (C(6')), 37.68 (C(2')), 12.65 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₃ H ₃₅ O ₇ N ₂ ([M+H] ⁺ ) 571.2439, experiment 571.2421.

EXAMPLE 39

(3'S,5'R,7'R)-1-{5,-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4,-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}thymine (39)

To a solution of nucleoside 38 (200 mg, 0.350 mmol) and 5-(ethylthio)-1H-tetrazole (59 mg, 0.46 mmol) in dry DCM (7 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.17 mL, 0.53 mmol) at room temp. After stirring for 1 h, the reaction mixture was diluted with DCM (50 mL) and washed with sat. NaHCO ₃ (2 X 25 mL) and sat. NaCl (25 mL). The aqueous phases were combined and extracted with DCM (30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (2% MeOH in DCM, +0.5% _Et3N ) to give 39 (220 mg, mixture of two isomers, 81%) as a white solid.

Data for 39: R _f = 0.44 (4% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.03 (br, 1H, HN(3)), 7.36 (d, J=8.1 Hz, 2H, H-arom), 7.30 -7.07 (m, 7H, H-arom.), 6.84 (s, 1H, H-C(6)), 6.80-6.69 (m, 4H, H-arom.), 5 .95, 5.88 (2dd, J=6.6, 4.8 Hz, 1H, H-C(1')), 4.70, 4.61 (2dd, J=7.3, 4.3 Hz, 1H, H-C(4')), 4.41-4.20 (m, 1H, H-C(5')), 3.94-3.82 (m, 1H, H-C(7')), 3.81-3.62 (m, 8H, MeO, OCH ₂ CH ₂ CN), 3.59-3.40 (m, 2H, (Me ₂ CH) ₂ N), 2.61-2.46 (m, 2H, OCH ₂ CH ₂ CN), 2.28 (ddd, J=14.1, 13.2, 7.3 Hz, 1H, H- C(3')), 1.91-1.73 (m, 5H, Me-C(5), H-C(6'), H-C(2')), 1.72-1.46 (m, 2H, H-C(6'), H-C(2')), 1.16-1.00 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.01, 163.98 (C(4)), 158.70 (MeO-C-arom), 150.39, 150.17 (C(2)) , 145.52, 136.84, 136.78 (C-aromatic), 135.44, 135.39 (C(6)), 130.21, 128.36, 128.32, 128.00, 127 .03 (CH-aromatic), 118.02, 117.76 (OCH ₂ CH ₂ CN), 113.32 (CH-aromatic), 110.91, 110.59 (C(5)), 88.31, 88.06 (С(Ph) ₃ ), 87.11, 87.06 (С(1')), 85.44, 85.39 (J _C,P = 4.6, 3.1 Hz, С(4')), 78.25, 78.13 (С(7')), 74.70, 74.34 (J _C,P =13.5, 18.5 Hz, С(5' )), 58.73, 58.47 (J _C,P = 18.9, 20.1 Hz, (OCH ₂ CH ₂ CN)), 55.35, 55.32 (MeO-DMTr), 48.80 , 48.64 (С(3')), 43.22, 43.06 (J _C,P =12.4, 11.0 Hz (Me ₂ CH) ₂ N), 39.68, 39.63 (C(6')), 38.06, 37.93 (C(2')), 24.81, 24.74 , 24.71, 24.68, 24.65, 24.59 (6s, Ме2СЯ)2Ш 20.37, 20.35 (J _C,P = 7.1, 6.8 Hz, OCH ₂ CH ₂ CN) , 12.66 (Me-C(5)).

³¹ P NMR (122 MHz, CDCl ₃ ) δ 148.18, 147.80.

ESI ⁺ -MSHR m/z, calculation for C ₄₂ H ₅₂ O ₈ N ₄ P ([M+H] ⁺ ) 771.3517, experiment 771.3517.

EXAMPLE 40

(3'S,5'R,7'R)-N4-benzoyl-1-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine (40)

To a solution of nucleoside 38 (268 mg, 0.470 mmol) in dry MeCN (5 mL) was added BSA (0.28 mL, 1.13 mmol) dropwise at 0 °C, and then the solution was stirred overnight at rt. In another flask, a suspension of 1,2,4-triazole (1.14 g, 16.5 mmol) in dry MeCN (50 mL) was cooled to 0 °C and POCl ₃ (0.35 mL, 3.8 mmol) was added, followed by Et ₃ N (2.62 mL, 18.8 mmol). The suspension was stirred for 30 min at 0 °C, and then the previously prepared solution of silylated compound 38 was added to the suspension and the mixture was stirred for an additional 7 h at rt. The reaction was quenched by adding sat. NaHCO ₃ (10 ml), MeCN was removed under reduced pressure and the resulting mixture was diluted with sat. NaHCO ₃ (30 ml) and extracted with DCM (3 X 30 ml). The combined organic phases were dried over MgSO ₄ , filtered and evaporated.

The crude product was then dissolved in a mixture of 1,4-dioxane (10 mL) and conc. _NH4OH (10 mL). After stirring for 3 h at room temp, the mixture was concentrated to half volume in vacuo, diluted with sat. _NaHCO3 (25 mL) and extracted with DCM (4 X 30 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated.

The crude product was then dissolved in dry DMF (10 mL). _Et3N (80 µL, 0.56 mmol) was added followed by _Bz2O (266 mg, 1.18 mmol) at rt and the solution was stirred overnight. The reaction in the resulting brownish solution was quenched by careful addition of sat. _NaHCO3 (40 mL) and extracted with DCM (4 X 40 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CHX (EtOAc/hexane 1:1, +0.5% _Et3N ) to give 40 (263 mg, 83%) as a white foam.

Data for 40: R _f = 0.53 (EtOAc/hexane 3:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 13.11 (br, 1H, NH), 8.30-8.10 (m, 2H, H-arom), 7.47-7.29 (m , 5H, H-arom.), 7.28-7.06 (m, 7H, H-arom.), 7.00 (d, J=0.8 Hz, 1H, H-C(6)), 6.74 (d, J=8.6 Hz, 4H, H-arom), 5.89 (dd, J=6.3, 4.6 Hz, 1H, H-C(1')), 4 .61 (dd, J=7.2, 4.5 Hz, 1H, Н-С(4')), 4.33-4.20 (m, 1H, H-C(5')), 3.87 (br., 1H, H-C(7')), 3.69 (s, 6H, MeO), 2.32-2.13 (m, 2H, H-C(3'), OH), 1.99 (s, 3H, Me-C(5)), 1.87-1.73 (m, 2H, H-C(2'), H-C (6')), 1.66-1.47 (m, 2H, H-C(2'), H-C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 179.61 (CONH), 159.76 (C(4)), 158.74 (MeO-C-arom.), 147.87 (C(2)), 145.47 (C-arom.), 137.17 (C(6)), 136.77, 136.68, 136.03 (C-arom.), 132.55, 130.21, 129.98, 128.34, 128.21, 128.03, 127.07, 113.35 (CH-arom.), 111.81 (C(5)), 88.74 (C(Ph) ₃ ), 87.13 (C(1')), 86.12 (C(4')), 78.17 (C(7')), 73.31 (C(5')), 55.35 (MeO-DMTr), 48.63 (C(3')), 40.35 (C(6')), 38.06 (C(2')), 13.78 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₄₀ H ₄₀ O ₇ N ₃ ([M+H] ⁺ ) 674.2861, experiment 674.2877.

EXAMPLE 41

(3'S,5'R,7'R)-N4-benzoyl-1-{5'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7,-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}-5-methylcytosine (41)

To a solution of nucleoside 40 (250 mg, 0.371 mmol) and 5-(ethylthio)-1H-tetrazole (73 mg, 0.56 mmol) in dry DCM (8 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.20 mL, 0.63 mmol) at room temp. After stirring for 30 min, the reaction mixture was diluted with DCM (30 mL) and washed with sat. NaHCO ₃ (2 X 20 mL) and sat. NaCl (20 mL). The aqueous phases were combined and extracted with DCM (20 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (EtOAc/hexane 1:1, +0.5% _Et3N ) to give 41 (260 mg, mixture of two isomers, 80%) as a white foam.

Data for 41: R _f = 0.57 (EtOAc/hexane 1:1);

^1H NMR (300 MHz, CDCl ₃ ) δ 13.26 (br, 1H, NH), 8.32 (d, J=7.2 Hz, 2H, H-arom), 7.58-7, 39 (m, 5H, H-arom.), 7.38-7.14 (m, 8H, H-arom., H-C(6)), 6.88-6.77 (m, 4H, H -arom.), 6.01, 5.96 (2dd, J=6.3, 4.6 Hz, 1H, H-C(1')), 4.82, 4.74 (2dd, J=7 ,3, 4.3 Hz, 1H, Н-С(4')), 4.42 (td, J=10.6, 6.0 Hz, 1H, H-C(5')), 3.97 (br., 1H, H-C(7')), 3.91-3.68 (m, 8H, MeO, OCH ₂ CH ₂ CN), 3, 59 (dtd, J=16.7, 6.7, 3.4 Hz, 2H, (Me ₂ CH) ₂ N)), 2.62 (dt, J=15.5, 6.4 Hz, 2H, OCH ₂ CH ₂ CN), 2.49-2.23 (m, 1H, H-C(3')), 2.11, 2.09 (2d, J=0.5 Hz, 3H, Me-C (5)), 2.00-1.82 (m, 2H, H-C(6'), H-C(2')), 1.82-1.55 (m, 2H, H-C(6'), H-C(2')), 1.17 (dd, J=16.3, 6.8 Hz, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 179.60 (CONH), 159.97 (C(4)), 158.76 (MeO-C-arom.), 147.81, 147.70 (C(2)), 145.54 (C-arom.), 137.34, 136.83 (C(6)), 136.77, 136.72, 136.65, 136.55 (C-arom.), 132.45, 130.22, 130.20, 129.96, 128.34, 128.31, 128.18, 128.00, 127.04 (CH-arom.), 117.89, 117,71 (OCH ₂ CH ₂ CN), 113,35 (СН-аром.), 111,60, 111,36 (С(5)), 89,24, 89,01 (С(Ph) ₃ ), 87,16, 87,12 (С(1')), 85,78, 85,62 (J _C,P =4,3, 3,2 Гц, С(4')), 78,20, 77,98 (С(7')), 74,68, 74,37(J _C,P =13,4, 18,2 Гц, С(5')), 58,70, 58,44 (J _C,P =18,5, 20,0 Гц, (OCH ₂ CH ₂ CN)), 55,36, 55,33 (MeO-DMTr), 48.65, 48.44 (С(3')), 43.27, 43.14 (J _C,P =12.4, 12.3 Hz (Me ₂ CH ₂ ) ₂ N), 39.87, 39.64 (J _C,P =3.4, 3.7 Hz (С(6')), 38.30, 38.22 (С (2')), 24.80, 24.72, 24.70, 24.67, 24.63 (Me ₂ CH) ₂ N), 20.39, 20.37 (J _C,P = 7.2, 6.8 Hz, OCH ₂ CH ₂ CN), 13.72 (Me-C (5)).

³¹ P NMR (121 MHz, CDCl ₃ ) δ 148.18, 147.96.

ESI ⁺ -NCBP m/z, calculation for C ₄₉ H ₅₇ O ₈ N ₅ P ([M+H] ⁺ ) 874.3939, experiment 874.3946.

EXAMPLE 42

(3'R,5'R,7'R)-N6-benzoyl-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}adenine (42)

Nucleoside 15 (1.74 g, 2.64 mmol) was dissolved in 0.15 M NaOH in THF/methanol/ _H2O (5:4:1, 80 mL) at 0 °C. The reaction mixture was stirred for 20 min and quenched by adding _NH4Cl (1.06 g). The solvents were then removed under reduced pressure and the product was purified by QC (5% isopropanol in DCM) to afford 42 (α-anomer, 836 mg, 51%) and 16 (β-anomer, 287 mg, 18%) as a white foam.

Data for 42: R _f = 0.35 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.34 (s, 1H, NH), 8.71 (s, 1H, H-C(2)), 8.02 (d, J=7.4 Hz , 2H, H-arom)), 7.92 (s, 1H, H-C(8)), 7.68-7.58 (m, 4H, H-arom), 7.58-7, 31 (m, 9H, H-arom), 6.23 (dd, J=6.7, 2.4 Hz, 1H, H-C(1')), 4.74 (dd, J=6, 6, 4.9 Hz, 1H, Н-С(4')), 4.49 (dt, J=12.5, 6.3 Hz, 1H, Н-С(5')), 4.10 (br., 1H, H-C(7')), 3.07 (d, J=6.1 Hz, 1H, OH), 2.92 (dd, J=15.4, 7, 3 Hz, 1H, H-C(3')), 2.52-2.35 (m, 1H, H-C(2')), 2.10-1.97 (m, 1H, H-C (6')), 1.94-1.77 (m, 2H, H-C(2'), H-C(6')), 1.06 (s, 9H, (CH ₃ ) ₃ -C -Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.98 (CONH), 152.65 (C(2)), 151.31 (C(4)), 149.69 (C(6)), 140.93 (C(8)), 135.74 (CH-arom.), 133.82, 133.68, 133.39 (C-arom.), 132.77, 130.02, 129.98, 128.76, 128.06, 127.87, 127.85 (CH-arom.), 123.38 (C(5)), 87.16 (C(1')), 85.35 (C(4')), 77.40 (C(7')), 72.79 (C(5')), 50.63 (C(3')), 40.86 (C(6')), 37.25 (C(2')), 26.94 ((CH ₃ ) ₃ -C-Si), 19.05 ((CH ₃ ) ₃ -C-Si).

ESI ⁺ -NCBP m/z, calculation for C ₃₅ H ₃₈ O ₄ N ₅ Si ([M+H] ⁺ ) 620.2688, experiment 620.2671.

EXAMPLE 43

(3'R,5'R,7'R}-N6-benzoyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}adenine (43)

To a solution of nucleoside 42 (1.09 g, 1.75 mmol) and 4-dimethylaminopyridine (321 mg, 2.63 mmol) in dry DCM (50 mL) was added acetic anhydride (0.83 mL, 8.8 mmol) at rt. After stirring overnight, the reaction was quenched by addition of _{saturated NaHCO3} (50 mL). The phases were separated and the aqueous phase was further extracted with DCM (2 X 80 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (2.5% MeOH in DCM) to give 43 (1.04 g, 90%) as a white foam.

Data for 43: R _f = 0.33 (EtOAc/hexane 4:1);

¹ H NMR (300 MHz, CDCl ₃ ) 5 8.99 (br, 1H, NH), 8.73 (s, 1H, H-C(2)), 8.09-7.99 (m, 2H , H-arom.), 7.98 (s, 1H, H-C(8)), 7.70-7.58 (m, 5H, H-arom.), 7.57-7.48 (m , 2H, H-arom.), 7.47-7.34 (m, 6H, H-arom.), 6.22 (dd, J=6.8, 3.2 Hz, 1H, H-C( 1')), 5.45-5.35 (m, 1H, H-C(5')), 5.01 (dd, J=6.7, 5.0 Hz, 1H, H-C(4')), 4.09 (d, J=4.1 Hz, 1H, H-C(7')), 3.02 (dt, J=9.5, 6.5 Hz, 1H, Н-С(3')), 2.55 (ddd, J=13.5, 10.0, 3.2 Hz, 1H, Н-С(2')), 2.15 (dd , J=13.2, 6.2 Hz, 1H, H-C(6')), 2.09 (s, 3H, MeCO ₂ ), 2.01 (dt, J=8.0, 3.5 Hz, 1H, H-C(2')), 1.88 (dt, J=13.6, 5.3 Hz, 1H, H-C(6')), 1.08 (s, 9H, ( CH ₃ ) ₃ -C-Si).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 170.61 (MeCO ₂ ), 164.75 (CONH), 152.67 (C(2)), 151.37 (C(4)), 149.64 (C(6)), 141.41 (C(8)), 135.85 (CH-arom.), 133.71, 133.38 (C-arom.), 132.91, 130.15, 130.10, 128.99, 128.02, 127.99, 127.97 (CH-arom.), 123.64 (C(5)), 87.37 (C(1')), 83.37 (C(4')), 76.63 (C(7')), 74.51 (C(5')), 51.19 (C(3')), 37.44 (C(2')), 37.32 (C(6')), 27.01 ((CH ₃ ) ₃ -C-Si), 21.08 (MeCO ₂ ), 19.14 ((CH ₃ ) ₃ -C-Si).

ESI ⁺ -NCBP m/z, calculation for C ₃₇ H ₄₀ O ₅ N ₅ Si ([M+H] ⁺ ) 662.2793, experiment 662.2787.

EXAMPLE 44

(3'S,5'R,7'R)-N6-benzoyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-α-D-ribofuranosyl}adenine (44)

To a solution of nucleoside 43 (990 mg, 1.50 mmol) in dry THF (50 mL) was added TBAF (1 M in THF, 3.0 mL, 3.0 mmol) at rt. After stirring for 3.5 h at rt, the solution was diluted with EtOAc (30 mL) and the THF was removed under reduced pressure. The mixture was then diluted with saturated NaHCO ₃ (50 mL) and extracted with DCM (4 X 50 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (6% MeOH in DCM) to give 44 (570 mg, 90%) as a white foam containing traces of TBAF.

Data for 44: R _f = 0.33 (10% MeOH in DCM);

^1H NMR (400 MHz, CDCl ₃ ) δ 9.60 (br, 1H, NH), 8.67 (s, 1H, H-C(2)), 8.09 (s, 1H, H-C (8)), 7.96 (d, J=7.4 Hz, 2H, H-arom), 7.51 (t, J=7.4 Hz, 1H, H-arom), 7.42 (t, J=7.5 Hz, 2H, H-arom.), 6.33 (dd, J=6.7, 3.1 Hz, 1H, H-C(1')), 5.25 ( ddd, J=9.7, 6.4, 5.3 Hz, 1H, Н-С(5')), 4.92 (dd, J=6.4, 5.4 Hz, 1Н, H-C(1')), 4.14 (br., 2H, H-C(7'), OH), 3.06 (dd, J=16.0, 6.6 Hz, 1H, H- С(3')), 2.87 (ddd, J=13.2, 9.9, 3.0 Hz, 1H, Н-С(2')), 2.26-2.17 (m, 1H , H-C2')), 2.10-1.98 (m, 5H, H-C(6'), MeCO ₂ ).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.64 (MeCO ₂ ), 165.27 (CONH), 152.49 (C(2)), 151.26 (C(4)), 149.58 (C(6)), 141.64 (C(8)), 133.60 (C-arom.), 132.82, 128.76, 128.06 (CH-arom.), 123.30 (C(5)), 87.30 (C(1')), 83.17 (C(4')), 74.67 (C(7')), 74.20 (C(5')), 50.41 (C(3')), 37.43 (C(2')), 36.92 (C(6')), 20.96 (MeCO ₂ ).

ESI ⁺ -NCBP m/z, calculation for C ₂₁ H ₂₂ O ₅ N ₅ ([M+H] ⁺ ) 424.1615, experiment 424.1623.

EXAMPLE 45

(3'S,5'R,7'R)-N6-benzoyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (45)

To a solution of nucleoside 44 (570 mg, 1.35 mmol) in dry pyridine (16 mL) was added DMTr-Cl (1.37 g, 4.04 mmol) at room temp. The solution was stirred for 1 day and then diluted with saturated NaHCO ₃ (100 mL) and extracted with DCM (3 X 80 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (2% MeOH in DCM, +0.5% Et ₃ N) to give 45 (876 mg, 89%) as a yellow foam.

Data for 45: R _f = 0.81 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.42 (d, J=14.6 Hz, 1H, NH), 8.73 (s, 1H, H-C(2)), 8.03 (d , J=7.6 Hz, 2H, H-arom.), 7.93 (s, 1H, H-C(8)), 7.66-7.55 (m, 1H, H-arom.), 7.55-7.45 (m, 4H, H-arom.), 7.45-7.22 (m, 7H, H-arom.), 6.87 (d, J=8.7 Hz, 4H , H-arom.), 6.25 (dd, J=6.6, 2.4 Hz, 1H, H-C(1')), 5.47-5.33 (m, 1H, H-C(5')), 4.89 (dd, J=6.7, 4.9 Hz, 1H, H-C(4')), 4.02 (d, J= 2.5 Hz, 1H, H-C(7')), 3.79 (s, 6H, MeO), 2.58 (dd, J=16.0, 6.9 Hz, 1H, H-C( 3')), 2.38 (ddd, J=12.7, 10.0, 2.4 Hz, 1H, H-C(2')), 2.11 (s, 3H, MeCO ₂ ), 2 .09-1.87 (m, 3Н, Н-С(2'), Н-С(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 170.40 (MeCO ₂ ), 164.84 (CONH), 158.66 (MeO-C-arom.), 152.45 (C(2)), 151.22 (C(4)), 149.51 (C(6)), 145.23 (C-arom.), 141.23 (C(8)), 136.51, 133.65 (C-arom.), 132.68, 130.12, 128.75, 128.33, 127.95, 127.90, 127.03 (CH-arom.), 123.55 (C(5)), 113.27 (CH-arom.), 87.19 (C(Ph) ₃ ), 87.12 (C(1')), 83.25 (C(4')), 77.16 (C(7')), 74.41 (C(5')), 55.23 (MeO-DMTr), 49.23 (C(3')), 37.61 (C(2')), 36.22 (C(6')), 20, 98 (MeCO ₂ ).

ESI ⁺ -NCBP m/z, calculation for C ₄₂ H ₄₀ O ₇ N ₅ ([M+H] ⁺ ) 726.2922, experiment 726.2905.

EXAMPLE 46

(3'S,5'R,7'R)-N6-benzoyl-9-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (46)

Nucleoside 45 (870 mg, 1.20 mmol) was dissolved in 0.1 M NaOH in THF/methanol/ _H2O (5:4:1, 50 mL) at 0 °C. The reaction mixture was stirred for 30 min at 0 °C and then quenched by adding _NH4Cl (321 mg). The solution was diluted with _{saturated NaHCO3} (100 mL) and extracted with DCM (4 X 80 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM, +0.5% _Et3N ) to give 46 (777 mg, 94%) as a white foam.

Data for 46: R _f = 0.26 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.39 (s, 1H, NH), 8.61 (s, 1H, H-C(2)), 7.93 (d, J=7.4 Hz , 2H, H-arom.), 7.75 (s, 1H, H-C(8)), 7.46 (t, J=7.3 Hz, 1H, H-arom.), 7.40- 7.31 (m, 4H, H-arom), 7.29-7.16 (m, 6H, H-arom), 7.11 (t, J=7.2 Hz, 1H, H-arom .), 6.73 (d, J=8.7 Hz, 4H, H-arom.), 6.12 (dd, J=6.5, 1.9 Hz, 1H, Н-С(1')), 4.53 (dd, J=7.5, 4.5 Hz, 1H, Н-С(4')), 4.32 (br., 1H, Н-С( 5')), 3.90 (t, J=4.5 Hz, 1H, H-C(7')), 3.66, 3.65 (2s, 6H, MeO), 3.31 (br. , 1H, OH), 2.36 (dd, J=16.5, 8.1 Hz, 1H, H-C(3')), 2.04 (ddd, J=12.0, 9.9, 2.0 Hz, 1H, H-C(2')), 1.92-1.69 (m, 3H, H-C(2'), H-C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.92 (CONH), 158.64 (MeO-C-arom.), 152.60 (C(2)), 151.28 (C(4)), 149.61 (C(6)), 145.44 (C-arom.), 140.71 (C(8)), 136.77, 133.65 (C-arom.), 132.72, 130.15, 130.12, 128.73, 128.39, 128.04, 127.96, 127.02 (CH-arom.), 123.27 (C(5)), 113.28 (CH-arom.), 87.11 (C(1')), 87.01 (C(Ph) ₃ ), 85.60 (C(4')), 78.16 (C(7')), 72.72 (C(5')), 55.28 (MeO-DMTr), 48.89 (C(3')), 39.93 (C(6')), 37.55 (C(2')).

ESI ⁺ -NCBP m/z, calculation for C ₄₀ H ₃₈ O ₆ N ₅ ([M+H] ⁺ ) 684.2817, experiment 684.2800.

EXAMPLE 47

(3'S,5'R,7'R)-N6-benzoyl-9-{5'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}adenine (47)

To a solution of nucleoside 46 (199 mg, 0.290 mmol) and 5-(ethylthio)-1H-tetrazole (57 mg, 0.44 mmol) in dry DCM (7 mL) was added 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.16 mL, 0.49 mmol) dropwise at room temp. After stirring for 60 min, the reaction mixture was diluted with _{saturated NaHCO3} (20 mL) and extracted with DCM (3 X 20 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CC (EtOAc, +0.5% _Et3N ) to give 47 (197 mg, mixture of two isomers, 77%) as a white foam.

Data for 47: R _f = 0.75 (5% MeOH in DCM);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.98 (br, 1H, NH), 8.68, 8.67 (2s, 1H, C(2)), 7.94 (d, J=7 ,6 Hz, 2H, H-arom.), 7.90, 7.84 (2s, 1H, C(8)), 7.56-7.49 (m, 1H, H-arom.), 7, 48-7.34 (m, 4H, N-arom.), 7.30-7.10 (m, 7H, N-arom.), 6.80-6.69 (m, 4H, N-arom. ), 6.21, 6.15 (2dd, J=6.8, 2.2 Hz, 1H, H-C(1')), 4.69, 4.59 (2dd, J=7.3, 4.5 Hz, 1H, H-C(4')), 4.44 (tt, J=12.3, 6.3 Hz, 1H , Н-С(5')), 3.90 (dd, J=9.0, 3.8 Hz, 1H, Н-С(5')), 3.82-3.63 (m, 8Н, MeO, OCH ₂ CH ₂ CN), 3.59-3.43 (m, 2H, (Me ₂ CH ₂ ) ₂ N), 2.61-2.49 (m, 2H, OCH ₂ CH ₂ CN), 2.47-2.07 (m, 2H, H-C(3'), H-C(2')), 1.98-1.66 (m, 3H, H-C(2'), H-C(6')), 1.15-1.03 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.67 (CONH), 158.77 (MeO-C-arom.), 152.58 (C(2)), 151.34, 151.29 (C(4)), 149.46 (C(6)), 145.55, 145.54 (C-arom.), 141.58, 141.50 (C(8)), 136.87, 136.85, 136.84, 133.85 (C-arom.), 132.85, 130.26, 130.23, 130.20, 128.97, 128.47, 128.43, 128.02, 127.96, 127.08 (CH-arom.), 123.62, 123.58 (C(5)), 117.91, 117.70 (OCH ₂ CH ₂ CN), 113.37 (CH-arom.), 87.80, 87.67 (C(1')), 87.20, 87.14 (C(Ph ) ₃ ), 85.29, 85.22 ((J _C,P =4.2, 3.1 Hz, C(4')), 78.16, 77.96 (C(7')), 74.28, 73.98 (J _C,P =14.8, 18.4 Hz, C(5')), 58.80, 58.61 (J _C,P =16.2, 17.3 Hz OCH ₂ CH ₂ CN), 55.37, 55.35 (MeO-DMTr), 49.02, 48.91 (C(3')), 43.29, 43.16 (J _C,P = 8.9, 9.0 Hz, ((Me ₂ CH) ₂ N), 39.09 (C(6')), 37.99, 5 (C(2')), 24.82, 24.77, 24.74, 24.70, 24.64 ((Me ₂ CH) ₂ N), 20.43, 20.42 (J _C,P = 1.4, 1.9 Hz, OCH ₂ CH ₂ CN).

³¹ NMR (121 MHz, CDCl ₃ ) δ 148.14, 148.11.

ESI ⁺ -NCBP m/z, calculation for C ₄₅ H ₅₆ O ₇ N ₈ P ([M+H] ⁺ ) 884.3895, experiment 884.3904.

EXAMPLE 48

(3'R,5'R,7'R)-2-amino-6-chloro-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}purine (48)

Nucleoside 20 (1.78 g, 3.01 mmol) was dissolved in 0.5 M NaOH in THF/methanol/ _H2O (5:4:1, 15 mL) at 0 °C. The reaction mixture was stirred for 20 min at 0 °C and quenched by adding _NH4Cl (484 mg). The suspension was then diluted with _{saturated NaHCO3} (100 mL) and extracted with DCM (4 X 75 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM) to give 48 (α-anomer, 992 mg, 60%) and 21 (β-anomer, 428 mg, 25%) as a white foam.

Data for 48: R _f = 0.34 (5% MeOH in DCM);

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.71-7.60 (m, 5H, H-arom., H-(C(8)), 7.49-7.34 (m, 6H, H -arom.), 6.08 (dd, J=6.9, 2.6 Hz, 1H, H-C(1')), 5.26 (s, 2H, NH ₂ ), 4.70 (dd , J=7.5, 4.8 Hz, 1H, Н-С(4')), 4.47 (dt, J=10.0, 5.1 Hz, 1H, Н-С(5')) , 4.11 (t, J=3.3 Hz, 1H, Н-С(7')), 2.87 (dd, J=16.5, 7.7 Hz, 1Н, H-C(3')), 2.57 (br, 1H, OH), 2.27 (ddd, J=14.0, 9.9, 2.6 Hz, 1H, H-C(2' )), 2.10-2.01 (m, 1H, H-C(6')), 1.92-1.76 (m, 2H, H-C(2'), H-C(6' )), 1.06 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 159.09 (C(2)), 153.05 (C(4)), 151.46 (C(6)), 139.91 (C(8)), 135.71 (CH-arom.), 133.96, 133.27 (C-arom.), 130.00, 129.96, 127.86, 127.83 (CH-arom.), 125.52 (C(5)), 86.46 (C(1')), 84.92 (C(4')), 77.40 (C(7')), 72.63 (C(5')), 50.55 (C(3')), 40.92 (C(6')), 36.78 (C(2')), 26.88 ((CH ₃ ) ₃ -C-Si), 19.01 ((CH ₃ ) ₃ -C-Si).

ESI ⁺ -NCBP m/z, calculation for C ₂₈ H ₃₃ O ₃ N ₅ ClSi ([M+H] ⁺ ) 550.2036, experiment 550.2019.

EXAMPLE 49

(3'R,5'R,7'R)-9-{7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-α-D-ribofuranosyl}guanine (49)

To a solution of nucleoside 48 (610 mg, 1.03 mmol) in dry DCM (15 mL) was added 3-hydroxypropanenitrile (0.28 mL, 4.12 mmol), then 1,5,7-triazabicyclo[4.4.0]dec-5-ene (287 mg, 2.06 mmol) at room temp. After stirring for 4 h at room temp, second portions of 3-hydroxypropanenitrile (0.28 mL, 3.23 mmol) were added, then 1,5,7-triazabicyclo[4.4.0]dec-5-ene (287 mg, 2.06 mmol). The reaction mixture was stirred for an additional 2 days and then directly purified by EC (10% MeOH in DCM) to give 49 (500 mg, 87%) as a white foam.

Data for 49: R _f = 0.30 (10% MeOH in DCM);

¹ H NMR (400 MHz, MeOD) δ 7.73-7.61 (m, 5H, H-arom, H-C(8)), 7.53-7.32 (m, 6H, H-arom .), 6.06 (dd, J=6.9, 3.7 Hz, 1H, H-C(1')), 4.74 (dd, J=7.0, 4.6 Hz, 1H, H-C(4')), 4.46-4.36 (m, 1H, H-C(5')), 4.11 (br., 1H, H-C(7')), 2, 91 (dd, J=16.2, 6.6 Hz, 1H, H-C(3')), 2.31 (ddd, J=13.8, 10.0, 3.7 Hz, 1H, H-C(2')), 1.98-1.78 (m, 3H, H-C(2'), H-C(3')), 1.07 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (101 MHz, MeOD) δ 159.30 (C(2)), 155.14 (C(6)), 152.38 (C(4)), 137.28 (C(8)), 136.93, 136.88 (CH-arom.), 135.13, 134.78 (C-arom.), 131.07, 131.06, 128.91, 128.89 (CH-arom.), 117.98 (C(5)), 87.72 (C(1')), 86.25 (C(4')), 79.21, (C(7')) 73.87 (C(5')), 52.13 (C(3')), 41.44 (C(6')), 38.35 (C(2')), 27.42 ((CH ₃ ) ₃ -C-Si), 19.82 ((CH ₃ ) ₃ -C-Si)).

ESI ⁺ -NCBP m/z, calculation for C ₂₈ H ₃₄ O ₄ N ₅ Si ([M+H] ⁺ ) 532.2386, experiment 532.2367.

EXAMPLE 50

(3'R,5'R,7'R)-N2-acetyl-9-{5'-O-acetyl-7'-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-39,59-ethano-α-D-ribofuranosyl}guanine (50)

To a solution of nucleoside 49 (500 mg, 0.940 mmol) and 4-dimethylaminopyridine (276 mg, 2.4 mmol) in dry DCM (15 mL) was added acetic anhydride (1.0 mL, 10.3 mmol) at rt. After stirring for 2 days, the reaction was quenched by addition of saturated NaHCO ₃ (30 mL). The mixture was then extracted with DCM (3 X 30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (3.5% MeOH in DCM) to give 50 (441 mg, 76%) as a white foam.

Data for 50: R _f = 0.62 (10% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 12.11 (br, 1H, NH-C(4)), 9.94 (br, 1H, HN(1)), 7.62 (d, J =6.7 Hz, 5H, H-arom., H-C(8)), 7.46-7.31 (m, 6H, H-arom.), 6.03 (dd, J=6.7 , 2.7 Hz, 1H, H-C(1')), 5.31 (dt, J=10.3, 5.2 Hz, 1H, H-(C5')), 4.99-4, 81 (m, 1H, H-C(4')), 4.02 (d, J=3.8 Hz, 1H, H-C(7')), 2.88 (dd, J=16.0, 6.6 Hz, 1H, H-C(3')), 2.44-2.20 (m, 4H, MeCONH, H-C(2')), 2.12-1 .73 (m, 6H, MeCO ₂ , H-C(6'), H-C(2')), 1.04 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 172.73 (MeCONH), 170.46 (MeCO ₂ ), 155.87 (C(6)), 148.09 (C(4)), 147.47 (C(2)), 137.13 (C(8)), 135.74 (CH-arom.), 133.62, 133.29 (C-arom.), 130.13, 130.09, 127.96, 127.93 (CH-arom.), 121.54 (C(5)), 86.47 (C(1')), 82.81 (C(4')), 76.60 (C(7')), 74.37 (C(5')), 51.23 (C(3')), 37.04, 37.01, (C(2'), C(6')) 26.92 ((CH ₃ ) ₃ -C-Si), 24.46 (MeCONH), 21.00 (MeCO ₂ ), 19.05 ((CH ₃ ) ₃ -C-Si).

ESI ⁺ -NCBP m/z, calculation for C ₁₇ H ₃₈ O ₆ N ₅ Si ([M+H] ⁺ ) 616.2586, experiment 616.2580.

EXAMPLE 51

(3'S,5'R,7'R)-N2-acetyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-hydroxy-α-D-ribofuranosyl}guanine (51)

To a solution of nucleoside 50 (440 mg, 0.714 mmol) in dry THF (5 mL) was added TBAF (1 M in THF, 1.1 mL, 1.1 mmol) at rt. The solution was stirred for 4 h at rt and then directly purified by CC (13% MeOH in DCM) to give 51 (235 mg, 87%) as a white foam. Crystals suitable for X-ray analysis were obtained by recrystallization in _H2O /MeOH.

Data for 51: R _f = 0.25 (13% MeOH in DCM);

¹ H NMR (300 MHz, MeOD) δ 8.03 (s, 1H, H-C(8)), 6.28 (dd, J=7.0, 3.8 Hz, 1H, H-C(1 ')), 5.21 (ddd, J=9.2, 6.8, 5.1 Hz, 1H, H-C(5')), 4.98 (dd, J=6.7, 5, 0 Hz, 1H, Н-(4')), 4.13-4.05 (m, 1H, Н-С(7')), 3.17-3.05 (m, 1H, Н-С( 3')), 2.86 (ddd, J=13.8, 10.0, 3.8 Hz, 1H, H-C(2')), 2.39-2.27 (m, 1H, H-C(2')), 2.24 (s, 3H, MeCONH), 2.16-2.00 (m, 5H, MeCO ₂ , H-C(6')).

¹³ C NMR (101 MHz, MeOD) δ 174.95 (MeCONH), 172.32 (MeCO ₂ ), 157.50 (C(6)), 149.96 (C(4)), 149.38 (C (2)), 139.66 (C(8)), 121.76 (C(5)), 88.23 (C(1')), 84.23 (C(4')), 75.83 (C(5'), C(7')), 51.65 (C(3')), 38.04, 37.93 (C(2'), C(6')), 23.83 ( MeCONH), 20.71 (MeCO ₂ ).

ESI ⁺ -NCBP m/z, calculation for C ₁₆ H ₂₀ O ₆ N ₅ ([M+H] ⁺ ) 378.1408, experiment 378.1419.

EXAMPLE 52

(3'S,5'R,7'R)-N2-acetyl-9-{5'-O-acetyl-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (52)

To a solution of nucleoside 51 (186 mg, 0.492 mmol) in dry pyridine (10 mL) was added DMTr-Cl (501 mg, 1.48 mmol) at room temp. The solution was stirred for 2 days and then diluted with saturated NaHCO ₃ (40 mL) and extracted with DCM (3 X 30 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM, +0.5% Et ₃ N) to give 52 (333 mg, 99%) as a yellow foam.

Data for 52: R _f = 0.56 (10% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 12.05 (br, 1H, NH-C(4)), 9.90 (br, 1H, HN(1)), 7.40 (s, 1H , H-C(8)), 7.38-7.31 (m, 2H, H-arom.), 7.28-7.08 (m, 7H, H-arom.), 6.75 (dd , J=9.0, 2.7 Hz, 4H, H-arom.), 5.95-5.85 (m, 1H, H-C(1')), 5.30-5.10 (m , 1H, H-C(5')), 4.70-4.58 (m, 1H, H-C(4')), 3.81 (br., 1H, H-C(7')), 3.68, 3.68 (2s, 6H, MeO), 2.25-2.07(m, 5H, MeCONH, H-C(3'), H- C(2')), 1.96-1.79 (m, 5H, MeCO ₂ , H-C(2'), H-C(6')), 1.74-1.59 (m, 1H , N-S(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 172.65 (MeCONH), 170.42 (MeCO ₂ ), 158.73, 158.70 (MeO-C-arom.), 155.86 (C(6)), 147.96 (C(4)), 147.43 (C(2)), 145.31 (C-arom.), 137.17 (C(8)), 136.69, 136.44 (C-arom.), 130.32, 130.21, 128.29, 128.05, 127.09 (CH-arom.), 121.53 (C(5)), 113.38, 113.35 (CH-arom.), 87.25 (C(Ph) ₃ ), 86.73 (C(1')), 82.77 (C(4')), 77.19 (C(7')), 74.37 (C(5')), 55.38 (MeO-DMTr), 49.28 (C(3')), 37.25 (C(2')), 36.06 (C(6')), 24, 40 (MeCONH), 21.01 (MeCO ₂ ).

ESI ⁺ -NCBP m/z, calculation for C ₃₇ H ₃₈ O ₈ N ₅ ([M+H] ⁺ ) 680.2715, experiment 680.2718.

EXAMPLE 53

(3'S,5'R,7'R)-N2-(N,N-dimethylformamidino)-9-{2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (53)

To a solution of nucleoside 52 (333 mg, 0.490 mmol) in dry MeOH (10 mL) was added K ₂ CO ₃ (305 mg, 2.20 mmol) at room temp. The suspension was stirred for 7 h at room temp. Then NH ₄ Cl (78 mg, 1.46 mmol) was added and the resulting mixture was filtered through a small pad of SiO ₂ . The SiO ₂ was washed with additional MeOH and then the solvent was evaporated.

The crude product was dissolved in dry DMF (10 mL) and N,N-dimethylformamide dimethyl acetal (0.33 mL, 2.5 mmol) was added. The solution was stirred for 2 h at 55 °C and then the solvents were removed under reduced pressure. The crude product was purified by CX (7% MeOH in DCM, +0.5% Et ₃ N) to give 53 (245 mg, 77%) as a white foam containing traces of Et ₃ N.

Data for 53: R _f = 0.32 (12% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.75 (br, 1H, HN(1)), 8.25 (s, 1H, NCHN(CH ₃ ) ₂ ), 7.37 (d, J= 7.3 Hz, 2H, H-arom.), 7.29-7.08 (m, 8H, H-arom., H-C(8)), 6.74 (d,J=8.1 Hz , 4H, H-arom.), 6.03 (dd, J=6.7, 2.8 Hz, 1H, H-C(1')), 4.57 (dd, J=7.5, 4 .6 Hz, 1H, H-C(4')), 4.37-4.26 (m, 1H, H-C(5')), 3.89 (t, J=3.9 Hz, 1H, H-C(7')), 3.67, 3.67 (2s, 6H, MeO), 3.24 (br, 1H, OH), 2.94 (s , 3H, NCHN(CH ₃ ) ₂ ), 2.87 (s, 3H, NCHN(CH ₃ ) ₂ ), 2.35 (dd, J=15.9, 7.6 Hz, 1H, H-C( 3')), 1.94-1.68 (m, 4H, H-C(2'), H-C(6')).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 158.61 (MeO-C-arom.), 158.28 (C(2)), 157.92 (NCHN(CH ₃ ) ₂ ), 156.69 (C(6)), 149.90 (C(4)), 145.52, 136.86, 136.77 (C-arom.), 135.50 (C(8)), 130.15, 128.32, 127.92, 126.95 (CH-arom.), 120.27 (C(5)), 113.24 (CH-arom.), 86.92 (C(Ph) ₃ ), 85.57 (C(1')), 85.12 (С(4')), 78,31 (С(1')), 72,69 (С(5')), 55,28 (MeO-DMTr), 49,28 (С(3')), 41,38 (NCHN(CH ₃ ) ₂ ), 39,77 (С(6')), 37,58 (С(2')), 35,04 (NCHN(CH ₃ ) ₂ ).

ESI ⁺ -NCBP m/z, calculation for C ₃₆ H ₃₉ O ₆ N ₆ ([M+H] ⁺ ) 651.2926, experiment 651.2921.

EXAMPLE 54

(3'S,5'R,7'R)-N2-(N,N-dimethylformamidino)-9-{5'-O-[(2-cyanoethoxy)diisopropylaminophosphanyl]-2',3'-dideoxy-3',5'-ethano-7'-O-[(4,4'-dimethoxytriphenyl)methyl]-α-D-ribofuranosyl}guanine (54)

To a solution of nucleoside 53 (245 mg, 0.377 mmol) and 5-(ethylthio)-1H-tetrazole (74 mg, 0.57 mmol) in dry DCM (15 mL) was added dropwise 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.20 mL, 0.64 mmol) at room temp. After stirring for 50 min, the reaction mixture was diluted with saturated _NaHCO3 (25 mL) and extracted with DCM (3 X 25 mL). The combined organic phases were dried over _MgSO4 , filtered and evaporated. The crude product was purified by CX (3% MeOH in DCM, +0.5% _Et3N ) to give 54 (212 mg, mixture of two isomers, 67%) as a white foam.

Data for 54: R _f = 0.42 (7% MeOH in DCM);

^1H NMR (300 MHz, CDCl ₃ ) δ 9.35 (br, 1H, HN(1)), 8.51, 8.49 (2s, 1H, NCHN(CH ₃ ) ₂ ), 7.41- 7.10 (m, 10H, H-arom., HC(8)), 6.83-6.70 (m, 4H, H-arom.), 6.15-6.00 (m, 1H, H -C(1')), 4.64-4.36 (m, 2H, H-C(4'), H-C(5')), 3.90-3.82 (m, 1H, H -C(7')), 3.80-3.62 (m, 8H, MeO, OCH ₂ CH ₂ CN), 3.59-3.43 (m, 2H, (Me ₂ CH) ₂ N), 3.04, 3.02 (2s, 6H, NCHN(CH ₃ ) ₂ ), 2.67-2.48 (m , 2H, OCH ₂ CH ₂ CN), 2.32 (ddd, J=24.1, 15.1, 6.7 Hz, 1H, H-C(3')), 2.02-1.63 ( m, 4H, H-C(2'), H-C(6')), 1.14-1.03 (m, 12H, (Me ₂ CH) ₂ N).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 158.76 (MeO-C-arom.), 158.17, 158.12 (C(2)), 158.03 (NCHN(CH ₃ ) ₂ ), 156.66, 156.59 (C(6)), 149.85, 149.79 (C(4)), 145.51, 145.49, 136.84, 136.77, 136.73, 136.71 (C-arom.), 135.76, 135.59 (C(8)), 130.24, 130.20, 128.41, 128.33, 128.02, 127.10, 127.08 (CH-arom.), 120.74, 120.70 (C(5)), 117.98, 117.72 (OCH ₂ CH ₂ CN), 113.34 (CH-arom.), 87.16, 87.10 (C(Ph) ₃ ), 86.00, 85.72 (C( 1')), 84.13, 84.10 (J _C,P =3.6, 2.5 Hz, C(4')), 78.02, 77.67 (C(7')), 74.15, 73.74 (J _C,P =15.3, 18.7 Hz, C(5')), 58.90, 58.67 (J _C,P =18.7, 19.7 Hz OCH ₂ CH ₂ CN), 55.38, 55.36 (MeO-DMTr), 49.20, 49.09 (C(3')), 43.20, 43.15 (J _C,P = 12.4, 12.6 Hz, ((Me ₂ CH) ₂ N), 41.42, 41.38 (NCHN(CH ₃ ) ₂ ), 38.68, 38.65 (C(6')), 37.97, 37.84 (C(2')), 35.25 (NCHN(CH ₃ ) ₂ ), 24.83, 24.75, 24.68, 24.60, 24.53 ((Me ₂ CH) ₂ N), 20.35, 20.28 ( _{OCH2CH2CN )} .

³¹ P NMR (121 MHz, CDCl ₃ ) δ 148.21, 148.01.

ESI ⁺ -MSHR m/z, calculation for C ₄₅ H ₅₆ O ₇ N ₈ P ([M+H] ⁺ ) 851.4004, experiment 851.4013.

EXAMPLE 55

(3aR,4R,6R,6aS)-4-((tert-butyldiphenylsilyl)oxy)-2-methoxyhexahydro-2H-cyclopenta[b]furan-6-yl-(4-nitrobenzoate) (55)

To a solution of sugar 6 (195 mg, 0.437 mmol) and 4-dimethylaminopyridine (70 mg, 0.568 mmol) in dry DCM (10 mL) was added 4-nitrobenzoyl chloride (158 mg, 0.850 mmol) at rt. After stirring overnight, the reaction was quenched by slow addition of sat. NaHCO ₃ (3 mL). The mixture was then diluted with sat. NaHCO ₃ (15 mL) and extracted with DCM (3 X 15 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CHX (EtOAc/hexane 1:5) to give a mixture of 55 (260 mg, 98%) with an anomer ratio of α/β ≈ 4:1 as a white solid.

Data for 55: R _f = 0.62 (EtOAc/hexane 1:2);

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.33-8.17 (m, 4H, H-arom.), 7.72-7.61 (m, 4H, H-arom.), 7.51 -7.32 (m, 6H, H-arom.), 5.65-5.47 (m, 1H, H-C(6)), 4.97 (dd, J=9.2, 5.6 Hz, 1H, H-C(2)), 4.87 (t, J=5.8 Hz, 1H, H-C(6a)), 4.18 (d, J=5.0 Hz, 0, 2Н, Н-С(4)), 3.98 (d, J=3.5 Hz, 0.8Н, Н-С(4)), 3.21 (d, J=15.1 Hz, 3H, MeO), 2.88 (dd, J=16.6, 7.9 Hz, 0.8H, H-C(3a)), 2.75-2.62 (m, 0.2H, H- С(3а)), 2.49-2.34 (m, 0.2Н, Н-С(5)), 2.24-1.83 (m, 2.8Н, Н-(5), Н- C(3)), 1.28 (ddd, J=13.0, 7.9, 4.9 Hz, 1H, H-C(3)), 1.09 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.46, 164.41 (CO ₂ R), 150.63 (O ₂ NC-arom.), 135.87, 135.82 (CH-arom.), 134.07, 133.75, 133.69 (CH-arom.), 130.98, 130.89, 129.98, 129.96, 129.91, 127.89, 127.87, 127.85, 123.59 (CH-arom.), 106.49, 106.39 (C(2)), 83.21, 79.87 (C(6a)), 76.54 (C(4)), 76.09 (C(6)), 54.55, 54.47 (MeO), 51.69, 50.30 (C(3a), 38.07 (C(3)), 37.17, 36.65 (C(5)), 27.04, 26.99 90 ((CH ₃ ) ₃ -C-Si), 19.14 ((CH ₃ ) ₃ -C-Si).

ESI ⁺ -MSHR m/z, calculation for C ₃₁ H ₃₅ O ₇ NaSi ([M+Na] ⁺ ) 584.2075, experiment 584.2085.

EXAMPLE 56

(3'R,5'R,7,'R)-1-{7,-[(tert-butyldiphenylsilyl)oxy]-2',3'-dideoxy-3',5'-ethano-5'-O-(4-nitrobenzoate)-α,β-D-ribofuranosyl}thymine (56)

To a solution of sugar 55 (260 mg, 0.463 mmol) and thymine (84 mg, 0.695 mmol) in dry MeCN (3 mL) was added dropwise BSA (0.34 mL, 1.4 mmol) at rt. After stirring for 30 min at rt, the solution was cooled to 0 °C and TMSOTf (0.10 mL, 1.3 mmol) was added dropwise. After additional stirring for 2 h at 0 °C and 18 h at rt, the reaction mixture was diluted with saturated NaHCO ₃ (30 mL) and extracted with DCM (4 X 40 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by chromatography (2% MeOH in DCM) to give mixture 56 (240 mg, 79%) with an anomer ratio of α/β≈88:12 as a white foam.

Data for 56: R _f = 0.56 (DCM + 3% MeOH);

¹ H NMR (300 MHz, CDCl ₃ ) δ 9.38 (br, 1H, (s, 1H, HN(3)), 8.32-8.23 (m, 2H, H-arom.), 8.22-8.11 (m, 2H, H-arom.), 7.65 (dd, J=7.7, 1.5 Hz, 4H, H-arom.), 7.50-7.36 (m, 6H, H-arom.), 6.95 (d, J=0.9 Hz, 1H, H-C(6)), 5.96 (t, J=6.3 Hz, 1H, H-C(1')), 5.55 (dt, J=9.9, 6.0 Hz, 1H, H-C(5')), 5.13 (dd, J=6.4, 5.4 Hz, 1H, H-C(4')), 4.20-4.05 (m, 1H, H-C(7')), 2.94-2.78 (m, 1H, H-C(3')), 2.22 (dd, J=13.3, 6.4 Hz, 1H, H-C(6')), 2.09-1.73 (m, 6H, H-C(6'), H-C(2'), Me-C(5)), 1.09 (s, 9H, (CH ₃ ) ₃ -C-Si).

¹³ C NMR (75 MHz, CDCl ₃ ) δ 164.32, 163.79 (C(4), CO ₂ R), 150.65, 150.39 (O ₂ NC-arom., C(2)), 135.70, 135.68 (CH-arom.), 135.13 (C-arom.), 134.83 (C(6)), 133.46, 133.10 (C-arom.), 130.91, 130.73, 130.11, 127.93, 123.60 (CH-arom.), 111.30 (C(5)), 87.26 (C(1')), 82.44 (C(4')), 76.40 (C(7')), 76.07 (C(5')), 50.76 (C(3')), 37.94 (C(6')), 36.68 (C(2')), 26.89 ((CH ₃ ) ₃ -C-Si), 19.03 ((CH ₃ ) ₃ -C-Si), 12.62 (Me-C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₃₅ H ₃₇ O ₈ N ₃ NaSi ([M+Na] ⁺ ) 678.2242, experiment 678.2254.

EXAMPLE 57

(3'R,5'R,7'R)-1-{2',3'-dideoxy-3',5'-ethano-7'-hydroxy-5'-O-(4-nitrobenzoate)-α,β-D-ribofuranosyl}thymine (57)

To a solution of nucleoside 56 (220 mg, 0.335 mmol) in dry THF (2 mL) was added TBAF (1 M in THF, 0.84 mL, 0.84 mmol) at rt. After stirring for 4 h at rt, the reaction mixture was diluted with saturated NaHCO ₃ (20 mL) and extracted with EtOAc (3 X 20 mL) and DCM (2 X 80 mL). The combined organic phases were dried over MgSO ₄ , filtered and evaporated. The crude product was purified by CC (5% MeOH in DCM) to give a mixture of anomers 57 (101 mg, 72%). Crystals suitable for X-ray analysis were obtained by recrystallization from EtOAc.

Data for 57: R _f = 0.50 (DCM + 7% MeOH);

^1H NMR (300 MHz, CDCl ₃ ) δ 8.96 (br, 1H, HN(3)), 8.34-8.17 (m, 4H, H-arom), 7.07 (d, J=1.1 Hz, 1H, H-C(6)), 6.11 (t, J=6.3 Hz, 1H, H-C(1')), 5.57-5.45 (m , 1H, Н-С(5')), 5.15 (dd, J=6.6, 5.4 Hz, 1H, Н-С(4')), 4.38-4.23 (m, 1H, H-C(7')), 2.96 (dd, J=13.5, 6.9 Hz, 1H, H-C(3')), 2.26 (ddd, J=13.1, 10.3, 5.4 Hz, 4H, H-C(2'), H-C(6')), 1.91 (d, J=0.9 Hz, 3H, Me -C(5)).

ESI ⁺ -MSHR m/z, calculation for C ₁₉ H ₁₉ O ₈ N ₃ Na ([M+Na] ⁺ ) 440.1064, experiment 440.1072.

EXAMPLE 58

Design and synthesis of oligomers from alpha-anomeric monomers

To evaluate the incorporation of the modifications into the natural β-DNA chain, five oligonucleotides (ON16-20) with insertions of one or more thymidine 39 building blocks were prepared. To match the geometry of β-DNA, the modifications were introduced with polarity inversion, resulting in 3'-7' and 5'-'5 nucleoside linkers (see FIGS. 1 and 4). To investigate the pairing properties of this new system with natural nucleic acid and with itself, a fully modified ON21 containing all four nitrogenous bases, as well as its antiparallel (ON22) and parallel (ON23) fully modified complements, were prepared. Finally, a fully modified phosphorothioate ligand-containing chain (ON24) was synthesized to investigate possible antisense properties. The synthesis was performed on an automated synthesizer according to classical phosphoramidite chemistry. Fully modified chains were synthesized in the 5'→7' direction. Thus, 5% dichloroacetic acid in dichloroethane was required for complete cleavage of the DMTr protecting group at the 7' position. Under these conditions, the nucleotide coupling yield was >98% as determined by trityl assay. Fully modified chains can be cleanly cleaved from the universal solid support by efficient treatment with concentrated ammonia at 55°C overnight (see below for further details of the synthesis and analysis).

Method for the synthesis, removal of protective groups and purification of alpha-anomeric oligonucleotide

Oligonucleotide synthesis was performed on a Pharmaci-Gene-Assembler-Plus DNA synthesizer at a 1.3 μmol scale. Phosphoramidites for natural DNA (dT, dC4bz, dG2DMF, dA6Bz) and solid support (dA-Q-CPG 500, dmf-dG-Q-CPG 500, Glen Unysupport 500) were purchased from Glen Research. Phosphoramidites for natural DNA were prepared as a 0.1 M solution in MeCN, the coupling step duration was 4 min. Phosphoramidites for 7',5'-α-bc-DNA were prepared as a 0.1 M solution in 1,2-dichloroethane, the coupling was performed in a long step for 12 min. 5-(ethylthio)-1H-tetrazole (0.25 M in MeCN) was used as a coupling agent. Detritylation of the modified nucleoside was performed using 5% dichloroacetic acid in dichloroethane. Sulfurization was performed using 0.2 M phenylacetyl disulfide in MeCN/pyridine (1:1) mixture; the reaction time was 3.5 min. Introduction of the terminal group and oxidation were performed under standard conditions. Cleavage from the solid support and removal of protective groups in the oligonucleotide were performed by treatment with concentrated ammonia at 55°C for 16 h. After centrifugation, the supernatant was collected, the granules were additionally washed with H ₂ O (0.5 ml X2), and the resulting solutions were evaporated to dryness. Crude oligonucleotides were purified by ion-exchange HPLC (Dionex - DNAPac PA200). 25 mM Trizma buffer in _H2O , pH 8.0, was used as mobile phase “A”, and 25 mM Trizma, 1.25 M NaCl in _H2O , pH 8.0, was used as mobile phase “B”. For the phosphorothioate chain, 10 mM NaOH buffer in _H2O , pH 12.0, was used as mobile phase “A”, and 10 mM NaOH, 2.50 M NaCl in _H2O , pH 12.0, was used as mobile phase “B”. Salts were then removed from the purified nucleotides in Sep-pack C-18 cartridges. Concentration was determined by measuring absorbance at 260 nm on a Nanodrop spectrophotometer using the extinction coefficient corresponding to natural DNA oligonucleotides. Oligonucleotide characterization was performed by ^ESI mass spectrometry or LC-MS.

EXAMPLE 59

Pairing properties of modified oligodeoxynucleotides synthesized from alpha-anomeric monomers with complementary DNA and RNA

The duplex stability of the oligonucleotides was assessed using UV melting curves at 260 nm, and the corresponding T _m values were compared with natural DNA analogs (Table 1). Oligonucleotides ON16-17, with a single insert, provided strong destabilization upon pairing with complementary DNA and slightly lower destabilization upon pairing with RNA. The degradation with insertion is probably progressive, and ON18, with modifications at the two previously mentioned positions, further reduced the T _m values. However, when two or five modifications were sequentially incorporated (ON19-20), the destabilization provided by a single modification was reduced to approximately -3°C for DNA and -1.3°C for RNA. These data suggest that DNA binding to 7',5'-α-bc-DNA initiates a strong destabilization, with a decrease in T _m ranging from -4 to -9°C depending on the sequence in question. This destabilization upon binding to heterocyclic backbones has been previously observed for α-DNA (Aramini et al., Nucleic Acids Res 1998, 26, 5644, Aramini et al., Biochemistry 1997, 36, 9715) and for α-LNA (Nielsen et al., Chemistry - A European Journal 2002, 8, 712). This destabilization can be compensated for by introducing block modifications.

EXAMPLE 60

Pairing properties of fully modified oligonucleotides synthesized from alpha-anomeric monomers

As expected, all three fully modified ON21-23 sequences had synergistic melting behavior with parallel DNA and RNA complements (Figure 5), but not with antiparallel complements. The resulting 7',5'-α-bc-DNA/DNA duplexes were slightly less stable than the natural counterparts, with destabilization ranging from -0.1 to -0.5 °C per modification (Table 2). Surprisingly, ON21-23 were found to form very stable duplexes with RNA, with stabilization increases ranging from 1.3 to 1.5 °C per modification. There is a rather unexpected difference in the stabilization effects between the fully modified strands and the natural oligodeoxynucleotide strands with a single insertion or multiple insertions. This highlights the importance of synthesizing fully modified strands for characterizing the novel mating system.

To investigate mismatch recognition, UV melting experiments were performed using ON21 and parallel DNA complements containing all three alternative nucleobases at position 4 (Table 3). This mismatch is highly destabilizing and decreases the _Tm by -9.6 to -14.3°C. Compared to the natural analog, 7',5'-α-bc-DNA has improved mismatch recognition ability, with an additional decrease in _Tm of -1.0 to -2.4°C. Improved mismatch recognition should reduce potential side effects and is thus a promising property for potential antisense agents. The modification with a phosphorothioate linker fits well into the 7',5'-α-bc DNA context and a destabilizing effect of about -0.5°C per nucleotide has been reported for this modification (Kurreck, J. European journal of biochemistry/FEBS 2003, 270, 1628). ON24 retains good affinity for RNA, with a stabilizing effect of 0.6°C compared to natural DNA.

As for the related oligonucleotides, ON21 formed a very stable duplex with its antiparallel complement ON22, which yielded an unexpected T _{m of} 83.6 °C. Because of this high T _m, the full classical bend in the sigmoidal curve could only be observed in the absence of sodium chloride, in which case the T _m decreased to 68.6 °C (Table 4). Interestingly, the duplex formation provided a low hypochromism of only 10% (Figure 5). This is indicative of base stacking that differs from the classical helix, and thus one would expect to form a duplex with a geometry different from the canonical A- or B-duplexes. On the other hand, for ON21 and its parallel complement ON23, the melting transition in the sigmoidal curve was not observed (Figure 5). The resulting change in hypochromism is not different from the results of UV melting experiments performed on the two single strands separately, which was attributed to stacking of bases within the single strands. To investigate the ability of 7',5'-α-bc DNA to fold into an A-like helix, a melting experiment was performed using tricyclo DNA (tc DNA), a conformationally constrained analog of RNA (Renneberg et al., Journal of the American Chemical Society 2002, 124, 5993). When ON21 was mixed with the complementary parallel strand of tc DNA (Tc1), an unexpectedly high T _{m of} 81°C was observed, demonstrating the ability of 7',5'-α-bc DNA to fit into this helical geometry.

EXAMPLE 61

Thermodynamic data on duplex formation from alpha-anomeric oligomers

Thermodynamic data for the formation of ON21 duplexes with DNA and RNA and their natural analogues were obtained by substitution into the experimental melting curves according to the established procedure (Petersheim et al., Biochemistry 1983, 22, 256) (Table 5). As expected, the free energy profile ΔG at 25 °C is similar to the T _m data, where ON21⋅RNA is the most preferred duplex. As for the natural system, ON21 binds to natural nucleic acids with lower enthalpy. However, this destabilization is compensated by an entropy gain. Such characteristics are typical for the bc-DNA class and are determined by the conformational rigidity imparted by the ethylene bridge. Interestingly, the selectivity of 7',5'-α-bc-DNA for RNA over DNA is determined mainly by the enthalpy factor.

EXAMPLE 62

CD spectroscopy of alpha-anomeric oligomer

CD spectra of ON21 duplexes with DNA, RNA, or ON22 were acquired and compared with the corresponding natural DNA/RNA duplex (Figure 6). ON21 duplexes with DNA or RNA had CD characteristics roughly similar to the natural A/B helix. However, the ON21/DNA duplex lacked the negative signal at 210 nm and showed a 5 nm blue shift in the ellipticity at 226 nm and a corresponding increase in amplitude. The ON21/RNA duplex also had a peak with increased positive amplitude at 226 nm, and the positive ellipticity at 266 nm was shifted down by 4 nm and had a sharper peak. On the other hand, the modified homo-duplex had a highly atypical CD profile, characterized by a broad negative ellipticity in the range from 275 to 300 nm with a small amplitude and two positive peaks at 259 nm and 218 nm. In accordance with the low hypochromism change upon duplex formation, the CD spectrum of the homo-duplex indicates the formation of a structure different from the traditional helix.

EXAMPLE 63

Biological stability of alpha-anomeric oligomers

The biological stability of the fully modified ON21 oligonucleotide was studied under in vitro physiological conditions and compared with the corresponding natural oligonucleotide. The oligonucleotides were incubated in a 1:1 mixture of H ₂ O and human serum at 37°C and the reaction products were analyzed by PAGE with 20% denaturation. Specifically, ON21 and the corresponding natural oligonucleotide were diluted to 10 μM in a 1:1 mixture of H ₂ O and human serum (from US-born male AB plasma, sterile filtered (Sigma)). The reaction was performed in a volume of 20 μl and the mixtures were incubated at 37°C. Control reactions (a, f) were performed by incubating the oligonucleotides at a concentration of 10 μM in H ₂ O at 37°C for 24 h. The reactions were stopped at specific time points by the addition of formamide (20 μl). The resulting mixtures were stored at -20°C, denatured by heating at 90°C for 5 min, and analyzed by PAGE with 20% denaturation. Visualization was performed using a solution with a universal dye.

The experiment demonstrated complete cleavage of the natural DNA chain after only 4 hours (d), while the modified oligonucleotide remained completely stable even after 24 hours (j) (Figure 7). It is likely that the 7',5'-α-bc-DNA modification provides significantly improved biological stability.

EXAMPLE 64

Activation of complement C3 by alpha-anomeric oligomers

Complement activation is an important toxic response frequently associated with the in vivo use of antisense ONs. Furthermore, in vivo studies using tc-DNA have occasionally shown this acute toxicity, which therefore limits their applicability. In this context, it is of particular interest to investigate complement activation using 7',5'-α-bc-DNA containing phosphorothioate nucleoside linkers and to compare it with well-characterized modified or natural ONs. Experiments were performed by incubating mouse serum samples with 4 mg/ml of the ONs under study at 37°C for 45 min. Mouse complement C3 activation was then analyzed by ELISA using the PanSpecific C3 reagent and the SC5b-9 kit.

Incubation with ON24 (PS-7',5'-α-bc-DNA backbone) resulted in lower levels of complement protein C3 compared to natural DNA but higher levels compared to PS-DNA (Figure 8). The protein level was similar to non-toxic PO-tc-DNA. These promising results indicate that ON24 does not activate complement to a significant extent.

EXAMPLE 65

Antisense activity

Duchenne muscular dystrophy (DMD) is a fatal muscle degenerative disease caused by a mutation in the DMD gene, resulting in out-of-frame dystrophin transcripts and, ultimately, loss of functional dystrophin protein. In DMD, the function of the defective disease-associated pre-mRNA transcripts can be restored using antisense oligonucleotides (AOs). These AOs can alter the splicing profile and correct the defective out-of-frame dystrophin transcripts by excluding specific dystrophin exons. This restores the open reading frame and produces a truncated but functional dystrophin protein product (Yang et al., PloS one 2013, 8, e61584). The ability of the 7',5'-α-bc DNA backbone to trigger exon skipping was assessed in vitro by transfecting mdx mouse myoblasts, a mouse model of Duchenne muscular dystrophy, with ON24 using lipofectamine LTX. Mdx myoblasts were co-incubated with 7',5'-α-bc DNA, RNA was isolated, amplified by nested RT-PCR and analyzed using gels (PAGE).

The results indicate a high level of skipping of exon 23, as well as a significant level of double skipping of exons 22 and 23 (Figure 9). This double exon skipping, which is often observed for compounds that effectively restore disrupted reading frames (Mann et al., Proceedings of the National Academy of Sciences of the United States of America 2001, 98, 42; Mann et al., The journal of gene medicine 2002, 4, 644; Yin et al., J. Molecular therapy. Nucleic acids 2013, 2, e124; Yang et al., PloS one 2013, 8, e61584), indicates that ON24 has high exon skipping activity for therapeutic purposes in muscular dystrophy. Furthermore, 7',5'-α-bc DNA initiated exon skipping to a higher extent compared to the tc DNA modification, which is known to provide significant therapeutic effect in mice (Goyenvalle et al., Nat Med 2015, 21, 270). Thus, the 7',5'-α-bc DNA backbone fulfills all the requirements to initiate highly active exon skipping for therapeutic purposes.

EXAMPLE 66

X-ray crystal structure determination of alpha-anomers

Crystals of the monomers were obtained primarily to confirm the relative configuration of the 7',5'-α-bc-DNA class, but also to compare the present structure with the minimum energy structure predicted by initial calculations. To obtain crystals of the thymidine and guanosine monomers, p-nitrobenzoate was inserted at the O3' position to allow crystallization of the T monomers (compound 57). Cocrystallization of the resulting molecule with EtOAc yielded low-quality crystals. The resulting structure (Figure 3a) adopted a C1'-endo,O4'-exo conformation of the sugar and a C6'-endo conformation of the carbocyclic ring. This conformation orients the substituent at C5' to a pseudoequatorial position and the C7'-hydroxyl group to a pseudoaxial position. The sugar conformation in the present structure differs from the minimum energy structures predicted by initial calculations. However, additional analysis demonstrated that the p-nitrobenzoate substituent perturbed the conformation of the sugar. Good quality crystals were obtained for the protected guanosine 51. In this case (Figure 3b), the furanose adopted a nearly perfect C1'-exo conformation, with the carbocyclic ring adopting a geometrical configuration as described above. In this case, the structure perfectly matches one of the minimum-energy conformers predicted from initial calculations.

Compound 57: A colourless transparent crystal of [C ₁₉ H ₁₉ N ₃ O ₈ ] ₂ [0.5(C ₄ H ₈ O ₂ )] was placed in air and used for ambient X-ray structure determination. All measurements were performed on an Oxford Diffraction SuperNova 2D detector (Dupradeau et al., Nucleic Acids Res 2008, 36, D360) using monochromatic Mo Kα radiation (λ=0.71073 Ǻ) passed through mirror optics and filtered by Al (Lu et al., Nature protocols 2008, 3, 1213). Unit cell edges and the orientation matrix for data collection were obtained from least-squares refinement of reflectance setting angles in the range 1.7°<θ<28.07°. A total of 440 frames were acquired using ω-scans, exposure time 15+15 sec, rotation angle 1° per frame, crystal-detector distance 65.1 mm, T=223(2) K. Data transformation was performed using the CrysAlisPro program (Dupradeau et al., Nucleic Acids Res 2008, 36, D360). Intensity correction was performed to account for the Lorentz force and polarization effect and absorption correction was introduced within the framework of the multiple scanning method using SCALE3 ABSPACK in the CrysAlisPro package. Data acquisition and refinement parameters are given in Table 6. The structure was solved by direct methods using the SHELXT program, which showed the positions of all non-hydrogen atoms in the title compound. Anisotropic refinement of the positions of all non-hydrogen atoms was performed. All H atoms were placed in geometrically calculated positions and refined using the rider model, where each H atom was assigned a fixed isotropic substitution parameter value of 1.2 Ueq of the parent atom. The structure was refined for F ² using a full-matrix least squares method, minimizing the function Σw(F _o ² -F _c ² ) ² . The weighting scheme was based on computational statistics and included a factor to reduce the weight of intense reflections. All calculations were performed with SHELXL-2014/7. The compound crystallizes to form a monoclinic space group C 2, where the monoclinic angle is almost 90 degrees, indicating pseudo-merohedral twinning, which cannot be easily reversed. In addition, the p-NO ₂ -benzoate group in the parent molecule is disordered into two conformations, and the co-crystallizing acetate solvent is disordered about the two-fold axis. For all these reasons, the determination and refinement of the structure was somewhat difficult, only short intermolecular contacts were calculated, and the absolute configuration could not be determined (the Flack parameter was unattainable), but it was assigned according to the reaction sequence.

Compound 57: A colourless transparent crystal of [C ₁₆ H ₁₉ N ₅ O ₆ ]⋅(CH ₄ O) was placed in air and used for ambient X-ray structure determination. All measurements were performed on an Oxford Diffraction SuperNova 2D detector diffractometer (Dupradeau et al., Nucleic Acids Res 2008, 36, D360) using monochromatic Mo Kα radiation (λ=0.71073 Ǻ) passed through mirror optics and filtered by Al. The unit cell edges and the orientation matrix for data collection were obtained from least-squares refinements of the reflectance setting angles in the range 1.5°<θ<27.2°. A total of 970 frames were acquired using ω-scans, exposure time 45+45 sec, rotation angle 1° per frame, crystal-detector distance 65.1 mm, T=123(2) K. Data transformation was performed using CrysAlisPro software. Intensity correction was performed to account for the Lorentz force and polarization effect and absorption correction was introduced within the framework of the multiple scanning method using SCALE3 ABSPACK in the CrysAlisPro package. Data acquisition and refinement parameters are presented in Table 7. The structure was solved by direct methods using SHELXT software, which showed the positions of all non-hydrogen atoms in the title compound. Anisotropic refinement of the positions of all non-hydrogen atoms was performed. All H atoms were placed in geometrically calculated positions and refined using the “rider” model, where each H atom was assigned a fixed value of the isotropic substitution parameter equal to 1.2 Ueq. of the initial atom. The structure was refined on F ² using the full-matrix least-squares method, in which the function Σw(F _o ² -F _c ² ) ² was minimized. The weighting scheme was based on computational statistics and included a factor to reduce the weight of intense reflections. All calculations were performed in the SHELXL-97 program (Lu et al., Nature protocols 2008, 3, 1213).

EXAMPLE 67

Design and synthesis of oligomers from beta-anomeric monomers

A series of oligonucleotides containing one or more of the building blocks 12, 14, 19, and 25, as well as fully modified sequences, were synthesized in an automated synthesizer according to classical phosphoramidite chemistry (for details of the synthesis and analysis, see the supplementary information). Oligonucleotides containing one or more modifications were prepared mainly to determine the effect of the modifications on the T _m during complex formation with complementary DNA and RNA, and to determine the selectivity of Watson-Crick base pairing. Fully modified oligonucleotides were synthesized to determine the characteristics of pairing not only with natural DNA and RNA, but also to study internal pairing in 7',5'-bc-DNA. The main attention in this case was paid to whether this new structural analogue of DNA can form an independent base pairing system that may be of interest as an alternative genetic system.

Method for the synthesis, removal of protective groups and purification of beta-anomeric oligonucleotide

Oligonucleotide synthesis was performed on a Pharmaci-Gene-Assembler-Plus DNA synthesizer at a 1.3 μmol scale. Phosphoramidites for natural DNA (dT, dC ^4bz , dG ^2DMF , dA ^6Bz ) and solid support (dA-Q-CPG 500, dmf-dG-Q-CPG 500, Glen Unysupport 500) were purchased from Glen Research. Phosphoramidites for natural DNA were prepared as a 0.1 M solution in MeCN, the coupling step duration was 4 min. Phosphoramidites for bc DNA were prepared as a 0.1 M solution in 1,2-dichloroethane, the coupling was carried out in a long step for 12 min. 5-(ethylthio)-1H-tetrazole (0.25 M in MeCN) was used as a coupling agent. Endcapping, oxidation and detritylation were performed under standard conditions. Cleavage from the solid support and removal of protecting groups in the oligonucleotides were performed by treatment with concentrated ammonia at 55°C for 16 h. After centrifugation, the supernatant was collected, the pellets were additionally washed with H ₂ O (0.5 ml X2) and the resulting solutions were evaporated to dryness. Fully modified sequences were additionally treated with NaOH (0.4 M in H ₂ O/MeOH 1:1) for 1 h at room temp to completely cleave the universal support from the linker and then filtered through spin columns (Amicon Ultra centrifuge filters 0.5 ml, NOMM 3 kDa). Crude oligonucleotides were purified by ion-exchange HPLC (Dionex-DNAPac PA200). 25 mM Trizma buffer in _H2O , pH 8.0, was used as mobile phase “A”, and 25 mM Trizma, 1.25 M NaCl in _H2O , pH 8.0, was used as mobile phase “B”. The purified nucleotides were then desalted in Sep-pack C-18 cartridges. Concentration was determined by measuring absorbance at 260 nm on a Nanodrop spectrophotometer using the extinction coefficient corresponding to natural DNA oligonucleotides. Oligonucleotide characterization was performed by ^ESI mass spectrometry.

EXAMPLE 68

Pairing properties of modified β-oligodeoxynucleotides with complementary DNA and RNA

To determine the effect of incorporation of one or more modifications on duplex stability, modified oligodeoxynucleotides ON1-ON11, listed in Table 8, were prepared and the _Tm values of duplexes with complementary DNA and RNA were measured by analyzing UV melting curves.

According to the _ΔTm /mod data, the effect of single or double modifications on the thermal stability of the duplex was relatively minor. Purine modifications (ON6, 7) led to destabilization of duplexes with complementary DNA, but slightly increased the _Tm of duplexes with complementary RNA. In the pyrimidine series, however, more differences were observed. Modified thymidine nucleosides (ON1-3) generally provided a slight decrease in _Tm when RNA was used as a complement, while no clear patterns were observed for duplexes with DNA. Interestingly, both cytosine modifications (ON4, 5, 8-10) significantly stabilized duplexes with complementary DNA and at the same time had a lesser effect on the _Tm of duplexes with complementary RNA. The stabilizing effect was more pronounced for 5-methylcytosine nucleosides, highlighting the possibility of enhancing stacking interactions between adjacent base pairs when they are used.

To determine the selectivity of base pairing, the _Tm values of ON2 complexes with complementary DNA containing all three possible mismatches opposite the modified region were measured (Table 9). The _Tm values of mismatched duplexes decreased by 5.1 to 13°C, with the GT ambiguous pair, as expected, providing the least destabilization. The data obtained are in good agreement with the results obtained for a completely natural series of mismatched nucleic acids, indicating that the base pairing selectivity does not change significantly upon the introduction of modifications.

EXAMPLE 69

Pairing properties of fully modified P-oligonucleotides with

complementary DNA and RNA

A fully modified nonamer consisting of only pyrimidine bases (ON12) as well as three dodecamers (ON13-15) containing all four bases were prepared to study the affinity for complementary DNA or RNA in antiparallel or parallel orientation. According to the corresponding T _m data (Table 10), it is clear that binding was generally weak. Even under high salt conditions (1 M NaCl), there was no transition in the melting curve for mixtures of ON12 with antiparallel complementary DNA or RNA at temperatures in the range 5-80°C, suggesting that duplexes were not formed. However, transitions for duplexes with complementary antiparallel DNA and RNA were observed for the longer ON13-15 dodecamers. The Typ values for these duplexes were approximately 30°C lower than those of the natural control duplexes, yielding an average _ΔTm /mod of -2.5°C. This result was unexpected given the _Tm experiments with the individual modifications (Table 8) and highlights the importance of fully modified oligonucleotides in characterizing novel mating systems. Importantly, no evidence of duplex formation was detected when the corresponding parallel complements of DNA and RNA were used. This clearly demonstrates that 7',5'-bc DNA can interact with natural nucleic acids within antiparallel duplex structures, albeit at a much lower affinity.

EXAMPLE 70

Internal pairing of 7',5'-β-bc-DNA

ON14, which is the antiparallel complement, and ON15, which is the parallel complement of ON13, were developed to allow the study of internal pairing of 7',5'-bc DNA in both orientations. It was shown that the antiparallel ON13⋅ON14 duplex had a classical sigmoidal melting profile (Figure 11) with a _Tm of 54.5°C. This value was 5.4°C higher than that of the natural duplex with the same sequence. Interestingly, the hyperchromism at 260 nm of the 7',5'-bc DNA duplex was only 20% of that of the natural duplex. This indicates significantly different base stacking for the two duplex conformations. In the case of parallel orientation (ON13-ON15), the transition in the curve was absent, demonstrating the inability to form a parallel duplex in 7',5'-bc-DNA.

EXAMPLE 71

Thermodynamic data on duplex formation of beta-anomeric oligomers

For the ON13⋅ON14 duplex and the corresponding natural duplex, the enthalpy and entropy of the transition were determined by substituting the data into the experimental melting curves similarly to known methods (Table 11) (Petersheim et al., Biochemistry 1983, 22, 256). The data obtained suggest that the natural duplex has enthalpic stabilization, while the 7',5'-bc-DNA duplex is stabilized by the entropic component. This is consistent with previously obtained results for a number of bc-DNAs and indicates that conformational constraints of the 7',5'-bc-DNA backbone underlie the entropic stabilization. The values of the free enthalpy of duplex formation (AG) are consistent with the T _m data, which allows them to be considered as a measure of the thermodynamic stability of the duplex.

EXAMPLE 72

CD spectroscopy of beta-anomeric oligomers

To elucidate the structural properties of the 7',5'-bc-DNA homo-duplex and the 7',5'-bc-DNA/DNA hybrid duplex, CD spectra were acquired and compared with the spectrum of the corresponding natural DNA duplex (Figure 12). As expected, the natural duplex had the classical features of a B-DNA duplex. The 7',5'-bc-DNA homo-duplex, however, had a CD profile that was significantly different from that of the A- or B-type double helices. It was characterized by two ellipticity minima at approximately 215 and 265 nm and a maximum at 243 nm. The hybrid complex had a profile that was more similar to the DNA duplex. Compared to B-DNA, a red shift of the ellipticity minimum at 260 nm by about 15 nm and a red shift of the ellipticity maximum at 288 nm by about 7 nm and a decrease in amplitude were observed. The differences in the CD spectra thus indicate a different arrangement of the bases in the stacking caused by the change in the backbone geometry in each strand. This is consistent with the different hyperchromism values for the two pairing systems in the corresponding UV melting curves (Figure 11).

EXAMPLE 73

X-ray crystal structure of 7',5'-β-bc-DNA monomer

An analogue of 7',5'-bc-T containing a p-nitrobenzoate group at O7' was prepared. The resulting compound could be crystallized and its structure was determined (Figure 10). In addition to virtually confirming the relative configuration for a series of 7',5'-bc-DNAs, the authors also obtained information on the preferred conformation of the bicyclic sugar backbone. It was clearly shown that the furanose moiety exists in the ideal 1'-exo conformation, while the carbocyclic ring is present in the 6'-endo configuration, so that the oxy substituent at C7' is in a pseudo-axial position and the 5'OH group in a pseudo-equatorial position. This structure corresponds exactly to one of the two low-energy conformers determined from the initial calculations.

Compound 33: A colourless transparent crystal of [C ₁₉ H ₁₉ N ₃ O ₈ ] was placed in the open air and used for X-ray structure determination under ambient conditions. All measurements were performed on an Oxford Diffraction SuperNova 2D detector diffractometer (Oxford Diffraction (2010)) using monochromatic Mo Kα=0.71073 radiation. ), passed through mirror optics and filtered with Al (Macchi et al., J. Appl. Cryst 2011, 44, 763). The unit cell edges and the orientation matrix for data collection were obtained from the results of least-squares refinement of the reflectance setting angles in the range 2°<θ<27°. A total of 530 frames were acquired using ω-scans, exposure time 2.5+2.5 seconds, rotation angle 1° per frame, crystal-detector distance 65.1 mm, T=173(2) K. Data transformation was performed using CrysAlisPro software (version 1.171.34.44, Oxford Diffraction Ltd., Y., Oxfordshire, UK). Intensity corrections were performed to account for the Lorentz force and polarization effect and absorption correction was introduced within the multiple scan method using SCALE3 ABSPACK in the CrysAlisPro package. The data acquisition and refinement parameters are given in Table 12. The structure was solved by direct methods using the SHELXS-97 program (Sheldrick, ActaCryst. 2008, A64, 112-122), which showed the positions of all non-hydrogen atoms in the title compound. Anisotropic refinement of the positions of all non-hydrogen atoms was performed. All H atoms were placed in geometrically calculated positions and refined using the riding model, where each H atom was assigned a fixed isotropic substitution parameter value of 1.211 equiv. of the parent atom. Structure refinement was performed using a full-matrix least-squares method in which the function ∑w(F _o ² -F _c ² ) ² was minimized. The weighting scheme was based on computational statistics and included a factor to reduce the weight of intense reflections. All calculations were performed in SHELXL-97 (Macchi et al., J. Appl. Cryst 2011, 44, 763).

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LIST OF SEQUENCES

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formula (V))

<400> 6

gggntgttctc ga 12

<210> 7

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 7

ggatnttctc ga 12

<210> 8

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric cytosine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 8

ggatgttntc ga 12

<210> 9

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric cytosine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 9

ggatgttctnga12

<210> 10

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric cytosine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric cytosine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 10

ggatgttntn ga 12

<210> 11

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (4)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 11

gcannnnnac cg 12

<210> 12

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (6)..(6)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (7)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 12

nnnnnnnnn 9

<210> 13

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (11)..(11)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (12)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 13

nnnnnnnnnnnn nn 12

<210> 14

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(1)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (2)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric 5'-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (11)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<400> 14

nnnnnnnnnnnn nn 12

<210> 15

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<220>

<221> other_feature

<222> (11)..(11)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (V))

<220>

<221> other_feature

<222> (12)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (V))

<400> 15

nnnnnnnnnnnn nn 12

<210> 16

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 16

ggatgttcncga 12

<210> 17

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 17

ggangttctc ga 12

<210> 18

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 18

ggangttcnc ga 12

<210> 19

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 19

ggatgnnctc ga 12

<210> 20

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (4)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 20

gcannnnnac cg 12

<210> 21

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(1)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (2)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (11)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 21

nnnnnnnnnnnn nn 12

<210> 22

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (11)..(11)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (12)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 22

nnnnnnnnnnnn nn 12

<210> 23

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(1)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (1)..(12)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (2)..(2)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (3)..(3)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (5)..(5)

<223> 7',5'-phosphodiester-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (6)..(7)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (8)..(8)

<223> 7',5'-phosphodiester-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (9)..(9)

<223> 7',5'-phosphodiester-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphodiester-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 23

nnnnnnnnnnnn nn 12

<210> 24

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> other_feature

<222> (1)..(1)

<223> 7',5'-phosphorothioate-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (2)..(3)

<223> 7',5'-phosphorothioate-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (4)..(4)

<223> 7',5'-phosphorothioate-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (5)..(6)

<223> 7',5'-phosphorothioate-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (7)..(7)

<223> 7',5'-phosphorothioate-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (8)..(9)

<223> 7',5'-phosphorothioate-linked beta-anomeric guanine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (10)..(10)

<223> 7',5'-phosphorothioate-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (11)..(11)

<223> 7',5'-phosphorothioate-linked beta-anomeric thymine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<220>

<221> other_feature

<222> (12)..(13)

<223> 7',5'-phosphorothioate-linked beta-anomeric 5-methylcytosine

nucleotide containing a bicyclic sugar (bicyclic sugar

shown in formula (IV))

<220>

<221> other_feature

<222> (14)..(15)

<223> 7',5'-phosphorothioate-linked beta-anomeric adenine nucleotide,

containing bicyclic sugar (bicyclic sugar is shown in

formula (IV))

<400> 24

nnnnnnnnnn nnnnn 15

<---

Claims (66)

1. Compound of formula (I) where one of T ₁ and T ₂ is OR ₁ or OR ₂ ; and the other of T ₁ and T ₂ is OR ₁ or OR ₂ ; where R ₁ is H or a hydroxyl protecting group, wherein said hydroxyl protecting group is independently in each occurrence selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl (DMTr), 4,4',4''-trimethoxytrityl (TMTr), 9-phenylxanthine-9-yl(pexyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX), and R ₂ is a phosphorus-containing moiety, wherein said phosphorus-containing moiety is a phosphoramidite moiety represented by formula (X) where R ₅ is C ₁ -C ₉ alkyl substituted with cyano; R ₆ and R ₇ are independently C ₁ -C ₉ alkyl; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein said heterocyclic ring is selected from pyrrolidinyl, piperidinyl or morpholinyl, wherein said heterocyclic ring is optionally substituted with C ₁ -C ₃ alkyl; and wherein Bx is a purine base or a pyrimidine base. 2. The compound of claim 1, wherein Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine. 3. A compound according to claim 1 or 2, wherein said compound of formula (I) is a compound of formula (II) where (i) T ₁ is OR ₁ and T ₂ is OR ₁ or OR ₂ ; or (ii) T ₁ is OR ₁ or OR ₂ , T ₂ is OR ₁ . 4. The compound of claim 3, wherein T ₁ is OR ₁ or OR ₂ and T ₂ is OR ₁ . 5. A compound according to claim 1 or 2, wherein said compound of formula (I) is a compound of formula (III) where (i) T ₁ is OR ₁ and T ₂ is OR ₁ or OR ₂ ; or (ii) T ₁ is OR ₁ or OR ₂ , T ₂ is OR ₁ . 6. The compound of claim 5, wherein T ₁ is OR ₁ and T ₂ is OR ₁ or OR ₂ . 7. A compound according to any one of claims 1 to 6, wherein said phosphorus-containing moiety R ₂ is a phosphoramidite moiety of formula (X), wherein R ₅ is 2-cyanoethyl, and each of R ₆ and R ₇ is isopropyl. 8. The compound of claim 1, wherein said compound is selected from: 9. An oligonucleotide containing at least one monomer unit of formula (IV) wherein, independently, in each of said at least one monomer unit of formula (IV), one of _T3 or _T4 is a nucleoside linker group; the other of _T3 and _T4 is _OR1 , _OR2 , a 5'-terminal group, a 7'-terminal group or a nucleoside linker group, wherein _R1 is H and _R2 is a phosphorus-containing moiety, wherein said phosphorus-containing moiety is a phosphoramidite moiety represented by formula (X) where R ₅ is C ₁ -C ₉ alkyl substituted with cyano; R ₆ and R ₇ are independently C ₁ -C ₉ alkyl; or together with the nitrogen atom to which they are attached form a heterocyclic ring, wherein said heterocyclic ring is selected from pyrrolidinyl, piperidinyl or morpholinyl, wherein said heterocyclic ring is optionally substituted with C ₁ -C ₃ alkyl; and Bx is a purine base or a pyrimidine base. 10. An oligonucleotide according to claim 9, wherein Bx is selected from uracil, thymine, cytosine, 5-methylcytosine, adenine and guanine. 11. An oligonucleotide according to claim 9 or 10, wherein said monomer unit of formula (IV) is a monomer unit of formula (V) where (i) T ₃ is a nucleoside linker group and T ₄ is a 7'-terminal group, OR ₁ or OR ₂ ; or (ii) _T3 is a 5'-terminal group, _OR1 or _OR2 and _T4 is a nucleoside linker group; or (iii) _T3 and _T4 independently of each other represent a nucleoside linker group. 12. The oligonucleotide according to claim 11, in which (i) _T3 is a nucleoside linker group and _T4 is a 7'-terminal group or _OR1 ; or (ii) _T3 is a 5'-terminal group or _OR2 and _T4 is a nucleoside linker group; or (iii) _T3 and _T4 independently of each other represent a nucleoside linker group. 13. An oligonucleotide according to claim 9, wherein said monomer unit of formula (IV) is a monomer unit of formula (VI) where (i) T ₃ is a nucleoside linker group and T ₄ is a 7'-terminal group, OR ₁ or OR ₂ ; or (ii) _T3 is a 5'-terminal group, _OR1 or _OR2 and _T4 is a nucleoside linker group; or (iii) _T3 and _T4 independently of each other represent a nucleoside linker group. 14. The oligonucleotide according to claim 13, in which (i) _T3 is a nucleoside linker group and _T4 is a 7'-terminal group or _OR2 ; or (ii) _T3 is a 5'-terminal group or _OR1 and _T4 is a nucleoside linker group; or (iii) _T3 and _T4 independently of each other represent a nucleoside linker group. 15. An oligonucleotide according to any one of claims 9 to 14, wherein each nucleoside linking group is independently selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, a phosphonothioate linking group, a phosphinate linking group, a phosphorothioamidate linking group, or a phosphoramidate linking group. 16. An oligonucleotide according to claim 15, wherein each nucleoside linker group, independently of the others, is a phosphodiester linker group or a phosphorothioate linker group. 17. An oligonucleotide according to any one of claims 9 to 16, comprising at least two adjacent monomer units of formula (IV), wherein each of said adjacent monomer units of formula (IV) is independently linked to an adjacent adjacent monomer unit of formula (IV) by a nucleoside linker group, wherein said nucleoside linker group links the 5'-end and the 7'-end of two adjacent monomer units of formula (IV). 18. An oligonucleotide according to any one of claims 9-17, wherein said oligonucleotide comprises at least one nucleic acid sequence, wherein said nucleic acid sequence comprises said at least one monomer unit of formula (IV) and wherein said nucleic acid sequence is selected from any one of SEQ ID NOs: 1-24. 19. The oligonucleotide of claim 18, wherein said nucleic acid sequence is SEQ ID NO: 24. 20. An oligonucleotide according to any one of claims 9-12 or 15-19, wherein said oligonucleotide comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent monomer units of formula (V), wherein said nucleic acid sequence comprises at the 5'-end and at the 7'-end at least one nucleotide or nucleoside different from a monomer unit of formula (V), wherein the 5'-end of said nucleic acid sequence is linked to the 5'-end of said nucleotide or nucleoside other than the monomer unit of formula (V), and wherein the 7'-end of said nucleic acid sequence is linked to the 3'-end of said nucleotide or nucleoside other than the monomer unit of formula (V). 21. An oligonucleotide according to any one of claims 9-10 or 13-19, wherein said oligonucleotide comprises a nucleic acid sequence, wherein said nucleic acid sequence consists of at least two adjacent monomer units of formula (VI), wherein said nucleic acid sequence comprises at the 5'-end and at the 7'-end at least one nucleotide or nucleoside different from a monomer unit of formula (VI), wherein the 5'-end of said nucleic acid sequence is linked to the 3'-end of said nucleotide or nucleoside other than the monomer unit of formula (VI), and wherein the 7'-end of said nucleic acid sequence is linked to the 5'-end of said nucleotide or nucleoside other than the monomer unit of formula (VI). 22. An oligonucleotide according to any one of claims 9-21, wherein said monomer unit of formula (IV) is selected from: 23. An oligonucleotide according to any one of claims 9-22 for use as a medicinal product for the prevention, treatment or diagnosis of a disease. 24. The oligonucleotide of claim 23, wherein said disease is muscular dystrophy.