BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a class of oligonucleotides, modified oligonucleotides, or oligonucleosides ⁽ i . e. , phosphate-free oligonucleotides) optionally modified at the ends and having in their structure at least one linker of a non-nucleosidic nature containing at least one ( aza) nitrogen as an achiral site for attachment of a DNA-binding group or a DNA-interacting group or carrier or targeting ligand.

2. Description of Related Art

Oligonucleotides can be of value as therapeutic agents for the treatment of a wide variety of diseases. Classical therapeutics has generally focused on interactions with proteins, which either acting directly or through their enzymatic functions, contribute in major proportion to many disease states in animals and man. Compared to classical therapeutics, oligonucleotides offer the potential for a highly efficient specificity due to their capability for base pairing with complementary nucleic acid strands in a Watson-Crick or Hoogsteen manner. In that sense oligonucleotides provide a unique opportunity for gene therapy or the regulation of translation or transcription. The function of a gene starts by transcription of its information to a messenger RNA (mRNA) which, by interaction with the ribosomal complex, directs the synthesis of a protein coded for by its sequence. The synthetic process is known as translation. Translation process requires the presence of various cofactors and building blocks, the amino acids, and their transfer RNAs (tRNA), all of which are present in normal cells.

Transcription initiation requires specific recognition of a promoter DNA sequence by the RNA-synthesizing enzyme, RNA polymerase. In many cases in procaryotic cells, probably in all cases in eucaryotic cells, this recognition is based on sequence-specific binding of protein transcription factor to the promoter. Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors.

Synthetic oligonucleotides could be used as "antisense" probes involved in binding to transcellular RNA in a sequence-specific fashion such as Watson-Crick base pairing interactions. Thus, synthetic DNA could suppress translation in vivo. It also may be possible to effect the genome by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents.

Natural oligonucleotides, however, are relatively ineffective as therapeutic agents due to their poor permeability into the cell, and their rapid degradation by enzymes inside the cell. Therefore, relatively high concentrations of natural oligonucleotides are needed in order to achieve therapeutic effect.

In order to improve half life as well as membrane penetration, a large number of variations in polynucleotide backbones has been undertaken, although so far not with desired results. These variations include the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphoramidates, bridged phosphorothioates, bridged methylenephosphonates, dephospho internucleotide analogues with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various "plastic" DNAs, alpha-anomeric bridges, and borane derivatives .

For example, United States Patent 5,216,141 relates to DNA analogs containing sulfides, sulfoxides and sulfones as linking groups between subunits capable of forming bonds with natural oligonucleotides.

United States Patent 5,034,506 relates to polymeric compositions containing morpholino subunits linked together by achiral linkages. Each subunit is said to contain a purine or pyrimidine base pairing moiety. United States Patents 5,405,938 and 5,166,315 relate to polymers containing an uncharged 5- or 6- membered cyclic backbone having selected bases attached to the backbone. The polymer is said to be able to bind in sequence specific manner to a target sequence of a duplex polynucleotide.

International applications WO 92/20702, 92/20703 and 94/25477 all relate to the so-called peptide nucleic acids (PNAs) which are said to bind complementary single stranded DNA and RNA strands more strongly than a corresponding DNA. The PNAs are said to comprise ligands linked to a peptide or polyamide backbone via an aza nitrogen.

United States Patent 5,378,825 is directed to oligonucleotide analogs in which the normal phosphorodiester inter-sugar linkages are replaced with four atom linking groups.

International application WO 95/14706 is directed to PNA-DNA-PNA chimeric macromolecules, wherein the PNA and DNA portions are connected by amide linkages, amine linkages or ester linkages. » Today it has been recognized that nuclease resistance, fidelity of binding, and efficiency of binding of antisense or antigen oligonucleotides to a target RNA strand or double-stranded DNA are of great importance. At the same time there has been a long-felt need for methods and materials improving hybridization properties, providing high nuclease resistance and membrane penetration properties. The compounds and compositions of the present invention fulfill this need.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a macromolecule, at least a portion of which is of the

structure :

FORMULA I wherein each B is independently hydrogen, hydroxy, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety; each B ¹ is independently hydrogen, hydroxy, a ino, mercapto, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, an aromatic moiety, a targeting group, a carrier, a reporter group, or a soluble or non-soluble polymer; n is an integer from 1 to 50; each X is independently a single bond, methylene, methylenecarbonyl, C ₇ -C ₁₂ aralkylene or substituted aralkylene, C ₇ -C ₁₂ aralkylenecarbonyl or substituted aralkylenecarbonyl, or a group of the formula:

wherein each Z is independently a single bond, 0, S, NR ^S , C(=0)NR ⁶ or C(=0)NR ⁶ ; each Z ¹ is independently 0, S, NR ⁵ , methylene, or

C(CH ₃ ) ₂ ; each of p, q, r, and s is independently an integer from 0 to 20; each of R ¹ , R ² , R ³ and R ⁴ is independently hydrogen; Ci-Ca _, alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen; each of R ⁵ and R ⁶ is independently hydrogen; C -

C ₈ alkyl, which may be hydroxy-, or alkoxy-, or alkylthio- substituted; hydroxy; alkoxy; alkylthio; or amino; each of Q ¹ or Q ² independently comprises at least three atoms, at least one of which is carbon; each V is independently oxygen, sulfur, NR ⁸ or methy1ene; and each J is independently hydrogen, azido, halogen, -OR ⁷ , -R ⁷ or -NR ⁷ R ⁸ , wherein each R ⁷ is independently

-NR ^β R ⁹ or R ⁸ , wherein each of R ⁸ or R ⁹ is independently hydrogen, C ₃ -C ₁₀ branched alkyl or substituted alkyl, C _j ^C^ unbranched alkyl or substituted alkyl, C^C^ unbranched oxaalkyl or substituted oxaalkyl, C ₆ -C ₁₀ aryl or substituted aryl, C ₇ -C ₁₂ aralkyl or substituted aralkyl, C ₁ -C ₁₀ unbranched aminoalkyl or substituted unbranched aminoalkyl; C _j^ -Ci _Q unbranched aminooxaalkyl or substituted unbranched amiήooxaalkyl, C ₃ -C ₁₀ and N _x -N ₄ branched (polyamino- or

polyaza-)alkyl or substituted (polyamino- or polyaza-) alkyl, C^C _K) and N _x -N ₄ unbranched (polyamino- or polyaza-) alkyl or substituted (polyamino- or polyaza-)alkyl, ^C^ and N _x -N ₄ unbranched (polyamino- or polyaza-)oxaalkyl or substituted unbranched (polyamino- or polyaza-)oxaalkyl, a natural or non-natural amino acid side chain radical, or a protecting group.

In another aspect, the present invention relates to pharmaceutical compositions comprising an effective amount of a compound above, and a pharmaceutically suitable carrier.

In another aspect, the present invention relates to methods for the treatment of diseases caused by pathogenic organisms, which comprises administering to a host organism in need of such treatment an effective amount of a compound or pharmaceutical composition described above. The host organism may be any organism in need of such treatment, and includes mammals and humans.

In a further aspect, the present invention relates to methods for the treatment of tumors, which comprises administering to an organism in need of such treatment an effective amount of a compound or pharmaceutical composition described above. The organism may be any organism in need of such treatment, and includes mammals and humans.

_I n an _othe r aspect, the present invention relates to a compound having the formula :

FORMULA II wherein each B is independently a naturally occurring nucleobase, a non-naturally occurring nucleobase, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group; each B ¹ is independently hydrogen, hydroxy, amino, mercapto, a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group; t n is an integer from 1 to 50; each X is independently an optionally protected group selected from a single bond, methylene group, methylenecarbonyl, C ₇ -C ₁₂ aralkylene or substituted aralkylene, C ₇ -C ₁₂ aralkylenecarbonyl or substituted aralkylenecarbonyl or a group of the formula:

wherein

each Z is independently a single bond, 0, S, NR ⁶ , C(=0)NR ⁶ , C(=S)NR ⁶ , S(=0)NR ⁶ , or S(=0) ₂ NR ⁶ ; each Z ¹ is independently 0, S, Se, NR ⁵ , methylene, or C(CH ₃ ) ₂ ; each of p, q, r, and s is independently an integer from 0 to 20; each of R ¹ , R ² , R ³ and R ⁴ is independently hydrogen; ^-0 ₈ alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen; each of R ^s and R ⁶ is independently hydrogen; C _χ -

C ₈ alkyl, which may be hydroxy-, or alkoxy-, or alkylthio- substituted; hydroxy; alkoxy; alkylthio; or amino; each of Q ¹ or Q ² independently comprises at least three atoms, at least one of which is carbon; each V is independently oxygen, sulfur, NR ⁸ or ethylene, wherein R ⁸ is independently hydrogen, C ₃ -C ₁₀ branched alkyl or substituted alkyl, C ₁ -C ₁₀ unbranched alkyl or substituted alkyl, C _x -C ₁₀ unbranched oxaalkyl or substituted oxaalkyl, C ₆ -C ₁₀ aryl or substituted aryl, C ₇ -C ₁₂ aralkyl or substituted aralkyl, C^Cio unbranched aminoalkyl or substituted unbranched aminoalkyl; C^C^ unbranched aminooxaalkyl or substituted unbranched aminooxaalkyl, C ₃ - C ₁₀ and _! -N ₄ branched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C^C^ and N _x -N ₄ unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C ₁ -C ₁₀ and N^N, _} unbranched

(polyamino- or polyaza-)oxaalkyl or substituted unbranched

(polyamino- or polyaza-)oxaalkyl, a natural or non-natural amino acid side chain radical, or a protecting group; each J is independently, hydrogen, OR ⁷ , halogen, azide- or R ⁷ , any of which is optionally protected, wherein

each R ⁷ is independently -NR ⁸ R ⁹ or R ⁸ , wherein R ⁹ is independently hydrogen, C ₃ -C ₁₀ branched alkyl or substituted alkyl, C ₁ -C ₁₀ unbranched alkyl or substituted alkyl, C^C^ unbranched oxaalkyl or substituted oxaalkyl, C ₆ -C _1Q aryl or substituted aryl, C ₇ -C ₁₂ aralkyl or substituted aralkyl, C _x -

C ₁₀ unbranched aminoalkyl or substituted unbranched aminoalkyl; C _x -C ₁₀ unbranched aminooxaalkyl or substituted unbranched aminooxaalkyl, C ₃ -C _l0 and N _α -N ₄ branched

(polyamino- or polyaza-) alkyl or substituted (polyamino- or polyaza-) alkyl, Ci-C^ and N _x -N ₄ unbranched (polyamino- or polyaza-) alkyl or substituted (polyamino- or polyaza-) alkyl, C^C^ and N _x -N ₄ unbranched (polyamino- or polyaza-) oxaalkyl or substituted unbranched (polyamino- or polyaza-) oxaalkyl, a natural or non-natural amino acid side chain radical, or a protecting group; each Q ³ is independently -OX-, -SX- or -NR ⁸ X-, any of which is optionally protected; each Q ⁴ is independently oxygen, sulfur or NR ⁸ , any of which is optionally protected; Y is a protecting group;

Y ¹ is a spacer group linked to a solid support. In yet another aspect, the present invention relates to a compound represented by the formula:

o r

wherein : each B is independently a naturally occurring nucleobase , a non-naturally occurring nucleobase, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group ; each B ¹ is independently hydrogen, hydroxy, amino , mercapto , a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, a covalent or non-covalent DNA-binding group, a heterocyclic moiety, or an aromatic moiety, any of which optionally contains a protecting group; n is an integer from 1 to 50 ; each X is independently one of the following optionally protected groups : a single bond, methylene , methylenecarbonyl , C ₇ -C ₁₂ aralkylene or substituted aralkylene, C ₇ -C ₁₂ aralkylenecarbonyl or substituted aralkylenecarbonyl or a group of formula :

wherein each Z is independently a single bond, O, S, NR ⁶ , C(=0)NR ⁶ , C(=S)NR ⁶ , S(=0)NR ⁶ , or S(=0) ₂ NR ⁶ ; each Z ¹ is independently O, S, Se, NR ⁵ , methylene, or C(CH ₃ ) ₂ ; each of p, q, r and s is independently an integer from 0 to 20; each of R ¹ , R ² , R ³ and R ⁴ is independently hydrogen; C ₁ -C ₈ alkyl, which may be hydroxy-, or alkoxy-, or alkylthio-substituted; hydroxy; alkoxy; alkylthio; amino or halogen; each of R ⁵ and R ⁶ is independently hydrogen; C _χ -

C ₈ alkyl, which may be hydroxy-, or alkoxy-, or alkylthio- substituted; hydroxy; alkoxy; alkylthio; or amino; each of Q ¹ or Q ² comprises at least three atoms, at least one of which is carbon; each V is independently oxygen, sulfur, .NR ⁸ or methylene; each J is independently one of the following optionally protected groups: hydrogen, OR ⁷ , halogen, azide or R ⁷ , wherein each R ⁷ is independently -NR ^β R ⁹ or R ^β , wherein each of R ⁸ or R ⁹ is independently hydrogen, C ₃ -C, ₀ branched alkyl or substituted alkyl, C^C^ unbranched alkyl or substituted alkyl, C ₁ -C ₁₀ unbranched oxaalkyl or substituted oxaalkyl, C ₆ -C ₁₀ aryl or substituted aryl, C ₇ -C ₁₂ aralkyl or substituted aralkyl, C^-C^ _Q unbranched aminoalkyl or substituted unbranched aminoalkyl; C ₁ -C ₁₀ unbranched

aminooxaalkyl or substituted unbranched aminooxaalkyl, C ₃ -

C ₁₀ and N _x -N ₄ branched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C^-C^ and N _x -N ₄ unbranched (polyamino- or polyaza-)alkyl or substituted (polyamino- or polyaza-)alkyl, C^C-^ and N _x -N ₄ unbranched

(polyamino- or polyaza-)oxaalkyl or substituted unbranched (polyamino- or polyaza-)oxaalkyl, a natural or non-natural amino acid side chain radical, or a protecting group; each of oX and Q ² _, comprising at least one atom, is independently selected from optionally protected or activated fragments of Q ¹ or Q ² .

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1-8 each depicts comparisons of prior art compounds (labeled (A) in each Figure) with various compounds of the present invention, labeled (B-l) through (B-24) and (C-l) through (C-6) .

Figures 9-11 each depicts compounds of the present invention. Figures 12-14 each depicts comparisons of prior art compounds (labeled (A) in each Figure) with various compounds of the present invention, labeled (E-l) through (E-13) .

Figure 15 depicts compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to macromolecules that are able to function like oligonucleotides and which also possess other useful properties. As is illustrated in the Examples and Schemes of this specification, the macromolecules are constructed from basic nucleoside units and specific linker units bearing nucleobase or other nucleic acid-binding elements. The nucleoside units, joined by a nucleobase bearing linker of the present invention (i.e., the Q ¹ -N-Q ² segment of Formula I), forms trimeric units. The trimeric units can be further extended

to pentameric, heptameric and other, higher order macromolecules by addition of further nucleosides. The trimeric units (and/or the higher order units) can be connected via linkages other than those of the invention, as for example, via a normal phosphodiester linkage, a phospho hioate linkage, a phosphodithioate linkage, a phosphoroamidate linkage, a phosphotriester linkage, a methyl or other alkylphosphonate linkage or other linkage. The linkage of the present invention contains two parts (or sublinkages, i . e. , Q ¹ and Q ² in Formula I) separated by an aza-nitrogen atom. The aza-nitrogen atom is an achiral site of attachment of a nucleobase or any other nucleic acid binding moiety. In certain embodiments a single type of sublinkage is used to join nucleosides and aza-nitrogen atom(s) . In other embodiments, two different sublinkages are used to form trimeric units, or two or more different sublinkages are used to form the higher order units. Within the same macromolecule of the invention, sublinkages of different units (trimeric or higher order units) may be the same or of different types as described in this specification. Some trimeric units (and/or the higher order units) carry naturally occurring or non-naturally occurring nucleic bases attached to an achiral aza-nitrogen atom within an internucleosidic linkage, other units carry a nucleobase-binding ligand other than a nucleobase, described below more fully.

The naturally occurring nucleobases which are referred to herein include the four main naturally occurring nucleobases, i.e. thymine, cytosine, adenine or guanine, or other naturally occurring nucleobases, e. g. hypoxanthine, uracil, thiouracil, 5-methylcytosine, etc.

The non-naturally occurring nucleobases which are referred to herein include, for example, fluorouracil, bromovinyluracil, triazolcarboxamide, benzimidazole, etc. The heterocyclic moieties which are referred to herein include any heterocycle containing at least one heteroatom fused or non-fused ring systems, for example, nitroindole

derivatives, nitroimidazole derivatives, nitrotriazole derivatives, etc.

The DNA-binding groups which are referred to herein include: 1) covalent DNA-binding groups, which interact with DNA by means of chemical modification (for example, mustard gas derivatives, psoralen and its derivatives, itomycin C, etc.); 2) non-covalent DNA-binding groups, which interact with DNA by means of hydrogen bond formation, intercalation, electrostatic forces, etc. Hydrogen bond formation between functional groups of DNA (or RNA) and functional groups of nucleic acid binding ligands plays an important role in specific recognition of nucleic acids (especially double stranded nucleic acids) by antibiotics (e.g., distamycin, netropsin, echinomycin, etc.), or proteins (e.g., repressors, restrictases, etc.) , or oligonucleotides (e.g., DNA double strand formation, or DNA triple strand formation) . Intercalation is a special kind of stacking observed in nucleic acids which is associated with separation of two adjacent base pairs to allow insertion of a planar aromatic (hetero)-cyclic group, known as the intercalator. The intercalators capable of binding to double stranded nucleic acids which are referred to herein include, for example, acridine or its derivatives, phenanthridine or its derivatives, etc. The intercalators capable of binding to preferably triple stranded clusters of nucleic acids (compare to duplexes) which are referred to herein include, for example, coralyne (a member of the protoberberine family of alkaloids) , propidium bromide, etc. The carriers which are referred to herein include, for example, a polyamine group (e.g., polyethylenei ine, sper ine, spermidine, poly-L-lysine, starburst dendrimers, etc.), or lipophilic groups (e.g., cholesterol, alkyl chain, etc.), or a soluble polymer (e.g., polyethylene glycols, polysaccharides, proteins, etc.), or by a non- soluble polymer ( e. g. , dextranes, polyacrylamide derivatives, polyvinyl derivatives, etc.), or a targeting

ligand ( e. g. , sugar or sugar phosphate residues which act as binding sites to receptors on the surface of target cell, antibodies, immunoglobulins, etc.) .

As used herein, "nucleoside" refers to a unit composed of a heterocyclic base and a sugar, and includes the natural occurring nucleosides, including 2' -deoxy and 2'- hydroxy1 forms, e. g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) . In naturally occurring nucleosides, the heterocyclic base typically is adenine, cytosine, guanine, thymine or uracil; the sugar is normally deoxyribose, i . e. , erythro- pentofuranosyl, or ribose, i.e., ribo-pentofuranosyl. "Analogues" in reference to nucleosides includes synthetic nucleosides having modified base moieties (for example, 5 (6) -nitroindole, 4-nitrotriazole, 3 (4) -nitrobenzimidazole, 2-aminopurine, benzimidazole, 5-fluorouracil, and the like) and/or modified sugar moieties (for example, arabino, xylo or lyxo pentafuranosyl sugars; or substituted arabino, erythro, ribo, xylo or lyxo pentafuranosyl sugars; or acyclic moieties mimicking sugar; or hexose sugars; etc.) e.g. described by Scheit, Nucleotide Analogues (John Wiley, New York, 1980) . Such analogues include the natural and synthetic nucleosides with or without an appropriate protecting group for synthesis. As used herein, "nucleotide" refers to a nucleoside having a phosphate group esterified to at least one of the sugar hydroxyl groups.

The term "oligonucleotide" as used herein includes linear oligomers of natural or modified nucleosides, including deoxyribonucleosides, ribonucleosides, alpha-anomeric forms thereof, and the like, usually linked by phosphodiester bonds or analogues thereof ranging in size from a few monomeric units, e.g. 2-3, to several hundreds of monomeric units. "Analogues" in reference to oligonucleotide refers to structures including modified portions such as modified sugar moieties, modified base moieties or modified sugar linking moieties. Exemplary

among these are phosphorothioate, phosphorodithioate, phosphoroamidate, phosphoroanilidate, phosphodiester internucleoside linkages used in place of phosphodiester internucleoside linkages; deaza or aza purines and pyrimidines may be used in place of natural purines or pyrimidines; pyri idine bases having a substituent group at, for example, the 4-, 5- or 6-positions; purine bases having altered or replacement substituent groups at, for example, the 2-, 6- or 8-positions; or sugars having substituent groups at their 2'-position, substitutions for one or more of the hydrogen atoms of the sugar, or carbocyclic sugar. Preferably, oligonucleotides of the present invention are oligomers of the natural nucleosides having a length in the range of 2 to 50, and more preferably, having a length in the range of 2 to 20 monomeric units.

As mentioned above, the compounds of the present invention are represented by an oligonucleotide, or modified oligonucleotide, or so-called "oligonucleoside" (i.e. phosphate-free oligonucleotide) optionally modified at the ends, at least a portion of which has the structure of formula la:

Formula la

In Formula la, subs ituents W and W ¹ represent the remainder of the macromolecule. W and W ¹ may be any substituents which do not detract from the utility of the

present compounds. Preferably, each of W and W ¹ are independently -H; -OH; optionally modified phosphate or phosphate analogs; nucleosides or analogs thereof; nucleotides or analogs thereof; oligonucleotides or analogs thereof; amino; mercapto; a DNA intercalator; a covalent or non-covalent DNA-binding group; a heterocyclic moiety; or an aromatic moiety, any of which optionally contains a protecting group; peptides or analogs thereof; chelating groups ( e. g. , EDTA) ; polymers ( e. g. polya ides, polycations, polyanions, etc.) sugars; saccharides; polysaccharides; or lipophilic groups.

Preferably, each B independently comprises naturally occurring nucleobases, non-naturally occurring nucleobases, heterocyclic moieties, aromatic moieties, DNA intercalators, covalent or non-covalent DNA-binding groups; more preferably each B is a naturally occurring nucleobase or non-naturally occurring nucleobase. The most preferred choice for each B is a naturally occurring nucleobase.

In certain preferred embodiments, at least one of B ¹ is a naturally occurring nucleobase; in other preferred embodiments at least one of B ¹ is a non-naturally occurring nucleobase; in other preferred embodiments at least one of B ¹ is a DNA intercalator (such as acridine derivatives, phenazine derivatives, etc.); in other preferred embodiments at least one of B ¹ is a covalent DNA-binding group (such as mustard gas derivatives, psoralen derivatives, etc. ) ; in other preferred embodiments at least one of B ¹ is a DNA-binding antibiotic (such as daunomycin, actinomycin D or another representative of the actinomycin family, netropsin and its derivatives, distamycin and its derivatives, etc.); in other preferred embodiments at least one of B ¹ is a reporter group (such as a fluorescent or che iluminescent label, biotin, etc.); in other preferred embodiments at least one of B ¹ is a targeting group for recognition of definite cells (such as an antibody or saccharide) ; in other preferred embodiments at least one of

B ¹ is a soluble or non-soluble polymer; in other preferred embodiments at least one of B ¹ is a reactive functional group suitable for postsynthetic modification of an oligonucleotide (such as amino, mercapto, aldehyde, carboxyl, etc.) .

In a preferred embodiment, n is from 1 to 20; most preferably n=l.

In certain preferred embodiments, when B ¹ is a naturally occurring nucleobase or a non-naturally occurring nucleobase, X comprises from 1 to 4 atoms; in other preferred embodiments, when B ¹ is selected from DNA intercalators, covalent or non-covalent DNA-binding groups, or DNA-binding antibiotics, X ¹ comprises from 1 to 12 atoms; in a most preferred embodiment, when B ¹ is a nucleobase, X ¹ is methylenecarbonyl.

In preferred embodiments, each of Q ¹ and Q ² independently contains at least one of the following groups: oxygen; sulfur; substituted carbon; carbonyl; thiocarbonyl; sulfone; sulfoxide; Ci-Cg alkylene; C ₂ -C ₈ alkenylene; C ₂ -C ₈ alkynylene; C^Cg oxaalkylene or thiaalkylene or azaalkylene each containing one or two different heteroatoms or heteroatoms of the same type; or NR ⁷ , or ⁺ NR ⁸ R ⁹ , or NR ⁷ C(=0)-, or -NR ⁷ C(=S)-, or -NR ⁷ S(=0)-, or -NR ⁷ S(=0),- in either orientation, wherein R ⁷ , R ⁸ and R ⁹ have been specified above; or X, wherein X has been specified above; or a group of:

wherein each of Z ⁴ or Z ⁵ is independently selected from the group consisting of a single bond, 0, S, and NR ⁷ , wherein R ⁷ has been specified above; each Z ³ is independently selected from the group

consisting of hydrogen, R ⁸ , OR ⁷ , SR ⁷ , and NR ⁷ R ⁸ , wherein R ⁷ and R ⁸ have been specified above; and each Z ² is independently selected from the group consisting of 0, S, and NR ⁷ , wherein R ⁷ has been specified above.

In certain preferred embodiments each of Q ¹ and Q ² independently contains from 2 to 8 atoms; in more preferred embodiments Q ¹ and Q ² each independently contains from 3 to 6 atoms; most preferably 4 or 5 atoms. Most preferably, each Q ¹ is independently selected from the following groups: -0-CH ₂ -CH ₂ -CH ₂ -, -0-CH ₂ -C(=0)-NH-, -NH-NH-C(=0)-CH ₂ -,

-NH-N=CH-CH ₂ -, -NH-NH-CH ₂ -CH ₂ -, -0-NH-C(=0) -CH ₂ -,

-0-N=CH-CH ₂ -, -0-NH-CH ₂ -CH ₂ -, -CH ₂ -NH-C(=0) -CH ₂ -, -NH-NH-C(=0)-NH-, -0-C(=0) -NH-CH ₂ -CH ₂ -,

-0-P(=Z ² )Z ³ -NH-CH ₂ -CH ₂ -, or -CH ₂ -NH-CH ₂ -CH ₂ -, in either orientation; and each Q ² is independently selected from the following groups: Q ¹ , -CH ₂ -0-CH ₂ -CH ₂ -CH ₂ -, -CH ₂ -0-CH ₂ -C(=0) -NH-, -CH ₂ -NH-NH-C(=0)-CH ₂ -, -CH ₂ -NH-N=CH-CH ₂ -,

-CH ₂ -NH-NH-CH ₂ -CH ₂ -, -CH ₂ -0-NH-C(=0) _r CH ₂ -, -CH ₂ -0-N=CH-CH ₂ -,

-CH ₂ -0-NH-CH ₂ -CH ₂ -, -CH ₂ -NH-C(=0) -CH ₂ -, -CH ₂ -NH-NH-C(=0) -NH-,

-0-C(=0)-NH-CH ₂ -CH ₂ -, -0-P(=Z ² )Z ³ -NH-CH ₂ -CH ₂ -, or -CH ₂ -CH ₂ -NH-CH ₂ -CH ₂ -, in either orientation. Preferably, at least one of V is oxygen. The most preferred choice for each V is oxygen.

In more preferred embodoments, at least one of J is independently selected from the group of hydrogen, fluorine or 0CH ₃ ; the most preferred choice for J is hydrogen. The compounds of the invention are synthesized by adaptation of standard oligonucleotide synthesis procedures, or by adaptation of standard peptide synthesis procedures, or by a combination of both mentioned

procedures in solution or on solid phase.

Using standard oligonucleotide synthesis procedures, the compounds of the present invention may be prepared by incorporation of fragments of formula I onto the 5' -end of a growing oligonucleotide or modified oligonucleotide chain, represented by the formula II:

Formula II

wherein each of B, B ¹ , Q ¹ , Q ² , V and J are as described above, any of which is optionally blocked with a protecting group if appropriate ( e. g. , acetyl, isobutyryl, phenoxyacetyl, benzoyl, cyanoethyl, 4- nitrophenylethyloxycarbonyl, 4-nitrophenylethyl, benzyloxycarbonyl, p-anisyldiphenylmethyl, di-p- anisylphenyl ethyl, pixyl; tert-butyloxycarbonyl, diphenylcarbamoyl, for amidino, acetamidino, trialkylsilyl having from 3 to 14 carbon atoms, 9-fluorenylmethyl carbamate, or the like; see Greene and Wuts, Protective Groups in Organic Synthesis, 2nd Edition (John Wiley, New York, 1991)); each Q ³ independently comprises a single bond, oxygen, sulfur, -NR ⁸ -, -0X-, -SX-, -X-, or -NR ⁸ X-, wherein X and R ⁸ have the meanings specified above in connection with formula I; more preferably each Q ³ independently comprises a single bond, -OCH ₂ -, -SCH ₂ -, -CH ₂ -, -NR ⁸ CH ₂ -, -CH ₂ CH ₂ -, and

-OCH ₂ CH ₂ -; the most preferred choice for Q ³ is -OCH ₂ ~; each Q ⁴ independently comprises a single bond, oxygen, sulfur and NR ⁸ , wherein R ⁸ has the meanings specified above for formula I; Y is a protecting group such as triphenylmethyl, p- anisyldiphenylmethyl, di-p-anisylphenylmethyl, pixyl, trialkylsilyl having from 3 to 14 carbon atoms, 9- fluorenylmethyl carbamate, trifluoroacetyl, or the like; more preferably Y is p-anisyldiphenylmethyl, di-p- anisylphenylmethyl, or pixyl;

Y ¹ is a spacer group linked to a solid support, which spacer comprises carbonyl, ester, carbamate, urethane, hydrazide, C ₁ -C ₁ alkylene or modified alkylene, C ₆ -C ₁₄ aralkylene or modified aralkylene, C ₆ -C ₁₄ alkylarene or modified alkylarene, C^C^o oxaalkylene or thiaalkylene or azaalkylene each containing from one to fifty different heteroatoms or hetroatoms of the same type, where aza groups are, optionally, protected by amino protecting groups, C ₁ -C ₁₄ alkylenecarbonyl or alkylenethiocarbonyl or alkylenesulfone or alkylenesulfoxide, C^C^o oxaalkylenecarbonyl or thiaalkylenecarbonyl or azaalkylenecarbonyl (or their thiocarbonyl or sulfone or sulfoxide analogues) each containing from one to fifty different heteroatoms or hetroatoms of the same type, where aza groups are, optionally, protected by amino protecting groups; or a group of the formula (III) :

^—-s ° ° P — N H ¹⁰ R ¹¹ ' O H ' P— O H • ^π - P — Z ⁶

Z ⁵ H Z ⁵

( 1 ) ( 4 ) ( 5 )

Formula (III) or salts thereof, wherein each of R ¹⁰ or R ¹¹ independently comprises C ₃ -C ₁₀ branched alkyl, C^C^ unbranched alkyl or oxaalkyl, C ₆ -C ₁₀

aryl, C ₇ -C ₁₂ aralkyl; a more preferred choice for each of R ¹⁰ or R ¹¹ is C ₂ -C ₃ branched alkyl or C _x -C ₄ unbranched alkyl; the most preferred choice for each of R ¹⁰ or R ¹¹ is isopropyl; R ¹² is C ₂ -C ₈ alkylene, C ₂ -C ₈ alkenylene or -C ₂ -C ₈ oxaalkylene, comprising one or two heteroatoms; most preferably R ¹² is a morpholino group;

Z ⁵ is any phosphate protecting group; preferably, 4-Cl-C ₆ H ₄ -0-, 2-Cl-C ₆ H ₄ -0-, 4-N0 ₂ -C ₆ H ₄ CH ₂ CH ₂ -0-, 2,4-N0 ₂ -C ₆ H ₃ CH ₂ CH ₂ -0-, 2, 4-Cl-C ₆ H ₃ -0-, 2,3-Cl-C ₆ H ₃ -0-,

NCCH ₂ CH ₂ -0-, NCCH ₂ C(CH ₃ ) ₂ -0-, CH ₃ 0-, (Z) ₃ CCH ₂ -0-, R ¹⁰ S-,

R ¹⁰ SCH ₂ CH ₂ -0-, or R ¹⁰ SO ₂ CH ₂ -O-, wherein Z is halogen and R ¹⁰ is independently selected from the meanings described above; z ⁶ is -F, -Cl, -Br, -I, imidazol-1-yl, tetrazol-1-yl, 1,2,4-triazol-l-yl and 1-hydroxy-benzotriazol-O-yl; in certain preferred embodiments Y ¹ is a functionalized solid suDport of CPG or polystyrene nature; in other preferred embodiments Y ¹ is -HP(=0)0H and salts thereof; in other preferred embodiments Y ¹ is -P(OCH ₂ CH ₂ CN) (N(i-Pr) ₂ ) or

-P(OCH ₃ ) (N(i-Pr) ₂ ) •

Compounds of Formula (III) are commonly known (with reference to the activable phosphorus group, Y ¹ ) as phosphoroamidites, (III-l and -2), phosphorous acids (III- 3), H-phosphonates (III-4) and activated phosphodiesters (III-5) . Structures (III-3) and (III-4) represent moderately strong acids, and the reagents represented by these structures are generally isolated and used as their organically soluble salts. The novel compounds for incorporation of fragment of formula I onto the 5'-end of a growing modified oligonucleotide chain, represented by the group of formula IV:

o r

Formula IV

wherein each of B, Bl, Q ¹ , Q ² , V, X and J are as described above, any of which is optionally blocked with a protecting group; each of Q ^x _x and Q _χ comprises optionally protected or activated fragments of Q ¹ or Q ² ; more preferably each of Q ¹ ^ and Q ² _χ contains from one to three atoms, any of which optionally contains a protecting group; in most preffered embodiments each Q^ independently comprises Y ² -NH-CH ₂ ~,

Y ² -NH-0-, Y ² -NH-NH-, Y ² -NH-, Y ² -NH-CH ₂ -CH ₂ ~, Y -NH-NH-CH ₂ -,

Y ² -NH-C(=0)-CH ₂ -, Y ² -NH-C(=0)-NH-, Y ² -NH-NH-C(=0) -, wherein

Y ² is a protecting group or hydrogen; more preferably Y ² comprises p-anisyldiphenylmethyl, di-p-anisylphenylmethyl, pixyl, 9-fluorenylmethyl carbamate, or trifluoroacetyl; and each Q ² _χ independently comprises E-CH ₂ -CH ₂ -, E-CH ₂ -, E-NH-,

E-0-, wherein E is independently halogen, aldehyde, acetal, -S(=0) ₂ R ¹³ , or -COR ¹³ , wherein R ¹³ is a hydroxyl or activated group; more preferably R ¹³ is selected from halogen, hydroxyl, pentafluorophenoxy, tetrafluorophenoxy, p-nitrophenoxy, or N-succinimidoxy.

The compounds of the present invention may be divided into five groups:

(1) oligonucleotide analogues, wherein the non- nucleosidic linkage bears a nucleic base; (2) oligonucleotide analogues, wherein the non- nucleosidic linkage bears a covalent DNA-binding group, or a non-covalent DNA-binding group, or a DNA-intercalator, or a DNA-binding antibiotic;

(3) oligonucleotide analogues, wherein the non- nucleosidic, preferrably non-phosphate containing linkage, is used as a new type of linker connecting clusters of oligonucleotides or modified oligonucleotides;

(4) oligonucleotide analogues, wherein the non- nucleosidic linkage bears a carrier, or polymer, or targeting ligand, or lipophilic group; and

(5) oligonucleotide analogues, wherein the non- nucleosidic linkage bears a reporter group, such as biotin.

The oligonucleotide analogues (l)-(5) are able to recognize both single stranded and double stranded nucleic acids. The oligonucleotide analogues (1) with fragments of formula I placed on the 3'- and/or 5'-end or distributed along the oligonucleotide chain demonstrate the ability to bind DNA fragments and at the same time possess increased enzymatic stability. The oligonucleotide analogues (2) with fragments of formula I placed in definite positions along the oligonucleotide chain bind efficiently to double stranded DNA or RNA, and attachment of a covalent DNA- binding group, or a non-covalent DNA-binding group, or a DNA-intercalator, or a DNA-binding antibiotic, provides another opportunity to overcome the problem of recognition of polypyrimidine tracts by triplex forming

oligonucleotides (TFO) . The oligonucleotide analogues (3) can be used as a new type of TFO. The oligonucleotide analogues (4) and (5) are useful in the synthesis of oligonucleotide conjugates. The improved enzymatic stability and binding ability and cell membrane penetration of the compounds of the invention render them efficient as antisense (binding to RNA) or antigene (binding to DNA) agents.

Also, the invention provides reagents and methods for inhibiting transcription and/or replication of particular genes or for degradation of particular regions of double stranded DNA in cells of an organism by administering to said organism a compound of invention as defined above.

Further, the invention provides reagents and methods for killing or mutating cells (such as tumor cells) or pathogenic organisms (such as viruses, bacteria, fungi, etc.) by contacting said cells or organisms with compounds or compositions of the present invention which have specificity for such cells or organisms. Viruses susceptable to treatment according to the present invention would be readily determined by one of ordinary skill, and could include herpes simplex virus (HSV) , human papillo avirus (HPV) , human immunodeficiency virus (HIV) , etc. For therapeutic or prophylactic treatment, the compounds of the present invention may be formulated in a pharmaceutical composition, which may include, in addition to an effective amount of active ingredient, pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like. Pharmaceutical compositions may also include one or more other active ingredients such as antimicrobial agents, antiinflammatory agents, and the like.

The pharmaceutical compositions of the present invention may be administered in a number of ways as will be apparent to one of ordinary skill. Administration may be done topically, orally, by inhalation, or parenterally.

for example.

Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Oral formulations include powders, granules, suspensions or solutions in water or non-aqueous media, capsules or tablets, for example. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be used as needed.

Parenteral formulations may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

The dose regimen will depend on a number of factors which may readily be determined, such as severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with a course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. One of ordinary skill may readily determine optimum dosages, dosing methodologies and repetition rates. In general, it is contemplated that unit dosage form compositions according to the present invention will contain from about 0.01 mg to about 100 mg of active ingredient, preferably about 0.1 mg to about 10 mg of active ingredient. Topical formulations (such as creams, lotions, solutions, etc.) may have a concentration of active ingredient of from about 0.01% to about 50%, preferably from about 0.1% to about 10%.

The following Figures and Schemes illustrate, but not limit, the compounds and utilities of the present invention. Figures I-VI represent some trimeric fragments of the present invention with the variations in structures of linking moieties d and Q ₂ (see Formula I of present

Description) . All depicted structures from B-l (Fig. 1) through B-24 (Fig. 6) represent some oligonucleoside trimeric fragments of the invention with variations in the linking moieties, wherein B ⁿ is preferably a nucleic base.

The structures C-1 (Fig. 7) through C-9 (Fig. 9) represent examples wherein the aza nitrogen atom in Q _! - -Q ₂ serves as a site of attachment for an intercalating moiety. Formula C-1 (Fig. 7) illustrates the case where an acridine residue is attached through an acyl-type spacer; in formula C-2 the acridine residue is attached through an alkyl spacer. In both structures the intercalator is bound to the N-atom in propyliminopropyl internucleoside linking moiety. The structures from C-3 through C-6 (Fig. 8) demonstrate examples of attachment of an intercalator to the aza-atom in a relatively long (20-30 atoms) phosphate-free internucleoside linking moiety. Figure 9 shows the structures of the present invention with an attached coralyne residue. As was shown (J.S. Lee, Biochemistiry, 1993, v.32, pp. 5591-5597), coralyne demonstrates considerable preference for triplex binding (binding constants are about 10 ⁶ M ^_1 ) compared to duplex binding (binding constants are about 10 ³ -10 ⁴ M ^_1 ) . Coralyne conjugated to a triple helix forming oligonucleotide can be favorable for self-stabilization of the formed triplex.

Figures 10 and 11 represent situations where a DNA covalent binding moiety (i.e., psoralen) is conjugated to a internucleoside linker of the present invention. Psoralen binds preferentially to double-stranded DNA molecules and attachment of psoralen to an abasic site within the triplex forming oligonucleotide can considerably increase accuracy of site modification of the DNA duplex.

Figures 12-14 demonstrate examples of trimeric units of the present invention bearing a functional group attached to the aza-nitrogen. These functional groups are used as a site for postsynthetic modification of oligonucleotides with DNA-active substances (such as intercalators, alkylators, DNA-binding antibiotics, or any other nucleic acid binding group as described above) . Various methods for the synthesis of compounds according to the present invention are summarized in the following schemes, which are described in detail below.

Scheme t

1-yl;

*-N-(ιc»tyl)cytldln»-1-yl;

6-HHp ancwyacttyl)td«nln»4-yl;

2-N-(prιβnoxyaetyl>g-»πln»-9-yl;

B and B ¹ la tβltctad Indaptndanlly Irem: βιy lna-1-yl; eyidlna-1-yl; adtnln*-β-yl; guanlna-9-yl;

Scheme II

wherein B _x and B ¹ _X is selected independently from: thymine-1-yl;

3-N-{ben2yloxymethyl)thymine-1 -yl;

4-N-(isobutyryl)cytidine-1 -yl;

6-N-(phenoxyacetyl)adenine-9-yl;

2-N-(phenoxyacetyl)guanine-9-yl;

B and B ¹ is selected independently from: thymine-1-yl; cytidine-1-yi; adenine-9-yl; guanine-9-yl.

Scheme IV

wherein Q ³ _x is selected from single bond, oxygen, - CH _r , -N(CH ₃ )-, -NfCOCFj)-, -N(Pac)-;

Q ³ is selected from single bond, oxygen, - CH _r , -N(CH ₃ )-, -NH-.

Scheme V

39 wherein Q ³ x is selected from single bond, oxygen, - CH ₂ -, -N(CH ₃ h -N(COCF ₃ h -N(Pac)-i

Q ³ Is selected from single bond, oxygen, • CH _r> -N{CH ₃ )-, -NH-.

Scheme VI

UMVEIOH

Scheme VII

55 56 57

1. CθCVNaBH</IMOH

2. CF _j COOEWEIOH

Scheme VIII

wherein B ¹ _x is selected from: thymine-1-yl;

4-N-{acetyl)cytidIne-1 -yl;

6-N-(phenoxyacetyl)adeπine-9-yl;

2-N-(phenoxyacetyl)guaπine-9-yl;

B ¹ Is selected from: thymine-1-yl; cytidine-1-yl; adenine-9-yl; guanine-9-yl;

Scheme IX

Scheme X

Scheme XI

Scheme XII

Scheme XIII

Scheme XIV

87 88

Scheme XV

92 93

Scheme XVI

1.3%TFAΛ>CM 1. βO%NaHDUF 2. CSr,/P(Ptι), 2. Compound β

Scheme I illustrates the synthesis of trinucleoside units containing fragments of formula B-l (Fig. 1) with -OCH _j CH _j CH^f -B ¹ ] -CH ₂ CH ₂ CH ₂ OCH ₂ - (3 '→4' ) intersugar linkage. Nucleoside with a protected base and partially protected sugar moiety (compound 1) is cyanoe hylated (as in Example 2 , infra) at the 3 '-position, reduced to the aminopropyl derivative (compound 5, Example 5) and then trifluoroacetylated (compound 6, Example 6) . The other chain of transformation is allylation of the 5'-position of the nucleoside followed by conversion to the 3-bromopropyl derivative (compound 9) . These reactions are exemplified in Examples 7, 8 and 9. Compound 6 is alkylated with compound 9 to yield compound 10, that after deprotection of the bases and aza-nitrogen of the oligonucleosidic backbone gives compound 12 (Example 5) . Compound 12 is a key substance for synthesis of various compounds of the present invention. For example, acylation of compound 12 with an activated ester of a carboxymethyl derivative of different heterocyclic bases ( e. g. , thy ine, cytosine, adenine and guanine) gives, respectively, trinucleosides 14a, 14b, 14c and 14d (as in Examples 16-18, infra) . Acylation of compound 12 with an N-protected ω-amino acid derivative ( e.g. , N-Fmoc-4-aminobutyric acid) and incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having an amino modified (abasic) site (motif E-2, Fig. 12) in its structure, wherein the aliphatic amino-group serves as a site of attachment for various DNA-active groups. Acylation of compound 12 with an S-protected ω-mercapto acid derivative (e.g. , 3-Tr-4-mercaptobutyric acid) and incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having a mercapto modified (abasic) site (motif E-3, Fig. 12) in its structure, wherein an aliphatic mercapto group serves as a site of attachment for various DNA-active groups.

Acylation of compound 12 with an activated derivative of

2,3-di-O-Fmoc-l-O-carboxymethyl glycerine and incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having a cis-diol modified (abasic) site (motif E-4, Fig. 12) in its structure, wherein the cis-diol group after periodate oxidation provides an aldehyde group as a site of attachment for various DNA-binding groups. Acylation of compound 12 with an activated derivative of N-Fmoc- protected monohydrazido derivative of a dicarboxylic acid ( e. g. , adipic, pimelic, suberic, etc.) and incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having a hydrazido modified (abasic) site (motif E-5, Fig. 12) in its structure, wherein the hydrazido group after deprotection serves as a site of attachment for various DNA-active groups.

Scheme II illustrates synthesis of intermediates for preparation of oligonucleosides containing fragments with a -OCH ₂ CH ₂ CH ₂ N[X-B ¹ ]CH ₂ C(=0)NHQ ³ CH ₂ - (3 '→4' ) intersugar . linkage. Such oligonucleotide analogs include compounds having formulas B-2, B-3, or B-4 (Fig. 1) . Compound 6 (see Scheme I and Example 6) is a starting substance for the synthesis of such intermediates. Compound 6 is alkylated with the tert-butyl ester of bromoacetic acid in DMF in the presence of sodium hydride to give compound 19 (Example

23), that after hydrogenation is converted to compound 20 (Example 24) . The trifluoroacetyl protecting group is removed from compound 20 by ammonolysis to give compound 21 (Example 25) . Compound 21 is acylated with an activated carboxymethyl derivative of a nucleic base to give compound 22 (Examples 26, 27, 28), which after deprotection and the switching of the 5'-O-protecting group from tetrahydropyranyl to monomethoxytrytil is converted to the carboxylic acid derivative 23 (Examples 29, 30, 31). Compound 23 can be used for condensation directly, or transformed, for example, into activated ester 24 (Examples 32, 33, 34) .

Scheme III illustrates synthesis of intermediates for preparation of oligonucleosides containing fragments with a -Q ³ NHC(=0)CH ₂ N[X-B ¹ ]CH ₂ CH ₂ CH ₂ OCH ₂ - (3'→4') intersugar linkage. The oligonucleosides include compounds having formulas B-5, B-6, or B-7 (Fig. 2). Compound 7 (see Scheme I, Example 7) is a starting material for the synthesis of those intermediates. Compound 7 is cyanoethylated to give compound 25 (Example 35) , that after reduction is trifluoroacetylated to compound 26 (Example 36) . Compound 26 is alkylated with the tert-butyl ester of bromoacetic acid in DMF in the presence of sodium hydride to give compound 27 (Example 37), which after acidic deprotection is converted into a carboxylic acid derivative 28 (Example 38) . Compound 28 is treated with a saturated solution of ammonia in ethanol and acylated with an activated carboxymethyl derivative of a nucleic base to give compound 29 (Examples 39, 40 and 41). Compound 29 is dimethoxytritylated to protect the 3 '-hydroxyl group and to give compound 30 (Example 42) , which can be used for condensation directly, or transformed, for example, into activated ester 31 (Example 43).

Scheme IV illustrates examples of synthesis of trinucleosides 33 containing motifs of formulas B-2, B-3, or B-4 (Fig. 1) starting from compounds of general formula 24. Also, Scheme IV shows a route of conversion of partially protected trinucleoside 32 into phoshoramidite 34 and CPG derivative 35.

Scheme V illustrates examples of synthesis of trinucleosides 37 containing motifs of formulas B-5, B-6, or B-7 (Fig. 2) starting from compounds of general formula 31. Also Scheme V demonstrates the conversion of partially protected trinucleoside 36 into phoshoramidite 38 and CPG derivative 39.

Scheme VI illustrates synthesis of peptide-like oligonucleosides 54 containing

-OCH ₂ CH ₂ CH ₂ N[X-B ¹ ]CH ₂ C(=0)NHCH ₂ CH ₂ ~ (3'→4' ) intersugar

linkages. The methodology of synthesis is similar to that described in Scheme I. Homonucleoside with a protected base and partially protected sugar moiety (compound 40, Example 43) is cyanoethylated at the 3 '-position to give compound 41 (Example 44), which after switching of the 5'- O-protecting group from monomethoxytrytil to tetrahydropyranyl, is converted to compound 42 (Example 45) . Compound 42 is reduced to the aminopropyl derivative (compound 43, Example 46) and then amino group is blocked with trifluoroacetyl protecting group to give compound 44 (Example 47) . Compound 44 is alkylated with the tert-butyl ester of bromoacetic acid in the presence of sodium hydride (compound 45, Example 48). The 6'-0-THP group is converted to the brσmo derivative (compound 46, Example 49) through the treatment with CBr ₄ /P(Ph) ₃ in THF.

Compound 46 is treated with the sodium salt of di-tert- butyliminodicarboxylate in DMF to give compound 47 (Example 50), which after hydrgenolysis and ammonia treatment is converted to compound 48. Compound 48 is acylated with an activated carboxymethyl derivative of a nucleic base to give modified dinucleoside 49 (Examples 52 and 53). Acid labile protecting groups (Boc- and tert- butyl protecting groups) are removed with 50% TFA/DCM and the 6'-amino function is blocked with a monomethoxytrytil protecting group to give compound 50 (Examples 54 and 55) . Compound 50 can be used as a building block for solid- phase peptide-like synthesis of oligonucleosides with regular repeating elements depicted in square brackets in formula of compound 54. Compound 50 also can be used as a building block for the synthesis of peptide-like oligonucleosides in solution as shown in Scheme VI (compounds 53 and 54, Examples 59-64) .

Scheme VII illustrates the synthesis of peptide-like oligonucleosides 69 containing -OCH ₂ CH ₂ CH ₂ N[X- B ¹ ]CH ₂ C(=0)NHN(CH ₃ )CH ₂ - (3'→4') intersugar linkages. The methodology of synthesis is similar to described in Scheme

VI .

Scheme VIII illustrates the synthesis of trinucleoside phoshoramidite 18 and trinucleoside CPG derivative 17, containing the motif of formula B-l (Fig. 1) starting from compounds of general formula 13 (see Scheme I) .

Scheme IX illustrates the synthesis of 5'- monomethoxytrytil protected trinucleoside (compound 71) with an abasic site containing an aminoalkyl linker, and conversion of that compound into phoshoramidite 72.

Incorporation of phoshoramidite 72 into an oligonucleotide chain gives, after deprotection, an oligonucleotide having an amino modified (abasic) site (motif E-2, Fig. 12) in its structure, wherein the aliphatic amino-group is utilized as a site of selective attachment for various DNA-active groups, e. g. , an acridine moiety (Structure C-1, Figure 7) ; a coralyne moiety (Structure C-8, Figure 9); or psoralen moiety (Structure D-l, Figure 10) .

Scheme X illustrates the synthesis of 5'- monomethoxytrytil protected trinucleoside (compound 74) with an abasic site containing an aliphatic mercapto group, and conversion of this compound into phoshoramidite 75. Incorporation of phoshoramidite 75 into an oligonucleotide chain gives, after deprotection, an' oligonucleotide having a mercapto modified (abasic) site (motif E-3, Fig. 12) in its structure, wherein the aliphatic amino-group is utilized as a site of selective attachment for various DNA- active groups, e. g. , a psoralen moiety (Structure D-2, Figure 10) . Scheme XI illustrates the synthesis of trinucleoside phoshoramidite 75 containing a Fmoc-protected hydrazido modified abasic site (motif E-5, Fig. 13) .

Scheme XII illustrates the synthesis of trinucleoside phoshoramidite 81 with a 2,3-di-O-Ac-l-O- carboxymethyl glycerol modified abasic site (motif E-4, Fig. 12) . Incorporation of that trimeric unit into an oligonucleotide chain gives, after deprotection, an

oligonucleotide having a cis-diol modified abasic site (motif E-4, Fig. 12) in its structure, wherein a cis-diol group, after periodate oxidation, provides an aldehyde group as a site of attachment for various DNA-active groups.

Scheme XIII illustrates the synthesis of phoshoramidite 83 with an abasic site containing a trifluoroacetyl protected aza-nitrogen in a trinucleoside backbone (motif E-l, Fig. 12) . Incorporation of this trimeric unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide with an abasic site, containing no substituents at the aza-nitrogen in the trinucleoside backbone (motif E-l, Fig. 12) in its structure. Scheme XIV illustrates the synthesis of an activated ester of a carboxyl derivative of coralyne 88. Compound 88 is used for postsynthetic modification of oligonucleotides containing an aminoalkyl linker with a coralyne moiety. Scheme XV illustrates the synthesis of an activated ester of a carboxyl derivative of psoralen 93. Compound 93 is used for postsynthetic modification of oligonucleotides containing an aminoalkyl linker with a psoralen moiety. Scheme XVI illustrates the synthesis of phoshoramidite 101 with several abasic sites containing trifluoroacetyl protected aza-nitrogens in an oligonucleoside backbone (motif E-9, Fig. 14) . Incorporation of that unit into an oligonucleotide chain gives, after deprotection, an oligonucleotide having regular phosphodiester oligonucleotide clusters connected with abasic polyoxaazaalkylene fragments containing no negatively charged phosphate groups in the fragment backbone (motif E-9, Fig. 14) in its structure. The following Examples illustrate, but not limit, the compounds and utilities of the present invention.

All temperatures are in degrees Celsius (25°C refers

to ambient or room temperature) . All parts not otherwise indicated are by weight, except for mixtures of liquids, which are by volume. The following abbreviations are employed: AcOEt for ethylacetate; DCE for 1,2- dichloroethane; DCM for dichlorom thane; DIPEA for diisopropylethylamine; DMF for dimethylformamide; DMSO for dimethylsulfoxide; EtOH for ethanol; MeOH for methanol; THF for tetrahydrofuran; Py for pyridine; Phe for a phenyl radical; PPTS for pyridium p-toluenesulfonate; TFA for trifluoroacetic acid; Thy for a thymin-1-yl radical; Me for a methyl radical; Et for an ethyl radical; Gly for a glycine amino acid residue; dT for deoxythy idine; DMT for a dimethoxytrytil protecting group; MMT for a monomethoxytrytil (p-anisyldiphenylmethyl) protecting group; BOM for a benzyloxy ethylene protecting group; CE for a cyanoethyl protecting group; Bzl for a benzyl protecting group; Boc for a tert-butoxycarbonyl protecting group; t-Bu for a tert-butyl protecting group; THP for a tetrahydropyranyl protecting group; NMR for nuclear magnetic resonance spectrum; MS for mass spectrum; TLC for thin layer chromatography on silica gel; HPLC for high pressure liquid chromatography; mp for melting point; mp d for melting point with decomposition; bp for boiling point. In reporting NMR data, chemical shifts are given in ppm and coupling constants (J) given in Hertz (Hz) . All melting points are uncorrected.

Example 1

3 -N-Benzyloxymβthyl-5 ' -O- (p-anisyldiphenylmethyl ) thymidine , 1

To a stirred solution of 5 ' -0- (p- anisy Idiphenylmethyl ) thymidine, (25.75 g, 50 mole) in anhydrous DMF (350 ml) is added a suspension of 60% NaH (2 . 00 g, 50 mmole) . The mixture is stirred at room temperature for 1 hour, and BOM-C1 (7 . 67 g, 49 mmole ) is added. The mixture is stirred at room temperature for 2 hours , and DMF is evaporated. The residue is dissolved in

1 liter of a mixture AcOEt-H ₂ 0. Organic layer is washed successively with water (2x250 ml) and brine (2x250 ml) , dried (MgS0 ₄ ) , evaporated in vacuo to dryness, and the residue is chromatographed over a silica gel column (8x35 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 40%) as eluent to give 3-N-3 '-0- (di-benzyloxymethy1) -5'-0- (p- anisyldiphenylmethyl) thymidine, 4.15 g (11%), as white foam, and compound 1, 27.00 (85%), as white foam. ^- M (CDC _j ), 1: 7.59 (q, 1H, ⁴ J=1.0 Hz, H-6, Thy); 7.45-7.10 (gm, 17H, Phe, BOM&MMT) ; 6.82 (m, 2H, Phe, MMT) ; 6.42 (m, 1H, H-l', dT) ; 5.45 (s, 2H, NCH ₂ , BOM); 4.67 (s, 2H, OCH ₂ ,

BOM); 4.53 (m, 1H, H-3' , dT) ; 4.05 (m, 1H, H-4' , dT) ; 3.75 (s, OMe, MMT); 3.45 ( , 1H, H-5', dT) ; 3.32 (m, 1H, H-5 ' , dT) ; 2.41 (m, 1H, H-2 ' , dT) ; 2.25 (m, 1H, H-2' , dT) ; 1.44 (d, 3H, CH ₃ , Thy). ¹³ C-NMR (CDC1 ₃ ) , 1: 163.5 (C-4, Thy);

158.6 (C-4, Phe-OMe, MMT); 150.9 (C-2, Thy); 143.8&143.6 (C-1, Phe, MMT); 137.7 (C-1, Phe, BOM); 134.7 (C-1, Phe- OMe, MMT); 134.5 (C-6, Thy); 130.3-125.2 (gs, C-2, C-3, C- 4, Phe, BOM&MMT); 113.3 (C-3, Phe-OMe, MMT); 110.3 (C-5, Thy); 87.0 (-C.(Phe) ₂ Phe-OMe) ; 86.0 (C-1', dT) ; 85.3 (C-4', dT); 72.1 (C-3', dT) ; 72.0 (0CH ₂ , BOM); 70.4 (NCH ₂ , BOM);

63.5 (C-5', dT) ; 55.1 (OMe, MMT); 41.0 (C-2', dT) ; 12.4 (CH ₃ , Thy) .

Example 2

3-N-Benzyloxymethyl-3 ' -O- (2-cyanoβthyl) -5' -O- (p- anisyldiphβnylmethyl) thymidine, 2

To a stirred solution of compound 1 (12.70 g, 20 mmole) in anhydrous THF (150 ml) is added a suspension of 60% NaH (60 mg, 50 mmole) in one portion and dropwise neat acrylonitrile (5 ml) over 5 minutes. The resulting mixture is stirred at ambient temperature for an additional 1 hour.

The mixture is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (500 ml) . The organic layer is

washed with brine (2x250 ml) , dried (MgS0 ₄ ) , evaporated in vacuo to dryness, and the residue is chromatographed over a silica gel column (4.5x30 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 15%) as eluent to give compound 2, 12.66 g (92%), as a colorless oil. ^-NMR (CDC1 ₃ ) : 7.56

(q, 1H, ⁴ J=1.0 Hz, H-6, Thy); 7.45-7.15 (gm, 17H, Phe, BOM&MMT); 6.85 (m, 2H, Phe, MMT); 6.34 (m, 1H, ³ J _ ₂ ,=5.7&8.0 Hz, H-l', dT) ; 5.49 (s, 2H, NCH ₂ , BOM); 4.71 (s, 2H, OCH ₂ , BOM); 4.19 (m, 1H, ³ J ₃ ,_ ₄ ,=2.3 Hz, H-3 ' , dT) ; 4.09 (m, 1H, ³ J ₄ ,_ ₅ ,=2.7 Hz, H-4' , dT) ; 3.79 (s, OMe, MMT) ;

3.90&3.76 (m&m, 1H+1H, H-5'); 3.60 ( , *, Oϋ ₂ CH ₂ CN) ; 3.52 ( , 3H with *, H-5', dT) ; 3.34 (dd, 1H, J ₅ ,_ ₅ ,=10.6 Hz, H- 5', dT) ; 2.54 (t, **, OCH ₂ £H. ₂ CN) ; 2.49 (m, 3H with **, ³ J ₂ ,_ ₃ ,=2.2 Hz, ² J ₂ ,_ ₂ ,=13.6 Hz,H-2', dT) ; 2.20 (m, 1H, ³ J ₂ ,_ ₃ ,=6.1 Hz, 1.52 (d, 3H, CH ₃ , Thy) . ¹ C-NMR (CDC1 ₃ ) : 163.4 (C-4,

Thy); 158.8 (C-4, Phe-OMe, MMT); 150.9 (C-2, Thy); 143.7 (C-1, Phe, MMT); 137.9 (C-1, Phe, BOM); 134.6 (C-1, Phe- OMe, MMT); 134.1 (C-6, Thy); 130.3-127.3 (gs, C-2, C-3, C- 4, Phe, BOM&MMT); 117.4 (- N) ; 113.3 (C-3, Phe-OMe, MMT) ; 110.5 (C-5, Thy); 87.2 (-£ (Phe) ₂ Phe-OMe ) ; 85.3 (C-1', dT) ;

83.7 (C-4', dT); 80.3 (C-3', dT) ; 72.1 (OCH ₂ , BOM); 70.4

(NCH ₂ , BOM); 63.9 (0£H ₂ CH ₂ CN) ; 63.5 (C-5', dT) ; 55.2 (OMe,

MMT); 37.6 (C-2', dT); 18.8 (OCH ₂ £H ₂ CN) ; 12.5 (CH ₃ , Thy).

Example 3

3-N-Benzyloxymethyl-3 '-O- ( 2 -σyanoethyl) thymidine, 3

A solution of compound 2 (12.04 g, 17.5 mmole) in 3% TFA/DCE is kept at ambient temperature for 15 minutes. The reaction mixture is washed successively with 5% NaHC0 ₃ (3x150 ml) and water (2x150 ml) , dried (MgS0 ₄ ) , evaporated in vacuo to dryness and the residue is chromatographed over

a silica gel column (4.5x20 cm) using 3% MeOH/CHCl ₃ as eluent to give compound 3, 6.90 g (95%), as a colorless oil. MS: [M+l]=416; [M+1+NH ₃ ]=433. ^-NMR (CDC1 ₃ ) : 7.43

(s, IH, H-6, Thy); 7.4-7.2 (gm, 5H, Phe, BOM); 6.12 (m, IH, H-l') ; 5.48 (s, 2H, NCH ₂ , BOM); 4.69 (s, 2H, 0CH ₂ , BOM);

4.24 (m, IH, H-3 ' ) ; 4.09 (m, IH, H-4'); 3.90&3.76 (m&m, 1H+1H, H-5'); 3.66 (m, 2H, OCiI ₂ CH ₂ CN) ; 2.97 (t, IH, 5' -OH);

2.61 (t, 2H, OCH ₂ Cϋ ₂ CN) ; 2.34 (m, 2H, H-2 ' ) ; 1.91 (s, 3H,

CH ₃ , Thy) . ¹³ C-NMR (CDC1 ₃ ) : 163.4 (C-4, Thy); 150.9 (C-2,

Thy); 137.8 (C-1, Phe, BOM); 135.6(C-6, Thy); 128.19& 127.59&127.56(C-2, C-3, C-4, Phe, BOM); 110.2 (C-5, Thy) ; 85.4 (C-1', dT); 84.8 (C-4', dT) ; 79.7 (C-3', dT) ; 72.1 (OCH ₂ , BOM); 70.4 (NCH ₂ , BOM); 63.9 (OCH ₂ CH ₂ CN) ; 62.4 (C-5', dT) ; 36.9 (C-2', dT); 19.0 (OCH ₂ £H ₂ CN) ; 13.1 (CH ₃ , Thy) .

Example 4 3-N-Benzyloxymethyl-3 ' -O- (2-cyanoethyl) -5 ' -O- (tβtrahydropyran-2-yl) thymidine, 4

To a stirred solution of compound 3 (6.90 g, 16.6 mmole) in anhydrous DCM (150 ml) is added PPTS (100 mg, 0.4 mmole) and dropwise DHP (5 ml) over 15 minutes. The resulting mixture is stirred at ambient temperature for an additional 3 hours. The reaction mixture is evaporated in vacuo to dryness, coevaporated with toluene (2x100 ml) , dissolved in AcOEt (250 ml) and washed successively with 5% NaHC0 ₃ (3x100 ml) and brine (2x100 ml) . The residue is chromatographed over a silica gel column (4.5x30 cm) using toluene/AcOEt (1/1) as eluent to give compound 4, 7.62 g ⁽ 92%), as a colorless oil. MS: [M+l]=500; [M+1+NH ₃ ] =517.

^X H-NMR (CDC1 ₃ ): 7.59&7.52 (m&m, IH, H-6, Thy); 7.40-7.20 ⁽ gm, 5 H, Phe ,B0M); 6.31 (m, IH, H-l'); 5.49 (s, 2H, NCH ₂ , BOM); 4.69 (s, 2H, 0Cji ₂ , BOM); 4.61 (m, IH, H-l, THP) _; 4.22 (m, 2H with H-3', H-4'); 4.20 (m, 2H with H-4', H-3 ' ) _;

4.10-3.50 (g , 6H, H-5' + H-5 (THP) + 0Cϋ ₂ CH ₂ CN) ; 2.62 (t,

2H, OCH ₂ Cϋ ₂ CN) ; 2.45 (m, IH, H-2 ' ) ; 2.05 (m, IH, H-2 ' ) ;

1.94&1.91(s&s, 3H, CH ₃ , Thy); 1.85-1.40 (gm, 6H, H-2,-3,-4,

THP) . ¹³ C-NMR (CDC1 ₃ ): 163.2 (C-4, Thy); 150.7 (C-2, Thy); 137.8 (C-1, Phe, BOM); 134.3&134.0 (C-6, Thy);

128.1&127.5&127.4(C-2, C-3, C-4, Phe, BOM); 109.91&109.85 (C-5, Thy); 99.9&99.0 (C-1, THP); 85.7&85.6 (C-1', dT) ; 83.5&83.3 (C-4' ₍ dT) ; 80.5&80.4 (C-3', dT) ; 71.9 (OCH ₂ ,

BOM); 70.3 (NCH ₂ ' BOM); 67.5&67.2 (C-5', dT) ; 63.83&63.77 (0£H ₂ CH ₂ CN) ; 63.3&62.8 (C-5, THP); 37.6&37.S (C-2', dT) ;

30.6&30.5 (C-2, THP); 25.02&24.97 (C-4, THP); 20.0&19.7 (C- 3, THP) ; 18.8 (OCH ₂ £H ₂ CN) ; 13.1&12.9 (CH ₃ , Thy) .

Exam le 5 3-N-Benzyloxymethyl-3 ' -O- ( 3-aminopropyl) -5 ' -O- (tetrahydropyran-2-yl) hymidine, 5

To a stirred solution of compound 4 (7.0 g, 14 mmole) and CoCl ₂ -6H ₂ 0 (6.66 g, 28 mmole) in MeOH (150 ml) is added NaBH ₄ (4.48 g, 140 mmole) in four portions over 15 minutes. The resulting mixture is stirred at ambient temperature for an additional 1 hou and 28% NH ₄ OH (70 ml) is added and stirred at ambient temperature for 15 minutes. The mixture is centrifuged, the supernatant is kept and the precipitate is washed twice with MeOH. The combined supernatants are evaporated, the residue is suspended in CHC1 ₃ , the insoluble material is filtered off and the filtrate is evaporated in vacuo to dryness. The residue is chromatographed over a silica gel column (4.5x25 cm) using CHCl ₃ /MeOH/28%NH ₄ OH (100/3/0.25) as eluent to give compound 5, 4.37 g (62%), as a yellowish oil. MS: [M+l]=504. ^X H-

NMR (CDC1 ₃ ) : 7.61&7.54 (q&q, IH, H-6, Thy); 7.45-7.20 (gm, 5 H, Phe ,BOM); 6.32 (m, IH, H-l', dT) ; 5.49 (s, 2H, N£H ₂ , BOM); 4.70 (s, 2H, OCJJ ₂ , BOM); 4.65&4.58 (m&m, IH, H-l,

THP) ; 4.20 (m, IH, H-4 ' , dT) ; 4.09 (m, *, H-3 ' , dT) ; 4.08&3.95&3.69&3.57 (dd&dd&dd&m, *, H-5 ' , dT) ; 3.90&3.56 (m&m, *, H-5, THP); 3.50 (m, 7H with *, OCH ₂ CH ₂ CH ₂ NH ₂ ) ; 2.81

(t, 2H, OCH ₂ CH ₂ £H ₂ NH ₂ ) ; 2.44 ( , IH, H-2 ' , dT) ; 2.00 (m, **, H-2', dT) ; 1.94&1.91(d&d, **, CH ₃ , Thy); 1.72 (m, **,

OCH ₂ CH ₂ CiJ ₂ NH ₂ ) ; 1.78&1.57 (m&m, 12H with **, H-2,

-3,-4, THP) ; 1.38 (bs, 2H, OCH ₂ CH ₂ CH ₂ NH ₂ ) . ¹³ C-NMR (CDCl ₃ ) :

163.4 (C-4, Thy); 150.8 (C-2, Thy); 137.9 (C-1, Phe, BOM); 134.4&134.2(C-6, Thy); 128.1&127.6&127.5 (C-2, C-3, C-4, Phe, BOM); 109.9 (C-5, Thy) ; 99.9&99.8 (C-1, THP) ;

85.9&85.8 (C-1', dT) ; 83.8&83.7 (C-4', dT) ; 79.9&79.8 (C- 3', dT); 72.0 (OCH ₂ , BOM) ; 70.3 (NCH ₂ , BOM); 67.9&67.4 (C-

5', dT) ; 67.3&67.2 (OCH ₂ CH ₂ CH ₂ NH ₂ ) ; 63.2&62.5 (C-5, THP);

39.2 (OCH ₂ CH ₂ CH ₂ NH ₂ ) ; 37.9&37.8 (C-2', dT) ; 33.29&33.26 (OCH ₂ £H ₂ CH ₂ NH ₂ ) ; 30.60&30.55 (C-2, THP); 25.12&25.09 (C-4,

THP); 20.0&19.5 (C-3, THP); 13.1&13.0 (CH ₃ , Thy) .

Example 6 3-N-Benzyloxymethyl-3 ' -O- (3- trif luoroacetamidopropyl) -5 ' -O- ( tetrahydropyran-2- yl) hymidine, 6

A solution of compound 5 (4.00 g, 8.0 mmole), DIPEA (0.5 ml) and CF ₃ COOEt (7 ml) in anhydrous EtOH is kept at ambient temperature overnight. The reaction mixture is evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (4.5x25 cm) using toluene/AcOEt (7/3) as eluent to give compound 6, 4.46 g (93%), as a colorless oil. MS: [M+l]=600; [M+1+NH ₃ ]=617.

^X H-NMR (CDC1 ₃ ) : 7.60&7.53 (q&q, IH, H-6, Thy); 7.40-7.20 (g , *, Phe , BOM); 7.30 (m, 6H with *, NHCOCF ₃ ) ; 6.29 (m,

IH, H-l', dT) ; 5.49 (s, 2H, NCH ₂ , BOM); 4.69 (s, 2H, OCH ₂ ,

BOM); 4.65&4.S8 (m&m, IH, H-l, THP); 4.17 (m, **, H-4', dT).; 4.14&4.09 (m&m, **, H-3 ' , dT) ; 4.07&3.96&3.71&3.57

(dd&dd&dd&m, **, H-5 ' , dT) ; 3.90&3.56 (m&m, **, H-5, THP) ; 3.58 (m, **, 0Cϋ ₂ CH ₂ CH ₂ NHC0CF ₃ ) ; 3.50 ( , lOH with **,

0CH ₂ CH ₂ £H ₂ NHC0CF ₃ ) ; 2.43 (m, IH, H-2 ' , dT) ; 2.03 ( , ***, H-

2', dT); 1.95&1.91(d&d, ***, CH ₃ , Thy); 1.88 (m, ***, OCH ₂ £H ₂ CH ₂ NHCOCF ₃ ) ; 1.57&1.37 (m&m, 12H with ***, H-2,-3,-4,

THP) . ¹³ C-NMR (CDC1 ₃ ) : 163.4&163.3 (C-4, Thy); 157.0 (q, J _C _ _F =61.7 Hz, £(=0)CF ₃ ); 150.8 (C-2, Thy); 137.8 (C-1, Phe,

BOM); 134.4&134.1 (C-6, Thy); 128.1&127.5 (C-2, C-3, C-4, Phe, BOM); 115.8 (q, Xr _c _ _F =287.4 Hz, C(=0)C_F ₃ ); 110.0&109.9 (C-5, Thy); 100.0&99.1 (C-1, THP); 85.8&85.7 (C-1', dT) ;

83.7683.6 (C-4', dT) ; 80.4&80.3 (C-3', dT) ; 72.0 (OCH ₂ ,

BOM); 70.3 (NCH ₂ , BOM); 67.9&67.8 (OQH ₂ CH ₂ CH ₂ NHCOCF ₃ ) ;

67.3&67.2 (C-5', dT) ; 63.3&62.9 (C-5, THP); 38.4 (OCH ₂ CH ₂ CH ₂ NHCOCF ₃ ) ; 37.6&37.5 (C-2', dT) ; 30.59&30.57 (C-2, THP); 28.3 (OCH ₂ C_H ₂ CH ₂ NHCOCF ₃ ) ; 25.1&25.0 (C-4, THP) ;

20.1619.7 (C-3, THP); 13.1&12.9 (CH ₃ , Thy). ¹⁹ F-NMR (CDC1 ₃ ) : -76.23 (s, CF_ ₃ ) .

Exatmple 7 3-N-Benzyloxymethyl-3'-0-(benzyjL)thymidine, 7

To a stirred solution of compound 1 (33.25 g, 50 mmole) in anhydrous DMF (150 ml) is added a suspension of 60% NaH. (2.4 g, 60 mmole) and after 1 hour dropwise benzyl bromide (12.0 g, 8.4 ml, 70 mmole) over a period of 5 minutes. The resulting mixture is stirred at ambient temperature overnight, and the DMF is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (600 ml), washed successively with water (1x200 ml) and brine (2x200 ml), dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is coevaporated with toluene (2x300) , dissolved in 3% TFA/DCE (700 ml) and kept at ambient temperature for 15 minutes. The reaction mixture is washed successively with 5% ~NaHC0 ₃ (3x250 ml) and water (2x250 ml), dried (MgS0 ₄ )

and evaporated in vacuo to dryness. The residue is crystallized from toluene/hexane to give compound 7, 17.42 g (77%), as white crystals. Another 2.15 g (9.5%) of compound 7 can be isolated by chromatography of mother liquor over silica gel column (2.5x25 cm) using toluene/AcOEt (7/3) as eluent. ^-NMR (CDC1 ₃ ) : 7.42 (q,

IH, ⁴ J=0.9 Hz, H-6, Thy); 7.45-7.17 (gm, 10H, Phe ,BOM&Bzl); 6.16 (m, IH, ³ J ₁ ,_ ₂ ,=6.3&7.6 Hz, H-l', dT) ; 5.48 (s, 2H, N£H ₂ , BOM); 4.69 (s, 2H, 0£H ₂ , BOM); 4.57&4.50 (d&d, 2H, ² J=11.6 Hz, 0CH ₂ , Bzl) ; 4.26 (m, IH, ³ J ₃ ,_ ₄ ,=2.9 Hz, ³ J ₃ ,_

₂ ,=2.9&6.4 Hz,H-3',dT) ; 4.15 (m, ³ J ₅ ._ ₄ , =2.8&2.9 Hz, H-4' , dT) ; 3.90&3.74 (dd&dd, 2H, ² J ₅ ,_ ₅ ,=11.8 Hz, H-5', dT) ; 2.42 (m, IH, ² J ₂ ,_ ₂ ,=13.6 Hz, H-2' , dT) ; 2.03 (m, IH, H-2 ' , dT) ;

1.90(d, 3H, CH ₃ , Thy) . ¹³ C-NMR (CDCl ₃ ) : 163.4 (C-4, Thy),-150.9 (C-2, Thy); 137.8 (C-1, Phe, BOM); 137.4 (C-1, Phe, Bzl), 135.6 (C-6, Thy); 128.5&128.2&127.9&127.6 (C-2, C-3, C-4, Phe, BOM&Bzl) ; 110.2 (C-5, Thy); 87.6 (C-1', dT) ; 85.0 (C-4', dT) ; 78.6 (C-3', dT) ; 72.1 (OCH ₂ , BOM); 71.5

(OCH ₂ , Bzl); 70.3 (NCH ₂ , BOM); 62.8 (C-5', dT) ; 37.2 (C-2', dT) ; 13.2 (CH ₃ , Thy) .

Example 8 3-N-Bβnzyloxymβthyl-3 '-o- (benzyl) -5 ' -O- (allyl) hymidine, 8 To a stirred solution of compound 7 (6.75 g, 15 mmole) in anhydrous DMF (75 ml) is added a suspension of 60% NaH (0.80 g, 20 mmole) and after 1 hour dropwise allyl bromide (3.02 g, 2.16 ml, 25 mmo-le) over 5 minutes. The resulting mixture is stirred at ambient temperature for 2 hours, and the DMF is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (300 ml) , washed successively with water (1x100 ml) and brine (2x100 ml), dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is

chromatographed over silica gel column (2.5x25 cm) using toluene/AcOEt (9/1) as eluent to give compound 8, 7.05 g (95%), as a colorless oil. MS: [M+l]=493; [M+1+NH ₃ ] =510.

^X H-NMR (CDC1 ₃ ) : 7.60 (q, IH, ⁴ J=1.0 Hz, H-6, Thy) ; 7.40- 7.15 (gm, 10H, Phe , BOM&Bzl ) ; 6.40 (m, IH, ³ J ₁ ._ ₂ ,=6.0&7.5

Hz, H-l', dT) ; 5.88 (m, IH, ³ J _CH2=CH =10.2&17.4 Hz, Hz, CH ₂ =C£-CH ₂ -) ; 5.48 (s, 2H, NCH ₂ , BOM); 5.24 (m, 2H, ϋ ₂ =CH-CH ₂ -) ; 4.69 (s, 2H, OCH ₂ , BOM); 4.57&4.4S (d&d, 2H,

² J=11.9 Hz, OCH ₂ , Bzl); 4.24 (gm, 2H, , H-3 ' , -4', dT) ; 4.01 (bd, 2H, CH ₂ =CH-£H ₂ -) ; 3.72&3.56 (dd&dd, 2H, ² J ₅ ,_ ₅ , = 10.2,

_Hz, H-5', ³ J ₅ ._ ₄ . =?.2 Hz, dT) ; 2.47 (m, IH, ² J ₂ ,_ ₂ .=12.2 Hz, ³ J ₃ ,_ ₂ ,=2.4 Hz H-2', dT) ; 2.03 (m, IH, ³ J ₃ ,_ ₂ ,=5.8 Hz, H-2 ' , dT) ; 1.89 (d, 3H, CH ₃ , Thy) . ¹³ C-NMR (CDC1 ₃ ) : 163.3 (C-4,

Thy);150.8 (C-2, Thy); 137.9 (C-1, Phe, BOM); 137.3 (C-1, Phe, Bzl), 134.5 (C-6, Thy); 133.8 (CH ₂ =£H-CH ₂ -) ;

128.3&128.0&127.9&127.7&127.5&127,4&127,3 (C-2, C-3, C-4, Phe, BOM&Bzl); 117.2 (£H ₂ =CH-CH ₂ -) ; 119.8 (C-5, Thy) ; 87.6

(C-1', dT) ; 83.7 (C-4', dT) ; 79.0 (C-3', dT) ; 72.1 (CH ₂ =CH-

CH ₂ -) ; 71.9 (OCH ₂ , BOM); 71.1 (OCH ₂ , _t Bzl) ; 70.2 (NCH ₂ , BOM); 70.1 (C-5', dT); _. 63.3&62.9 (C-5, THP); 37.8 (C-2' , dT) ; 13.1 (CH ₃ , Thy) .

Example 9 3-N-Bβnzyloxymethyl-3'-0-(benzyl)-5' -O- (3- bromopropyl) thymidine, 9

A dry 50-ml flask equiped with septum inlet, thermometer well, and magnetic stirrer is flashed with argon and then maintained under positive argon pressure. The flask is charged with a solution of compound 8 (4.5 g, 9.1 mmole) in anhydrous THF (10 ml) and cooled to 0°C. A 1M solution of BH ₃ /THF (3.1 ml, 9.3 mmole) is added

dropwise over a period of 5 minutes. The resulting mixture is stirred for 30 min at 0°C and 30 min at ambient temperature. Then MeOH (60 μl) is added to destroy excess of hydride. The solution is cooled to -10°C and bromine (1.94 g, 0.62 ml, 12.1 mmole) is added dropwise over 10 minutes. Then a 4.34 M solution of NaOMe (3.5 ml, 15.2 mmole) is added dropwise over 45 minutes at 0°C. The reaction mixture is diluted with AcOEt (150 ml) and washed successively with saturated NaHC0 ₃ (3x50 ml) and brine (2x50 ml) , dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is coevaporated with toluene (2x100 ml) . The residue is chromatographed over a silica gel column (4.5x25 cm) using toluene/AcOEt (gradient of AcOEt from 0 to 10%) as eluent to give compound 9, 2.10 g (40%), as a colorless oil. MS: [M+l] =573&575 [M+1+NH ₃ ]=590&592.

^-NM (CDC1 ₃ ) : 7.45 (q, IH, ⁴ J=1.0 Hz, H-6, Thy); 7.40- 7.10 (gm, 10H, Phe ,BOM&Bzl); 6.33 (m, IH, ³ J _χ ,_ ₂ ,=6.2&7.2 Hz, H-l', dT) ; 5.49 (s, 2H, NCH ₂ , BOM); 4.69 (s, 2H, OCH ₂ , BOM); 4.59&4.49 (d&d, 2H, ² J=11.8 Hz, OCH ₂ , Bzl); 4.19 (gm, 2H, , H-3', -4', dT) ; 3.74 (dd, IH, ² J ₅ ,_ ₅ ,=10.6 Hz, ³ J ₅ ,_ ₄ ,

=2.5 Hz, H-5', dT) ; 3.61 (m, *, OCJ3 ₂ CH ₂ CH ₂ Br) ; 3.57 (m, 3H with *, H-5'); 3.43 (t, 2H, OCH ₂ £H ₂ CH ₂ Br) , 2.50 (m, IH, ² J ₂ ,_

₂ ,=13.6 Hz, ³ J ₃ ,_ ₂ ,=2.8 Hz, H-2 ' , dT) ; 2.11 ( , **, OCH ₂ CH ₂ CH ₂ Br) ; 1.97 (m, 3H with **, H-2' , dT) ; 1.91 (d, 3H, CH ₃ , Thy) . ¹³ C-NMR (CDC1 ₃ ) : 163.3 (C-4, Thy);150.8 (C-2,

Thy); 137.9 (C-1, Phe, BOM); 137.3 (C-1, Phe, Bzl), 134.2 (C-6, Thy); 128.4&128.2&127.9&127.9&127.6& 127,5 (C-2, C-3, C-4, Phe, BOM&Bzl); 109.8 (C-5, Thy) ; 85.8 (C-1', dT) ; 83.7 (C-4', dT) ; 78.6 (C-3', dT) ; 72.1 (OCH ₂ , BOM); 71.4 (OCH ₂ , Bzl); 70.8 (C-5', dT) ; 70.4 (NCH ₂ , BOM); 69.9

(OCH ₂ CH ₂ CH ₂ Br) ; 37 . 9 (C-2 ' , dT) ; 32 . 4 (OCH ₂ CH ₂ CH ₂ Br ) ; 29 . 82

(OCH ₂ CH ₂ £H ₂ Br) ; 13.3 (CH ₃ , Thy) .

Example 10 3-N-Benzyloxymβthyl-5 ' -O- ( etrahydropyran-2 -yl ) -3 ' - O- [ (4-N-trif luoroacetyl) -1, 7-heptaza-4- nediyl] thymidylyl- (3 '→5' ) -3-N-bβnzyloxymethyl-3 ' -O-

(bβnzyl) thymidine, 10

To a stirred solution of compound 6 (2.00 g, 3.3 mmole) in anhydrous DMF (10 ml) is added a suspension of 60% NaH (140 mg, 3.5 mmole) and after 1 hour a solution of compound 9 (2.00 g, 3.5 mmole) in anhydrous DMF (5 ml) . The resulting mixture is stirred overnight at 50°C, and cooled to ambient temperature. Another portion of 60% NaH (40 mg, 1 mmole) is added and after one hour a solution of compound 9 (600 mg, 1.0 mmole) in anhydrous DMF (2 ml) is added. The resulting mixture is stirred overnight at 50°C, cooled to ambient temperature and the DMF is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (100 ml) , washed successively with water (1x30 ml) and brine (2x30 ml) , dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is chromatographed over a silica gel column (2.5x25 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 50%) as eluent to give cOmpound 10, 2.99 g (83%) , as a white foam. ^-NMR (CDC1 ₃ ) : 7.61&7.53 (m&m, IH, H-6, dT _x ); 7.48&7.36 (m&m, *, H-6, dT ₃ ) ;7.40-7.20 (gm, 16H with *, Phe ,B0M); 6.34 (m, 2H, H-l', dT _x &dT ₃ ) ; 5.48 (s, 4H, N£H ₂ , BOM); 4.69 (s, 2H, 0£fi ₂ , BOM); 4.62 (m, **, H- 1, THP); 4.54 (m, 3H with **, OCH ₂ , Bzl); 4.19 (m, ***, H- 4', d _i &dT ₃ ) ; 4.19&4.08&4.03 (m&m&m, 4H with***, H-3', dT _i &d ^; 4.00-3.40 (gm, 14H, H-5', dTi&dT^ H-5, THP;

OCϋ ₂ CH ₂ CH ₂ N(COCF ₃ )CH ₂ CH ₂ CH ₂ 0);; 2.45 (m, 2H, H-2 ' , dT^dT-,); 2.00 (m, ****, H-2', dT _i &dT^; 1.95&1.93&1.91 (s&s&s, ****, CH ₃ , Thy); 1.85 (m, ****, OCH ₂ ςH ₂ CH ₂ N(COCF ₃ )CH ₂ CH ₂ CH ₂ 0) ;

1.80&1.56 (m&m, 18H with ****, H-2,-3,-4, THP) . ^X3 C-NMR (CDC1 ₃ ) : 163.36&163.31&163.26 (C-4, dTi&dT^ ; 156.6 (m, £<=0)CF ₃ ); 150.87&150.85&150.81&150.75 (C-2, dT^dT- _j );

137.88&137.86&137.84 (C-1, Phe, BOM); 137.4&137.3 (C-1, Phe, Bzl); 134.42&134.30&134.1& 134.06 (C-6, dT _α &dT ₃ ) ;

128.4-127.5 (gs, C-2, C-3, C-4, Phe, BOM); 116.4 (q, ¹ J _C _

_F =288.1 Hz, C(=0)£F ₃ ); 110.10&100.04&109.98&109.87 (C-5, d i&dT-,); 100.0&99.1&98.9 (C-1, THP); 85.82&85.72&85.70 (C- 1', dTi&d^); 83.67&83.62&85.55&83.47 (C-4', dT _j &dT ,) ; 80.55&80.46 (C-3', dT _x ) ; 79.13&78.83&78.74 (C-3', dT ₃ ) ;

72.0 (OCH ₂ , BOM); 70.3 (NCH ₂ , BOM); 71.34&71.30&71.30 (OCH ₂ , Bzl); 71.12&70.92&70.86 (C-5', dT ₃ ) ; 70.3 (NCH ₂ , BOM); 69.44&68.42&68.36 (0£H ₂ CH ₂ CH ₂ N, dT ₃ ) ; 68.0&67. &67.6 (0£H ₂ CH ₂ CH ₂ N, dT _x ); 66.44&66.39&65.91&65.84 (C-5', dT _x ) ; 63.42&62.86& 62.71 (C-5, THP) ; 45.2&44.8&44.1

(£H ₂ N(C0CF ₃ )£H ₂ ) ; 37.78&37.63&37.58 (C-2', dT _χ &dT ₃ ) ;

32.23&29.03&28.90&27.14 (OCH ₂ £H ₂ CH ₂ N(COCF ₃ )CH ₂ £H ₂ CH ₂ 0) ; 30.6&30.5 (C-2, THP); 25.11&25.06 (C-4, THP); 20.1&19.7&19.6 (C-3, THP) ; 13.3&13.2&13.1&13.0 (CH ₃ , Thy) . ¹⁹ F-NMR (CDC1 ₃ ) : -69.12&69.16 (s&s,'CF_ ₃ ) .

Example 11 5'-θ- (tβtrahydropyran-2-yl) -3 '-o- [ (4-N- trif luoroacβtyl) -1, 7-heptaza-4-nediyl] thymidylyl- (3 '→5') -thymidine, 11

To a stirred solution of compound 10 (2.18 g, 2.0 mmole) in a mixture of AcOEt/MeOH (1/1) (50 ml) is added 10% Pd/C (Degussa type, Aldrich) (1.1 g) . The flask is evacuated and then flushed 3 times with hydrogen and finally filled with hydrogen at 40 to 50 psi. The resultant suspension was stirred vigrously at 23°C for 14 hours. The suspended material is filtered off through a

pad of Celite in a Buchner funnel and then the tunnel is rinsed several times with a small portion of AcOEt and MeOH. The combined filtrate and washings is evaporated in vacuo to dryness. The residue is chromatographed over a silica gel column (2.5x25 cm) using 3% MeOH/CHCl ₃ as eluent to give compound 11, 1.32 g (87%), as a white foam. MS: [M+l]=762; [M+1+NH ₃ ] =779. ^- MR (CDCl ₃ ) :

9.96&9.87&9.75&9.67 (bs&bs&bs&bs, 2H, NH, Thy); 7.64&7.56&7.54&7.4S (bs&bs&bs&bs, 2H, H-6, dTi&dT^ ; 6.32 (gm, 2H, H-l', dT _x &dT ₃ ) ; 4.6S&4.57 (bs&bs, *, H-l, THP) ;

4.51 ( , 2H with *, H-3 ' , dT ₃ ) ; 4.19 (m, IH, H-4', dT ;

4.09 (m, **, H-4', dT ₃ ) ; 4.07 (m, **, H-3 ' , dT^ ; 4.1-3.40

(gm, 16H with **, H-5', dTi&dT ₃ ; H-5 , THP;

0£H ₂ CH ₂ £H ₂ N(COCF ₃ )£H ₂ CH ₂ £H ₂ 0) ; 2.41 (m, 2H, H-2 ' , dT) ; 2.15&2.00 (m&m, ***, H-2', dT) ; 1.94&1.90 (s&s, ***, CH ₃ , dT _! &dT ₃ ); 1.90 (m, ***, OCH ₂ £H ₂ CH ₂ N(COCF ₃ ) CH ₂ Cϋ ₂ CH ₂ 0) ;

1.80&1.59 (m&m, 18H with ***, H-2,-3,-4, THP) . ¹³ C-NMR (CDC1 ₃ ) : 164.1&164.0 (C-4, dT _x &dT ₃ ) ; 156.8 (m, £(=0)CF ₃ ); 150.9&150.6 (C-2, T _i &dT ₃ ) ; 135.9&135.7&135.6& 135.3 (C-6, ^&dT- _j ); 116.4 (q, ^^=290.3 Hz, C(=0)£F ₃ );

111.3&111.2&109.98&110.8&110.7 (C-5, dT^dT-,) ;

100.1&99.1&99.0 (C-1, THP); 85.7&85.6 (C-4', dT ₃ ) ;

85.22&85.12&85.08 (C-1', dT _x &dT ₃ ) ; 83.65&83.49 (C-4', dT^ ;

80.94&80.77 (C-3', dT _j _) ; 71.9&71.6 (C-3', dT ₃ ) ; 71.0&70.5&70.86&68.4&68.2&67.8&66.5&66.0&65.5 ( C-5', dT ₃ ;

0£H ₂ CH ₂ CH ₂ N, dT _i &dT- _j ); 66.44&66.39&65.91&65.84 (C-5', dT _x ) ;

63.5&63.0& 62.8 (C-5, THP); 45.6&45.2&44.5 (£H ₂ N(COCF ₃ )£H ₂ ) ;

40.5 (C-2', dT ₃ ); 37.9&37.7 (C-2', dT _x ) ; 30.72&30.66 (C-2,

THP) ; 29.14&29.05&27.09& 26.98 (OCH ₂ £H ₂ CH ₂ N(C0CF ₃ )CH ₂ £H ₂ CH ₂ O) ; 25.2&25.1 (C-4, THP) ;

20.2&19.8&19.7 (C-3, THP); 12.6&12.5&12.3 (CH ₃ , Thy) . ¹⁹ F-

NMR (CDCI ₃ ) : -69.25 (s, CF ₃ ) .

Example 12 5 ' -O- (tetrahydropyran-2-yl) -3 ' -O- (1, 7-heptaza-4- nβdiyl) thymidylyl- (3 '→S' ) -thymidine, 12

A solution of compound 11 (1.14 g, 1.5 mmole) in 20 ml of a 3:1 mixture of 12% NH ₃ /EtOH - 28% NH ₄ 0H is kept at

65°C overnight. The reaction mixture is cooled and evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (4.5x25 cm) using CHCl ₃ /MeOH/28%NH ₄ OH (95/5/0.5) as eluent to give compound

12, 0.96 g (96%) , as a white solid. MS: [M+l]=666. *H- NMR (CDC1 ₃ ) : 7.64&7.55 (bs&bs, 2H, H-6, dT ₁ &dT ₃ ); 6.30 (g , 2H, H-l', dT _i &dT^; 4.66&4.59 (bs&bs, IH, H-l, THP); 4.49 (m, IH, H-3', dT ₃ ) ; 4.20 ( , IH, H-4\ dT _x ) ; 4.06 (m, *, H-

4', dT ₃ ); 4.00 (m, *, H-3 ' , άT _x ) ; 3.94&3.55 (m&m, *, H-5', dT _j ); 3.80&3.52 (m&m,*,; H-5, THP); 3.58 (m, *, H-5 ' , dT ₃ ) ; 3.52 (m, 12H with *, OCH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ £H ₂ 0) ; 2.75 (gm, 4H, OCH ₂ CH ₂ Cϋ ₂ NH£H ₂ CH ₂ CH ₂ 0) ; 2.38 (gm, 2H, H-2 ' , d^&d^); 2.20 (m, IH, H-2', dT ₃ ) ; 2.01 (m, **, H-2 ' , dT _x ) ; 1.93&1.90

(s&s, **, CH ₃ , dT _x &dT ₃ ); 1.75 (m, **,

OCH ₂ u ₂ CH ₂ NHCH ₂ CH ₂ CH ₂ 0) ; 1.70&1.57 (m&m, 17H with **, H-2,- 3,-4, THP) . ¹³ C-NMR (CDCl ₃ ) : ^' 164.4&164.3 (C-4, ^&dT- _j ); 150.83&150.75 (C-2, 136.0 (C-6, dT ₃ ); 135.7&135.5(C-6, dT _x ) ; 111.02&110.95 (C-5, dT _x ) ; 110.7 (C- 5, dT ₃ ); 100.0&99.0 (C-1, THP); 85.6 (C-4', dT ₃ ) ; 85.2&85.1 (C-1', dTi&dT^; 83.8&83.7 (C-4', dT _x ) ; 80.5&80.3 (C-3', dT _χ ) ; 71.7 (C-3', dT ₃ ); 70.6&69.9 (C-5', 0CH ₂ CH ₂ CH ₂ N, dT ₃ ) ; 68.16&67.8 ( 0£H ₂ CH ₂ CH ₂ N, dT _x ) ; 67.72&67.67 (C-5', dT _x ) ; 63.3&62.8 (C-5, THP); 47.1&47.0 (CH ₂ NH£H ₂ ) ; 40.6 (C-2', dT ₃ ) ; 37.84&37.78 (C-2', dT _χ ) ; 30.7 (C-2, THP) ; 29.45

(NCH ₂ £H ₂ CH ₂ 0, dT ₃ ); 29.2&27.09&26.98 (OCH ₂ £H ₂ CH ₂ N, dT _x ) ; 25.2 (C-4, THP) ; 20.1&19.7 (C-3, THP); 12.7 (CH ₃ , dT ₃ ) ; &12.6&12.4 (CH ₃ , dT _x ) .

Example 13

5'-0- (tetrahydropyran-2-yl) -3'-0-{4-N- [ (thymine-1- yl) acβtyl] -1, 7 -heptaza-4-nediyl) thymidylyl- (3'→5 ' ) - thymidine, 13a

A solution of compound 12 (333 mg, 0.50 mmole), pentaf luorophenyl ester of thymine-1-ylacetic acid (210 mg, 0.60 mmole) and DIPEA (0.6 mmole) in anhydrous DMF (3.0 ml) is kept at ambient temperature for 2 hours and the DMF is evaporated. The residue is chromatographed over a silica gel column (2.5x25 cm) using CHCl ₃ /MeOH/28%NH ₄ OH (97/3/0.1) as eluent to give compound 13a, 407 mg (98%) , as a white solid. MS: [M+l]=832, [M+NH ₃ ]=848, [M+1+NH ₃ ] =849. ^X H-NMR

(CD ₃ OD) : 7.76&7.68&7.65&7.58 (m&m&m&m, 2H, H-6, dT _x &dT ₃ ) ;

7.31&7.28 (IH, H-6, Thy ₂ ) ; 6.25 (gm, 2H, H-l', dTi&dT ₃ ) ;

4.67&4.62 (s&s, *, C (=0)£H ₂ Thy) ; 4.64 ( , 3H with *, H-l, THP); 4.39 (m, IH, H-3 ' , dT ₃ ) ; 4.20 (m, **, H-4', dT _x ) ;

4.15 (m, 2H with **, H-3 ' , dT _x ) ; 4.60 (m, ***, H-4 ' , dT ₃ ) ;

3.95&3.64 (m&m, ***, H-5', dT _x ) ; 3.89&3.52 (m&m,***, H-5,

THP); 3.60 (m, ***, H-5 ' , dT ₃ ) ; 3.70-3.40 (g , 15H with

***, OCϋ ₂ CH ₂ CiJ ₂ N) ; 2.40 (m, IH, H-2 ' , dT _j .) ; 2.24 (m, ****, H-2', dT ₃ ); 2.14 (m, 3H with ****, H-2 ' , dT _x ) ; 1.90 (gm,

*****, OCH ₂ Cϋ ₂ CH ₂ N) ; 1.89&1.88&1.86 (s&s&s, *****, CH ₃ ,

Thy); 1.76&1.56 (m&m, 19H with *****, H-2,-3,-4, THP) . ¹³ C- NMR (CD ₃ OD) : 175.5&175.3 (C (=0) CH ₂ Thy) ; 168.7&168.7,

167.0&166.9, 166.2&166.1 (C-4, Thy); 155.0&154.6, 153.02&152.98, 152.25&152.19 (C-2, Thy) ; 144.1 (C-6, Thy ₂ ) ;

137.9&137.8&137.5 (C-6, dT _L &dT ₃ ) ; 111.6&111.4&110.7 (C-5,

Thy) ; 101.4&100.3 (C-1, THP) ; 87.5&87.1 (C-4', dT ₃ ); 86.6&86.4, 85.6&85.4 (C-1', dT _x &dT ₃ ) ; 8S.2&83.7 (C-4', dT _: ) ; 81.8&81.7 (C-3', dT _j .) ; 72.9&72.5 (C-3', dT ₃ ); 71.9&69.9&68.9&67.7&66.5 (C-5', dT _x &dT ₃ , 0£H ₂ CH ₂ CH ₂ N, ) ; 64.4&63.6&63.5 (C-5, THP); 50.0 (C (=0)£H ₂ Thy) ; 45.3&44.8&44.7 (-£H ₂ N (C (=0) CH ₂ Thy)£H ₂ -) ;

41.2&41.0&38.4&36.4&36.3&34.9&34.3 (C-2' , dT _x &dT ₃ ) ; 31.8 (C- 2, THP); 29.8&29.6&28.8 (NCH ₂ £H ₂ CH ₂ 0, d^&dT- _j ); 26.5 (C-4, THP); 21.3&20.7 (C-3, THP); 12. &12.7&12.6&12.4 (CH ₃ , Thy) .

Example 14 5 '-O- (tetrahydropyran-2-yl) -3 '-0-{4-N- [ (4-N- (isobutyryl ) cytidine-1-yl) acβtyl] -1, 7-heptaza-4- nediyl} thymidylyl- (3 5' ) -thymidine, 13b A solution of compound 12 (333 mg, 0.50 mmole), pentafluorophenyl ester of [4-N- (isobutyryl)cytidine-l- yl] acetic acid (243 mg, 0.60 mmole) and DIPEA (0.6 mmole) in anhydrous DMF (3.0 ml) is kept at ambient temperature for 2 hours and the DMF is evaporated. The residue is chromatographed over a silica gel column (2.5x20 cm) using CHCl ₃ /MeOH (gradient of MeOH from 0 _T to 7%) as eluent to give compound 13b, 422 mg (95%) , as a white solid. The structure is confirmed by ¹ H- and ¹³ C-NMR.

Example 15

5 '-O- (tetrahydropyran-l-yl) -3'-0-{4-N- [ (6-N- (phenoxyacβtyl) adenine-9-yl) acetyl] -1, 7-heptaza-4- nediyl} thymidylyl- (3' →5' ) -thymidine, 13c

A solution of compound 12 (333 g, 0.50 mmole) , pentafluorophenyl ester of [6-N- (phenoxyacetyl)adenine-7- yl] acetic acid (296 mg, 0.60 mmole) and DIPEA (0.6 mmole) in anhydrous DMF (3.0 ml) is kept at ambient temperature for -2 hours and the DMF is evaporated. The residue is

chromatographed over a silica gel column (2.5x20 cm) using CHCl ₃ /MeOH (gradient from 0 to 7%) as eluent to give compound 13c, 412 mg (87%) , as a white solid. The structure is confirmed by ^X H- and ¹³ C-NMR.

Example 16 3 ' -0-{ [4-N- (thyminβ-l-yl)acetyl] -1, 7-heptaza-4- nediyl} thymidylyl- (3'→5' ) -thymidine, 14a

A solution of compound 13a (300 mg, 0.40 mmole) and PPTS (300 mg, 0.4 mmole) in anhydrous EtOH (6 ml) is kept at 35°C overnight. The reaction mixture is chromatographed over a Sephadex LH-20 column (3.0x120 cm) using MeOH as eluent with a flow rate of 10 ml/h to give compound 14a, 287 mg (96%) , as a white solid. MS: [M+l]=748, [M+1+NH ₃ ]=763. ^-N R (CD ₃ OD) : 7.68&7.59&7.50 (m&m&m, 2H,

H-6, dT ₁ &dT ₃ ); 7.23&7.20 (IH, H-6, Thy ₂ ) ; 6.14 (gm, 2H, H- 1', d _i &d - _j ); 4.S8&4.55 (s&s, 2H, C ( =0 ) £H ₂ Thy ) ; 4.30 ( , IH, H-3', dT ₃ ); 4.09&3.98 (m, *, H-3 ' , dT _χ ), - 3.95-3.85 (gm, 3H with *, H-4', dT _! &dT ₃ ); 3.70-3.50 (gm, **, H-5', dTi&dT ₃ ) ; 3.60-3.30 (gm, 12H with **, 0£H ₂ CH ₂ £H ₂ N) ; 2.35-1.95 (gm, 4H, H-2', dT _! &d ₃ ); 1.95-1.65 (gm, ***, _t OCH ₂ Cϋ ₂ CH ₂ N) ; 1.78&1.77&1.76 (s&S&S, ***, CH ₃ , Thy) . ¹³ C-NMR (CD ₃ OD) :

168.9&168.8, 167.1&167.0, 166.4&166.3 (C-4, Thy); 153.14&153.03, 152.39&152.34&152.27 (C-2, Thy) ; 144.2 (C-6, Thy ₂ ); 139.1&138.0 (C-6, dT _x &dT ₃ ) ;

111.70&111.67&111.61&111.46&110.76 (C-5, Thy); 87.6&87.1&86.6 (C-4', dT _x &dT ₃ ) ; 86.5&86.4&86.3 (C-1', dT _i &dT- _j ); 85.2&83.7 (C-4', d _χ ) ; 81.2&81.0 (C-3', dT _χ ) ; 72.9&72.4 (C-3', dT ₃ ) ; 71.9&69.9&68.8&67.8&66.7 (C-5', dT ₃ , OCH ₂ CK ₂ CH ₂ N, ) ; 63.3 (C-5', dT _L ) ; 50.1&49.9 (-C (=0)£H ₂ Thy) ;

45.4&45.2&44.8 (-£H ₂ N(C (=0) CH ₂ Thy)£H ₂ -) ; 41.2&41.0&38.1 (C- 2' ,. dT ₁ &dT ₃ ); 29.8&29.5&28.8 (NCH ₂ CH ₂ CH ₂ 0, dT _x &dT ₃ ) ;

12 . 9&12 . 6&12 . 4 (CH ₃ , Thy) .

Example 17 3 ' -O- { [4-N- (cytidine-1-yl) cetyl] -1, 7-heptaza-4- nβdiy1} hymidylyl- (3 '→5' ) -thymidine, 14b

A solution of compound 13b (355 mg, 0.40 mmole) in 5 ml of EtOH saturated with NH ₃ is kept at ambient temperature overnight. The reaction mixture is evaporated in vacuo to dryness. The residue and PPTS (300 mg, 0.4 mmole) in anhydrous EtOH (6 ml) is kept at 35°C overnight. The reaction mixture is chromatographed over a Sephadex LH- 20 column (3.0x120 cm) using MeOH as eluent with a flow rate of 10 ml/h to give compound 14b, 280 mg (87%) .

Example 18

3 ' -0-{4-N- [ (adenine-9-yl) acetyl] -1, 7-he taza-4- nediyl)thymidylyl- (3'→5' ) -thymidine, 14c

Compound 13c (380 mg, 0.40 mmole) is treated as described in Example 17 to give compound 14c, 240 mg (82%) . The structure is confirmed by ¹ H-NMR.

Example 19 5' -O- (p-anisyldiphenylmethyl) -3 ' -0-{ [4-N- (thymine- 1-yl) acetyl] -1, 7-heptaza-4-nediyl}thymidylyl- (3 '→5' ) -thymidine, 15

A solution of compound 13a (225 mg, 0.30 mmole) and PPTS (300 mg, 0.4 mmole) in anhydrous EtOH (6 ml) is kept at 35°C overnight. Anhydrous Py (20 ml) was added to the reaction mixture. The reaction mixture is evaporated in vacuo and then coevaporated with anhydrous Py (3x10 ml) , dissolved in anhydrous Py (5 ml) and MMT-C1 (110 mg, 0.36 mmole) is added. The reaction mixture is kept overnight at ambient temperature, then MeOH (100 μl) is added and after 30 minutes the reaction mixture is poured in CHC1 ₃ (50 ml) .

The organic layer is washed with NaHC0 ₃ (3x15 ml) , brine

(2x15 ml) , dried (MgS0 ₄ ) , evaporated in vacuo to dryness, and the residue is chromatographed over a silica gel column (2.5x10 cm) using CHCl ₃ /Me0H/28%NH ₄ 0H (97/2/0.1) as eluent to give compound 15, 279 mg (91%) , as a white foam. ^X H- NMR (acetone-d ₆ ) : 7.66&7.60&7.59 (m&m&m, 2H, H-6, dT _x &dT ₃ ) ;

7.50-7.20 (gm, *, Phe, MMT); 7.24 (m, 13H with*, H-6, Thy ₂ ); 6.92 (m, 2H, Phe, MMT); 6.29 (gm, 2H, H-l', dT ₂ &dT ₃ ) ;

4.65 (m, 2H, C(=0)£H ₂ Thy) ; 4.48 (m, IH, H-3 ' , dT ₃ ) ; 4.34&4.27 (m, IH, H-3 ' , dT ₂ ) ; 4.17&4.11 (m&m, IH, H-

-i'.dT _i ); 4.00 (m&m, IH, H-4',dT ₃ ); 3.78 (s, 3H, OCH ₃ , MMT); 3.75&3.62 (m&m, **, H-5', dT ₃ ) ; 3.75-3.30 (gm, 12H with **, 0£H ₂ CH ₂ CH ₂ N) ; 3.38 ( , **, H-5' , dT ₃ ) ; 2.38 (m, ***, H-2 ' , dT _x ); 2.22 (m, 4H with ***, H-2', dT ₃ ). 2.05-1.75 (gm, ****, OCH ₂ £iJ ₂ CH ₂ N) ; 1.83&1.81&1.79&1.77&1.48 (s&s&s, 13H with

****, CH ₃ , Thy) . ^-NMR (DMF-d ₇ ) : 7.68&7.66 (m&m, 2H, H-6, dT _! &dT ₃ ); 7.60-7.20 (g , 13H, Phe, MMT; H-6, Thy ₂ ) ; 6.98 (m, 2H, Phe, MMT); 6.34 (gm, 2H, H-l', dT _x &dT ₃ ) ; 5.44 (t, IH, 3' -OH, dT ₃ ); 4.72 (bs, 2H, C (=0)£H ₂ Thy) ; 4.43 (m, IH, H-3 ' , dT ₃ ); 4.36&4.30 (m, IH, H-3 ' , dT _x ) ;' 4.18&4.11 (m&m, IH, H- 4',dT ₁ ); 4.01 (m&m, IH, H-4',dT ₃ ); 3.82 (s, *, 0CH ₃ , MMT); 3.78&3.62 (m&m, *, H-5', dT ₃ ) ; 3.40 (m, *, H-5', dT _x ) ; 3.75-3.30 (gm, 15H with *, 0£H ₂ CH ₂ £H. ₂ N) ; 2.44 (m, 2H, H-2 ' , dT _x ); 2.25 (m, 2H, H-2 ' , dT ₃ ) . 2.00-1.70 (gm, **, OCH ₂ £H ₂ CH ₂ N) ; 1.86&1.84&1.81&1.79&1.56 (s&s&s&s&ε, 13H with

**, CH ₃ , Thy) . ¹³ C-NMR (DMF-d ₇ ) , only proton correlated carbons: 142.8 (C-6, Thy ₂ ) ; 136.2&136.0 (C-6, dT _x &dT ₃ ) ;

130.6&128.6&128.2&127.3&113.5 (Phe, MMT) ; 86.3&86.1 (C-4', dT ₃ ); 84.7&84.5 (C-1', dTi&dT- _j ) ; 83.7 (C-4', dT _x ) ; 80.0&79.8 (C-3', dΥ _x ) ; 71.8&71.6 (C-3', dT ₃ ) ; 71.1 (C-5', dT ₃ ) ;

&68.9&67.9&67.0&65.9 (0£H ₂ CH ₂ CH ₂ N, ) ; 64.6&64.5 (C-5', dT _x ) ; 55.2 (0CH ₃ , MMT) ; 48.6

(-C(=0)CH ₂ Thy) ; 43.9&43.4&43.2 ( -£H ₂ N(C (=0) CH ₂ Thy)£H ₂ -) ; 39.9 (C-2', dT ₃ ); 36.9 (C-2', dT^ ; 29.0&28.8&28.0 (NCH ₂ £H ₂ CH ₂ 0, dT _j &d^); 12.2&11.9&11.8 (CH ₃ , Thy) .

Example 20 5 ' -O- (p-anisyldiphenylmβthyl) -3 '-0-{ [4-N- (thymine- 1-yl) acetyl] -1, 7-heptaza-4-nediyl} thymidylyl - (3 '→5' ) -3' -O-(succinoyl) hymidine, 16

Compound 15 (252 mg, 0.25 mmole) is coevaporated with anhydrous Py (3x5 ml) and dissolved in Py (3 ml) . Succinic anhydride (100 mg, 1 mmole) and DMAP (30 mg, 0.25) is added, and reaction mixture is kept at ambient temperature overnight. A 1:3 mixture of H ₂ 0/Py (4 ml) is added and kept 1 hour at ambient temperature. The reaction mixture is evaporated, the residue is dissolved in CHC1 ₃

(25 ml), washed successively with cold 0.5 M citric acid ⁽ 3x10 ml) and brine (2x10 ml) , dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is coevaporated with anhydrous dioxane (2x5 ml) and lyophylized from anhydrous dioxane (3 ml) to give compound 16, 270 mg (97%) . ^- R ⁽ acetone-d ₆ ) : 7.66&7.64&7.60 (m&m&m, 2H, H-6, dT _x &dT ₃ ) ; 7.49

(m, 4H, Phe, MMT); 7.40-7.20 (gm, *, Phe, MMT); 7.23 ( , 9H with *, H-6, Thy ₂ ) ; 6.92 (m, 2H, Phe, MMT); 6.29 (gm,

2H, H-l', dT ₁ &dT ₃ ); 5.35 (m, IH, H-3 ' , dT ₃ );4.65 (m, 2H,

C(=0)£H ₂ Thy); 4.32 (m, IH, H-3', dT^ ; 4.16 (gm, 2H, H-

4',dT _x ); 4.00 (m&m, IH, H-4 ' , dT _x &dT ₃ ) ; 3.79 (s, **, 0CH ₃ ,

MMT); 3.90-3.20 (gm, 15H with **, H-5', d^&dT- _j ,-

OCH ₂ CH ₂ £H ₂ N) ; 2.63&2.62 (s&s, 4H, -OOCCH ₂ CH ₂ C00H) ; 2.35 (gm,

4H, H-2', dT ₁ &dT ₃ ). 1.95-1.75 (gm, ***, OCH ₂ CH ₂ CH ₂ N) ;

1.84&1.82&1.79&1.77&1.48 (s&s&s, 13H with ***, CH ₃ , Thy) .

¹³ C-NMR (acetone-d ₆ ) , only proton correlated carbons: 142.7

(C-6, Thy ₂ ); 136.0 (C-6, d^&d^);

129.9&128.9&128.4&127.5&113.7 (Phe, MMT); 85.2&85.1&84.9&84.1&83.9 (C-1', C-4', dT _x &dT ₃ ) ; 80.4&80.2 (C-3', dT _x ); 76.2&75.6 (C-3', dT ₃ ) ; 71.2 (C-5', dT ₃ ) ;

69.2&67.9&67.2&66.0 (0£H ₂ CH ₂ CH ₂ N, ) ; 64.7&64.6 (C-5', dT _x ) ;

55.2 (OCH ₃ , MMT); 48.8 (-C(=0)£H ₂ Thy) ; 44.3&44.2&43.7&43.5

(-£H ₂ N(C(=0)CH ₂ Thy)£H ₂ -) ; 37.5 (C-2', dT ₁ &dT ₃ );

29.30&29.28&29.20&28.74&28.26 ( -NCH ₂ £H ₂ CH ₂ 0- & - 00C£H ₂ £H ₂ C00H) ; 12.4&12.30&11.90&11.80 (CH _3/ Thy).

Example 21 CPG bound trinucleoside 16, 17

Trinucleoside 16 is converted to the succinimide ester and treated with long chain alkylamine CPG (500 A, Sigma) . The resulting CPG 17 with a loading of 15-20 μmole/g is used for solid phase oligonucleotide synthesis according standard protocols.

Example 22

5 / _o- (p-anisyldiphenylmβthyl) -3/ -0-{ [4-N- (thymine-

1-yl) acetyl] -1, 7 -hβptaza-4-nediyl} hymidylyl -

(3 '→5 ' ) -3 ' - [ (β-cyanoethoxy) -N-

(diisopropyl)phosphiryl]thymidine, 18 Compound 15 (504 mg, 0.5 mmol) is dissolved in anhydrous dichloromethane (7 ml) under an argon atmosphere.

Diisopropylethylamine (0.22 ml, 1.23 mmol) is added and the reaction mixture cooled to -30°C.

Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.75 mmol) is added to the reaction mixture and the reaction mixture is allowed to warm to 20°C and stirred for 4 hours.

Ethylacetate (40 ml) and triethylamine (0.5 ml) are added and the solution is washed with brine solution (3 x 20 ml) .

The organic phase is separated and dried over sodium

sulfate. After filtration of the solids the solvent is evaporated in vacuo at 20°C to an oil that is then purified by column chromatography using silica and a solvent such as toluene-ethyl acetate-triethylamine (20:80:1) as eluent. The fractions are then evaporated in vacuo (1 torr) at 20°C and the residue is evaporated with anhydrous pyridine and the rest is lyophilized from benzene-acetonitrile (1:1) to yield title compound 18.

Tϋxam l ft ^ 3

3-N-Benzyloxymethyl-3 '-O- [3-N- (trifluoroacetyl) -3- N-(tβx-t-butoxycarbonylmethyl)-3-aminopropyl] -5'-0- (tβ rahydropyran-2-yl)thymidine, 19

To a stirred solution of compound 6 (2.00 g, 3.3 mmole) in anhydrous DMF (10 ml) is added a suspension of 60% NaH (140 mg, 3.5 mmole) and after 1 hour tert-butyl ester of bromoacetic acid (F 195.06, d 1.321) (0.68 g, 0.52 ml, 3.5 mmole) . The resulting mixture is stirred overnight at 50°C and cooled to ambient temperature. Another portion of 60% NaH (40 mg, 1 mmole) is added and after 1 hour tert-butyl ester of bromoacetic acid (0.20 mg, 0.15 ml, 1 mmole) is added. The resulting mixture is stirred overnight at 50°C, cooled to ambient temperature and DMF is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (100 ml) , washed successively with water (1x30 ml) and brine (2x30 ml), dried (MgS0 ₄ ) and evaporated in vacuo to dryness. The residue is chromatographed over silica gel column (2.5x25 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 50%) as eluent to give compound 19, 2.10 g (89%), as a white foam. ^-NMR (CDC1 ₃ ) :

7.60&7.53(m&, IH, H-6, dT) ; 7.40-7.20 (gm, 5H, Phe , BOM); 6.30 ( , IH, H-l', dT) ; 5.49 (s, 2H, N£H ₂ , BOM); 4.85 (m,

2H, N£H ₂ C0OBu ^t ) ; 4.70 (s, 2H, 0£H ₂ , BOM); 4.64&4.59 (m&m,

IH, H-l, THP); 4.18 (m, *, H-4', dT) ; 4.15&4.08 (m&m, *, H-3' , dT) ; 4.10-3.40 (gm, 10H with *, H-5' , dT; H-5, THP;

0£H ₂ CH ₂ £Ii ₂ N(COCF ₃ )-) ; 2.44 (m, IH, H-2 ' , dT) ; 2.02 (m, **,

H-2', dT); 1.95&1.91(d&d, **, CH ₃ , Thy); 1.87 (m, **,

OCH ₂ £H ₂ CH ₂ N(COCF ₃ )-) ; 1.80&1.56&1.45 (m&m&m, 21H with ****,

H-2,-3,-4, THP; NCH _j COOBj^ ) . ¹⁹ P-NMR (CDC1 ₃ ) : -69.30 (C£ ₃ ) .

Example 24 3 ' -O- [3-N- (trifluoroacetyl) -3-N- (tβrt- butoxycarbonyl eth l) -3-aminopropyl] -5'-O- (tβtrahydropyran-2-yl)thymidine, 20 To a stirred solution of compound 19 (2.14 g, 3.0 mmole) in AcOEt (50 ml) is added 10% Pd/C (Degussa type, Aldrich) (1.1 g) . The flask is evacuated and then flushed 3 times with hydrogen and finally filled with hydrogen at 40 to 50 psi. The resultant suspension was stirred vigorously at 23°C for 14 hours. The suspended material is filtered off through a pad of Celite in a Buchner funnel and then the funnel is rinsed several times with a small portion of AcOEt and MeOH. The combined filtrate and washing is evaporated in vacuo to dryness. The residue is chromatographed over a silica gel column (2.5x25 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 70%) as eluent to give compound 20, 1.59 g (87%), »as a white foam. ¹ H-

NMR (CDC1 ₃ ) : 9.96&9.87 (bs&bs, IH, N2, Thy) ;7.60&7.53(m&, IH, H-6, dT) ; 6.30 (m, IH, H-l', dT) ; 4.80 (m, 2H, NCI^COOBu ; 4.64&4.59 (m&m, IH, H-l, THP); 4.18 (m, *, H-

4', dT) ; 4.15&4.08 (m&m, *, H-3', dT) ; 4.10-3.40 (gm, 10H with *, H-5', dT; H-5, THP; 0£H ₂ CH ₂ ϋ ₂ N(COCF ₃ )-) ; 2.44 (m,

IH, H-2', dT) ; 2.02 (m, **, H-2' , dT) ; 1.95&1.91(d&d, **, CH ₃ , Thy); 1.87 (m, **, OCH ₂ £H ₂ CH ₂ N(COCF ₃ )-) ; 1.80&1.56&1.45 (m&m&m, 21H with **, H-2, -3,-4, THP; NC^COOSu . ¹⁹ F-NMR (CDC1 ₃ ) : -69.30 (C£ ₃ ) .

Example 25 3 ' -O- [ 3 -N- ( tert-butoxycarbonylmethyl ) - 3 - aminopropyl] -5 ' -O- ( tetrahydropyran-2-yl ) thymidine, 21 A solution of compound 20 (1.82 g, 3.0 mmole) in 20 ml of 3:1 mixture of 12% NH ₃ /EtOH - 28% NH ₄ OH is kept at

65°C overnight. The reaction mixture is cooled and evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (4.5x25 cm) using CHC1 ₃ /28%NH ₄ 0H (99.5/0.5) (gradient of MeOH from 0 to 7%) as eluent to give compound 21, 1.47 g (96%), as an oil. ^ ^■ H-NMR (CDC1 ₃ ) : 9.95 (bs, IH, NH, Thy); 7.60 (m, IH, H-6, dT) ; 6.30 (m, IH,

H-l', dT) ; 4.64&4.59 (m&m, IH, H-l, THP); 4.30-4.00 (gm, 4H, H-4', dT;H-3', dT; - H^COOBu*) ; 4.00-3.40 (gm, 6H, H-

5', dT; H-5, THP; 0£H ₂ CH ₂ CH ₂ NH- ) ; 2.75 (m, 2H, OCH ₂ CH ₂ £H ₂ NH) ;

2.40 ( , IH, H-2', dT) ; 2.02 (m, *, H-2 ' , dT) ; 1.93 (d, *, CH ₃ , Thy); 1.75 (m, *, OCH ₂ £H ₂ CH ₂ NH-) ; 1.70&1.56&1.45

(m&m&m, 21H with *, H-2, -3,-4, THP; -NHCH ₂ COOBu ^t ) .

Example 26 3'-θ-{3-N-[ (thymine-1-yl) acetyl] -3-N-( tert- butoxycarbonylmethyl ) -3 - minopropyl } - 5 ' -O- ( t β t r ahydr opyran- 2 -yl ) thymidine , 22a A solution of compound 21 (1.02 g, 2.00 mmole), pentafluorophenyl ester of thymine-1-ylacetic acid (0.77 g, 2.10 mmole) and DIPEA (2.10 mmole) in anhydrous DMF (10.0 ml) is kept at ambient temperature for 4 hours and the DMF is evaporated. The residue is chromatographed over a silica gel column (2.5x25 cm) using CHCl ₃ /MeOH/28%NH ₄ OH

(97/3/0.1) as eluent to give compound 22, 1.33 g (98%), as a white solid. ^Η-NMR (CD ₃ OD) : 7.65&7.58 (m&m, IH, H-6, dTi); 7.30&7.27 (m&m, IH, H-6, Thy ₂ ) ; 6.23 (m, IH, H-l',

dT _j ); 4.85-4.60 (g , 5H, -J H ₂ COOBu ^t ; -C (=0) £H ₂ Thy; H-l, THP); 4.20 (m, *, H-4', dT _x ) ; 4.15 (m, 2H with *, H-3 ' , dT _χ ) ; 4.00-3.40 (gm, 8H, H-5', dT _ι; H-5, THP; 0£ϋ ₂ CH ₂ £H ₂ N) ; 2.38 ( , IH, H-2 ' , dT _x ) ; 2.10 (m, IH, H-2 ' , dT _x ) ; 2.00- 1.40 (g , 23H, OCH ₂ £H. ₂ CH ₂ N; CH ₃ , dT^Thy^- H-2, -3,-4, THP; -NHCH ₂ COOSn ^t ) .

Example 27 3 ' -0-{3-N- [ ( (4-N- (isobutyryl)cytidine-l-yl) acetyl] - 3-N- (tert-butoxycarbonylmethyl) -3 -aminopropyl} -5 ' - O- (tβtrahydropyran-2-yl) thymidine, 22b

A solution of compound 21 (1.02 g, 2.00 mmole), pentafluorophenyl ester of [4-N- (isobutyryl)cytidine-l- yl] acetic acid (0.85 g, 2.10 mmole) and DIPEA (2.10 mmole) in anhydrous DMF (10.0 ml) is kept at ambient temperature for 4 hours and the DMF is evaporated. The residue is chromatographed over a silica gel column (2.5x25 cm) using CHC1 ₃ /MeOH/ (gradient of MeOH from 0 to 7%) as eluent to give compound 22b, 1.42 g (97%), as a white solid.

Example 28 3 * _o- {3-N- t ( (6-N- (phenoxyacetyl) adenine- 9- yl) acetyl] -3-N- ( tert-butoxycarbonylmethyl) -3- aminopropyl } - 5 ' -O- ( tetrahydropyran-2 -yl ) thymidine , 22c

A solution of compound 21 (1.02 g, 2.00 mmole), pentafluorophenyl ester of [6-N- (phenoxyacetyl) adenine-9- yl] acetic acid (1.04 g, 2.10 mmole) and DIPEA (2.10 mmole) in anhydrous DMF (10.0 ml) is kept at ambient temperature for 4 hours and the DMF is evaporated. The residue is chromatographed over a silica gel column (2.5x25 cm) using CHC1 ₃ /MeOH/ (gradient of MeOH from 0 to 7%) as eluent to give compound 22c, 1.51 g (92%), as a white solid.

Example 29 3'-θ-{3-N- [ (thyminβ-1-yl)acetyl] -3-N- (carboxymβ hyl) -3-aminopropyl)-5'-O- (p- anisyldiphenylmethyl)thymidine, 23a A solution of compound 22a (1.69 g, 2.5 mmole) in

50% TFA/DCM (50 ml) is kept at ambient temperature for 30 minutes. The reaction mixture is evaporated in vacuo and the residue is coevaporated with toluene (2x50 ml) , anhydrous Py (3x50 ml) , dissolved in anhydrous Py (25 ml) , and MMT-Cl (0.93 g, 3.0 mmole) is added. The reaction mixture is kept at ambient temperature overnight and evaporated. The residue is suspended in CHC1 ₃ (150 ml) and washed successively with water (2x50 ml) , cold citric acid (3x30 ml) and brine (2x50 ml) , dried (MgS0 ) and evaporated in vacuo. The residue is reprecipitated from CHCl ₃ /hexane. The precipitate is collected and dried in vacuo to give compound 23a, 1.92 g (95%), as a white solid. ^- MR (CD ₃ 0D) : 7.65&7.58 (m&m, IH, H-6, dT^ ; 7.45-7.10 (gm, 13H,

Phe, MMT; H-6, Thy ₂ ) ; 6.82 (m, 2H, Phe, MMT); 6.20 (m, IH, H-l', dT _x ) ; 4.90-4.60 (gm, 4H, -N£fl ₂ COOH; -C(=0)£H_ ₂ Thy) ; 4.20 (m, *, H-4', dT _x ) ; 4.15 (m, 2H with *, H-3' , dT^ ; 4.00-3.40 (gm, 9H, H-5', dT _x ; 0CH ₃ ,,MMT; 0£H ₂ CH ₂ £H. ₂ N) ; 2.40

( , IH, H-2', dT _x ) ; 2.08 (m, IH, H-2', dT _x ) ; 2.00-1.40

(g , 8H, OCH ₂ £H ₂ CH ₂ N; CH ₃ , d^Thy^ .

Example 30

3 ' -0- { 3 -N- [ ( ( 4 -N- ( isobutyryl ) cytidine-l-yl ) cetyl ] -

3 -N- ( carboxymethyl ) - 3 -aminopropyl } -5 ' -O- (p- anisy ldiphenylme thyl ) thymidine , 23b Compound 22b ( 1.46 g, 2 . 00 mmol ) is treated as described in Example 29 to give the title compound 23b,

1 . 47 g ( 85% ) .

Example 31 3 ' -0-{3-N- [ (6-N- (phenoxyacetyl )adenine-l- yl) acetyl] -3-N- (carboxymethyl) -3 -aminopropyl} -5 ' -O- (p-anisyldiphenylmethyl) thymidine, 23c Compound 22c (1.64 g, 2.00 mmol) is treated as described in Example 29 to give the title compound 23c, 1.49 g (78%) .

Example 32 3 ' -0-{3-N- [ (thymine-1-yl) acetyl] -3-N-

(pentaf luorophenoxycarbonylmethyl) - 3 -aminopropyl } - 5 ' -O- (p-anisyldiphenylmethyl) thymidine, 24a

A solution of compound 23a (1.00 mmol), pentaf luorophenol (1.1 mmol) and N,N'- dicyclohexylcarbodimide (1.1 mmol) in DMF (7 ml) is kept at ambient temperature for 1 hour. N,N'-dicyclohexylurea is filtered off and the resulting solution of compound 24a is used without purification.

Example 33

3' -0-{3-N- [ (4-N- (isobutyryl)cytidinβ-l-yl)acetyl] -

3-N-(pentafluorophenoxycarbonylmethyl)-3- aminopropyl}-5'-0-(p- anisyldiphenylmβthyl) thymidine, t 24b Compound 23b (1.00 mmol) is treated as described in

Example 32 to give a solution of the title compound 24b.

Example 34 3 ' -0-{3-N- [ (6-N- (phenoxyacetyl) adβninβ-1- yl) cetyl] -3-N- (pentaf luorophenoxycarbonylmethyl) - 3 -aminopropyl} -5' -O- (p- anisyldiphenyl ethyl) thymidine, 24c

Compound 23c (1.00 mmol) is treated as described in Example 32 to give a solution of the title compound 24c.

Example 35 3-N-Benzyloxymethyl-5 ^, -0- (2-cyanoethyl) -3'-O- (benzyl)thymidine, 25

To a stirred solution of compound 7 (2.0 mmole) in anhydrous THF (150 ml) is added a suspension of 60% NaH (60 mg, 1.5 mmole) in one portion and dropwise neat acrylonitrile (5 ml) over 5 minutes. The resulting mixture is stirred at ambient temperature for an additional 1 hour. The mixture is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (500 ml) . The organic layer is washed with brine (2x250 ml), dried (MgS0 ₄ ), evaporated in vacuo to dryness, and the residue is chromatographed over a silica gel column (4.5x30 cm) using toluene/AcOEt (gradient of AcOEt from-10 to 15%) as eluent to give compound 25, (92%), as a colorless oil. ^-N R (CDC1 ₃ ) : 7.45-7.15 (gm,

11H, Phe, BOM&Bzl H-6, dT) ; 6.40 (m, IH, H-l', dT) ; 5.48 (s, 2H, NCH ₂ , BOM); 4.68 (s, 2H, OCH ₂ , BOM); 4.53 (m, 2H,

OCH ₂ , Bzl); 4.24 (m, H-3' , dT) ; 4.16 (m, H-4', dT) ; 3.80- 3.55 (gm, 4H, H-5' , dT; 0£H ₂ CH ₂ CN) ; 2.56 (t, 2H, OCH ₂ £H _.2 CN) ; 2.45 (m, IH, H-2' , dT) ; 2.09 (m, IH, H-2' , dT);1.94 (d, 3H, CH ₃ , Thy). ¹³ C-NMR (CDC1 ₃ ): 163.2 (C-4, Thy); 150.7 (C-2,

Thy); 137.8&137.3 (C-1, Phe, BOM; C7I, Bzl); 134.1 (C-6, Thy); 128.3-127.4 (gs, C-2, C-3, C-4, Phe, BOM&Bzl); 117.4 (~£N) ; 110.1 (C-5, Thy); 85.4 (C-1', dT) ; 83.1 (C-4', dT) ; 78.4 (C-3', dT); 71.9 (OCH ₂ , BOM); 71.4 (0CH ₂ , Bzl); 70.7

(C-5', dT) ; 70.3 (NCH ₂ , BOM); 65.6 (0£H ₂ CH ₂ CN) ; 55.2 (OMe, MMT); 37.4 (C-2', dT) ; 18.7 (0CH ₂ £H ₂ CN) ; 13.2 (CH ₃ , Thy).

Example 36 3-N-Benzyloxymβthyl-5'-0-(3- trif luoroacetamidopropyl ) - 3 ' -O- (benzyl ) thymidine , 2 6

Compound 25 is treated as described in Example 5 to give 3 -N-benzyloxymethyl-5 ' -0- ( 3 -aminopropyl ) -3 ' -0- ( benzyl ) thymidine, ( 62%) , as a yellowish oil . ^- MR

(CDC1 ₃ ) : 7.52 (m,lH, H-6, Thy); 7.40-7.20 (gm, 10H, Phe

, BOM&Bzl) ; 6.35 (m, IH, H-l', dT) ; 5.47 (s, 2H, N£H ₂ , BOM) ;

4.68 (s, 2H, 0£H ₂ , BOM); 4.52 (m, IH, 0£H ₂ , Bzl) ; 4.16 (g ,

2H, H-4', H-3X dT) ; 3.70 (m, IH, H-5', dT) ; 3.60-3.40 (gm, 3H, 0£H ₂ CH ₂ CH ₂ NH ₂ ; H-5 ' , dT) ; 2.74 (t, 2H, OCH ₂ CH ₂ £H ₂ NH ₂ ) ;

2.44 (m, IH, H-2 ' , dT) ; 2.03 (m, IH, H-2 ' , dT) ; 1.92 (d, 3H, CH ₃ , Thy); 1.71 (m, ** , OCH ₂ CH ₂ £H ₂ NH ₂ ) . ¹³ C-NMR (CDC1 ₃ ) :

163.3 (C-4, Thy); 150.8 (C-2, Thy); 137.9&137.4 (C-1, Phe, BOM&Bzl); 134.3 (C-6, Thy); 128.4-127.4 (C-2, C-3, C-4, Phe, BOM&Bzl); 109.9 (C-5, Thy) ; 85.8 (C-1', dT) ; 83.7 (C- 4' , dT) ; 78.7 (C-3', dT) ; 72.0 (OCH ₂ , BOM); 71.2 (OCH ₂ ,

Bzl); 70.8 (C-5', dT) ; 70.3 (NCH ₂ , BOM); 69.4 (OCH ₂ CH ₂ CH ₂ NH ₂ ) ; 39.1 (OCH ₂ CH ₂ £H ₂ NH ₂ ) ; 37.8 (C-2', dT) ; 32.2 (OCH ₂ £H ₂ CH ₂ NH ₂ ) ; 13.2 (CH ₃ , Thy) . 3-N-Benzyloxymethyl-5 ' -O- ( 3 -aminopropyl ) -3 ' -O-

(benzyl) thymidine is treated as described in Example 6 to give the title compound 26, (93%) . ^-NMR (CDC1 ₃ ) : 7.40-

7.20 (gm, 12H, Phe , BOM&Bzl H-6, Thy; NHCOCF ₃ ) ; 6.29 (m,

IH, H-l', dT); 5.47 (s, 2H, N£H ₂ , BOM); 4.68 (s, 2H, 0£H ₂ , BOM) ; 4.52 (m, 2H, O £ ₂ , Bzl); 4.17 (m, 2H, H-4\ H-3 ' , dT) ; 3.68 (m, IH, H-5 ' , dT) ; 3.60-3.30 (gm, 5H, 0£H ₂ CH ₂ CH ₂ NHCOCF ₃ ; H-5 ' , dT) ; 3.50 ( , 1 OH with **,

OCH ₂ CH ₂ £H ₂ NHCOCF ₃ ) ; 2.48 (m, IH, H-2 ' , dT) ; 2.06 ( , IH, H- 2' , dT) ; 1.89 (s, 3H, CH ₃ , Thy); 1.83 (m, 2H, OCH ₂ £H ₂ CH ₂ NHCOCF ₃ ) . ¹³ C-NMR (CDCl ₃ ) : 163.3 (C-4, Thy); 157.1 (q, ² J _C _ _F =36.9 Hz, £(=0)CF ₃ ); 150.7 (C-2, Thy); 137.7&137.3

(C-1, Phe, BOM&Bzl); 134.4 (C-6, Thy) ; 128.4-127.0 (C-2, C- 3, C-4, Phe, BOM); 115.7 (q, ^l J _c _ _F =281.9 Hz, C(=0)£F ₃ );

109.9 (C-5, Thy); 86.0 (C-1', dT) ; 83.4 (C-4', dT) ; 78.6 (C-3', dT) ; 72.0 (OCH ₂ , BOM) ; 71.4 (OCH ₂ , Bzl) ; 71.0 (C-5' , dT) ; 70.3 (NCH ₂ , BOM); 69.2 (0CH ₂ CH ₂ CH ₂ NHC0CF ₃ ) ; 37.6&37.5

(C-2', dT) ; 28.6 (OCH ₂ £H ₂ CH ₂ NHCOCF ₃ ) ; 13.1 (CH ₃ , Thy) . ¹⁹ F- NMR (CDC1 ₃ ) : -76.06 (s, CF ₃ ) .

Example 37 3-N-Benzyloxymethyl-5 ' -O- [3-N- (trifluoroacetyl) -3 - N- ( ert-butoxycarbonylmethyl) -3 -aminopropyl] -3 ' -O- ( benzyl) thymidine, 27

Compound 26 (2.00 mmol) is treated as described in Example 23 to give the title compound 27, (85%) .

Example 38 5 ' -O- [3-N- (trifluoroacetyl) -3-N- (carboxymethyl) -3- aminopropyl] -thymidine, 28

Compound 27 (2.00 mmol) is treated as described in Example 24 to give 5' -0- [3 -N- (trifluoroacetyl) -3 -N- ( tert- butoxycarbonylmethyl) -3 -aminopropyl] thymidine, (82%) . 5'- O- [3-N- (Trifluoroacetyl) -3-N- ( tert-butoxycarbonylmethyl) -3- aminopropyl ] hymidine is treated then as described in Example 29 to remove the tert-butyl protecting group from the carboxyl moiety and to give the title compound 28, (95%) .

Example 39, 5'-θ-{3-N- [ (thymine-1-yl) acetyl] -3-N- (carboxymethyl) -3 -aminopropyl} -thymidine, 29a

Compound 28 is treated as described in Example 25 to give 5 ' -O- [ 3 -N- ( carboxyme hyl ) -3 -aminopropyl ] thymidine , (97%) . 5 '-0-.3-N- (Carboxymethyl) - 3 -aminopropyl ] - thymidine is treated then as described in Example 26 to give the title compound 29a, as a white solid (92%) .

Example 40 5 ' -0-{3-N- [ (4-N- (isobutyryl) cytidine-1-yl) acetyl] - 3-N- (carboxymethyl) -3 -aminopropyl } thymidine, 29b 5 '-0- [3 -N- (Carboxymethyl) -3 -aminopropyl ] - thymidine is treated then as described in Example 27 to give the

title compound 29b, as a white solid (91%) .

Example 41 5 * _o- {3-N- [ ( 6-N- (phenoxyacetyl) adenine-1- yl) acetyl] -3-N- (carboxymethyl) -3- aminopropyl} thymidine, 29c

5 ' -0- [ 3 -N- ( Carboxymethyl ) -3 -aminopropyl ] - thymidine is treated then as described in Example 28 to give the title compound 29c, as a white solid (93%) .

Example 42

5 '-0-{3-N-[ (thymine-1-yl) acetyl] -3-N-

( carboxymethyl) -3 -aminopropyl) --3 ' -O- (di-p- anisyldiphenylmethyl) thymidine, 30a; 5 ^/ _o-{3-N- [ (4-N- (isobutyryl) cytidine-1-yl ) acetyl] -

3-N- (carboxymethyl) -3 -aminopropyl} -3 ' -O- (di-p- anisyldiphenylmethyl) thymidine, 30b;

5 ' -O- {3-N- [ ( 6-N- (phenoxyacetyl) adβnine-1- yl) cetyl] -3-N- (carboxymethyl) -3 -aminopropyl} -3 " -O- (di-p-anisyldiphenylmethyl) thymidine, 30c

Compound 29 is treated with DMT-C1 in the same way as described in Example 29 to give the title compounds

30a-c.

Example 43

5'-0-{3-N- [ (thymine-1-yl) cetyl] -3-N-

( succxnimidoxycarbonylmethyl) -3 -aminopropyl} --3 ' -O-

( di-p-anisyldiphenylmethyl) thymidine, 31a;

5' -0-{3-N- [ (4-N- ( isobutyryl )cytidine-l-yl) acetyl] - 3-N- ( succinimidoxycarbόnylmethyl) - 3 -aminopropyl } - 3 ' -O- (di-p-anisyldiphenylmethyl) thymidine, 31b; 5 ^/ _o- {3-N- [ (6-N- (phenoxyacetyl) denine-1- yl) acetyl] -3-N- ( succinimidoxycarbonylmethyl) -3- aminopropyl} -3 ' -O- (di-p-anisyldiphenylmethyl ) thymidine, 31c

A solution of compound 30 (1.00 mmol), N- hydroxysuccini ide (1.1 mmol) and N,N'-

dicyclohexylcarbodimide (1.1 mmol) in DMF (7 ml) is kept at ambient temperature for 1 hour. N,N' -dicyclohexylurea is filtered off and resulting solution of compound 31 is used without purification.

3-N-Benzyloxymethyl-6' -O- (p-anisyldiphenylmethyl ) - 5'- homo thymidine, 40

5 ' -Homo thymidine (5.12 g, 20 mmol) (Etzold, G. et al., Chemical Communications, (1968), p. 422) is monomethoxytritylated as per the procedure of Example 19 and then benzyloxymethylated and purified as per the procedure of Example 1 to give entitled compound 40 (87%) .

Example 44

3-N-Benzyloxymethyl-6-3 ' -O- (2-cyanoethyl) ' -O- (p- anisyldiphenylmethyl) -5 ' -homo thymidine, 41

Compound 40 is cyanoethyla ed and purified as per the procedure of Example 2 to give compound 41.

Example 45 3-N-Benzyloxymethyl-3 ' -O- (2-cyanoethyl) -6 ' -O- (tetrahydropyran-2-yl) -5 ' -homothymidine, 42

Compound 41 is deprotected as per the procedure of Example 3 and then treated as per the procedure of Example 4 to give compound 42.

Example 46 3-N-Benzyloxymethyl-3 ' -O- (3 -aminopropyl) -6' -O- (tetrahydropyran-2-yl) -5 ' -homothymidine, 43

Compound 42 is reduced and purified as per the procedure of Example 5 to give compound 43.

Example 47 3-N-Benzyloxymethyl-3'-0- (3- trif luoroacetamidopropyl) -6' -O- ( tetrahydropyran-2- yl) -5 ' -homothymidine, 44 Compound 43 is trifluoroacetylated and purified as per the procedure of Example 6 to give compound 44.

Example 48 3-N-Benzyloxymβthyl-3' -O- [3-N- (trifluoroacetyl) -3- N- ( tert-butoxycarbonylmethyl) -3 -aminopropyl) -6 ' -O- ( e r ahydropyran- 2 -y 1 ) - 5 ' -homothymidine , 45

Compound 44 is alkylated with tert-butyl ester of bromoacetic acid and purified as per the procedure of

Example 23 to give compound 45.

Example 49

3-N-Benzyloxymethyl-3'-0- [3-N- (trifluoroacetyl) -3-

N- ( ert-butoxycarbonylmethyl) -3 -aminopropyl) -6 ' - bromo- 5 ' -homothymidine , 46 The solution of compound 45 (5 mmol), CBr ₄ (7 mmol) and P(Ph) ₃ (14 mmol) in anhydrous THF (40 ml) is stirred at ambient temperature for 24 hours. The reaction mixture is evaporated in vacuo to dryness, the residue coevaporated with toluene (2 x 100 ml) and chromatographed over a silica gel column (4.5x25 cm) using toluene/AcOEt ⁽ 7:3cl:l ⁾ as eluent to give compound 46, (95%) .

Example 50 3-N-Benzyloxymethyl-3'-0- [3-N- (trifluoroacetyl) -3- N- ( ert-butoxycarbonylmethyl) -3 -aminopropyl] -6 ' -N- (di-tert-butyloxycarbonyl) -6' -amino-5'- homo thymidine, 47

To a stirred solution of di-tert- butyliminodicarboxylate (1.09 g, 5.0 mmole) in anhydrous DMF (20 ml) is added a suspension of 60% NaH (200 mg, 5.0 mmole) and after 1 hour compound 46 (4.0 mmole) . The

resulting mixture is stirred overnight at 50°C, cooled to ambient temperature and DMF is evaporated in vacuo to dryness. The residue is dissolved in AcOEt (150 ml), washed successively with water (1x30 ml) and brine (2x30 ml), dried (MgS0 ) and evaporated in vacuo to dryness. The residue is chromatographed over silica gel column (2.5x25 cm) using toluene/AcOEt (gradient of AcOEt from 10 to 50%) as eluent to give compound 35.

Example 51

3 '-o- [3-N- ( ert-butoxycarbonylmethyl) -3- aminopropyl] -6 ' -N- ( ert-butyloxycarbonyl) -6 ' -amino- 5 ' -homothymidine , 48

Compound 47 (2.0 mmole) is hydrogenated and purified as per the procedure of Example 11. The product of reaction is treated withlO ml of 3:1 mixture of 12% NH ₃ /EtOH - 28% NH ₄ 0H at ambient temperature overnight. The reaction mixture is cooled and evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (2,5x25 cm) using CHCl ₃ /MeOH/28%NH ₄ OH (95/1/0.5) as eluent to give compound 48.

Example 52 3'-0-{3-N- [ (thymine-1-yl) acetyl] -3-N-(tert- butoxycarbonylmethyl) -3-aminopropyl}-6 ' -N- ( ert- butyloxycarbonyl) -6' -amino- 5' -homothymidine, 49a

Compound 48 is treated as described in Example 26 to give the title compound 49a, as a white solid (95%) .

Example 53

3'-0-{3-N- [ (4-N- (isobutyryl) cytidine-1-yl ) acetyl] -

3-N- ( ert-butoxycarbonylmethyl) -3 -aminopropyl} -6' -

N- ( ert-butyloxycarbonyl) -6 ' -amino-5 ' - homothymidine, 49b Compound 48 is treated as described in Example 27 to give the title compound 49b, as a white solid (95%) .

Example 54

3 '-0-{3-N- [ (thymine-1-yl) acetyl] -3-N- ( carboxymethyl) -3 -aminopropyl} -6 ' -N- (p- anisyldiphenyl ethyl) -6 ' -amino-5 ' -homothymidine, 50a

Compound 49a is treated as described in Example 29 to give the title compound 50a, as a white solid (93%) .

Example 55

3 ' -O- {3-N- [ (4-N- (isobutyryl) cytidine-1-yl) acetyl] - 3-N- (carboxymethyl) -3-aminopropyl}-6 ' -N- (p- anisyldiphβnylmethyl) -6 ' -amino- 5 ' -homothymidine, 50b Compound 49b is treated as described in Example 30 to give the title compound 50b, as a white solid (95%) .

Example 56 3'-0-{3-N- [ (thymine-1-yl) acetyl] -3-N- (pentaf luorophenoxycarbonylmethyl) -3-aminopropyl} -

6' -N- (p-anisyldiphenylmβthyl) -6'-amino-5' - homothymidine, 51a

Compound 50a is treated as described in Example 32 to give a solution of the title compound 51a.

Example 57

3 _o-{3-N- [ (4-N- (isobutyryl)cytidine-1-yl)acetyl] - 3-N-(pentafluorophenoxycarbonylmethyl)-3- aminopropyl}-6'-N-(p-anisyldiphenylmβthyl) -6'- amino-5'-homothymidine, 5lb

Compound 50b is treated as described in Example 33 to give a solution of the title compound 51b.

Example 58 3'-0-{3-N-[ (thymine-1-yl) acetyl] -3-N- (N- (3-N- (diethylamino) propyl)carboxamidomethyl) -3- aminopropyl}-6' -N- (p-anisyldiphenylmβthyl) -6 ' -

amino-5 '-homothymidine, 52a

A solution of compound 51a (1 mmole) in DMF (5 ml) is treated with 3-N-(diethylamino)propylamine (1.5 mmole) at ambient temperature for 2h. The reaction mixture is evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (2.5x25 cm) using CHC1 ₃ /28%NH ₄ 0H (99.9/0.1) (gradient of MeOH from 0 to 9%) as eluent to give compound 52a, (85%), as an oil.

Example 59

Oligonucleoside 53 (n=l)

A solution of compound 52 (1 mmole) in 80% AcOH 100 ml is kept overnight at ambient temperature. The reaction mixture is evaporated in vacuo to dryness and the residue is coevaporated with Py (3x50 ml) and anhydrous DMF (3x25 ml) . The residue is dissolved in anhydrous DMF (5 ml) and solution of compound 51 (1.2 mmole) and DIPEA (1.1 mmole) in anhydrous DMF (5 ml) is added. The reaction mixture is kept at ambient temperature for 2h. The reaction mixture is evaporated in vacuo to dryness and the residue is chromatographed over a silica gel column (2.5x25 cm) using CHC1 ₃ /28%NH ₄ 0H (99.9/0.1) (gradient of MeOH from 0 to 10%) as eluent to give compound 53 , (89%) .

Example 60

Oligonucleoside 53 (n=2)

Compound 53 (n=l) is treated as described in Example 59 to give the title compound 53 (n=2) , as a white solid (95%) .

Example 61 Oligonucleoside 53 (n=3)

Compound 53 (n=2) is treated as described in Example 59 to give the title compound 53 (n=3), as a white solid (94%) .

Example 62 Oligonucleoside 53 (n=4)

Compound 53 (n=3) is treated as described in Example 59 to give the title compound 53 (n=4) , as a white solid (90%) .

Example 63 Oligonucleoside 53 (n=5)

Compound 53 (n=4) is treated as described in Example 59 to give the title compound 53 (n=5) , as a white solid (91%) .

Example 64 Oligonucleoside 54 A solution of compound 53 (1 mmole) in 80% AcOH 100 ml is kept overnight at ambient temperature. The reaction mixture is evaporated in vacuo to dryness and the residue is dissolved in water and extracted twice with ether. The water layer is evaporated in vacuo to dryness and the residue is lyophilized twice from water.

Example 65

Once phosphoramidites of modified nucleosides and oligonucleoside synthetic blocks have been prepared, they can be incorporated into oligonucleotide analogues, which are synthesized by a stantard solid phase approach, using automated nucleic acid synthesizer such as Applied Biosysterns, Inc. Model 392. Standard phosphoramidite coupling chemistries (see, e.g. M.J. Gait, Oligonucleotide Synthesis. A practical approach., pp. 35-81) are used with these synthesizers to furnish desired oligonucleotides. The solution of tetraethylthiura disulfide (Applied Biosystems, Inc.) can be used to provide phosphorthioate oligonucleotides.

Example 66 Hybridization Analysis

The macromolecules of the invention can be compared in their ability to bind to complementary nucleic acids by determining the melting temperature of a particular double- stranded (ds) or triple-stranded (ts) complex. Upon formation of dsDNA from two single strands, due to base stacking the extinction coefficient decreases ⁽ hypochromicity) . Consequently, the denaturating of DNA can be followed by measuring changes in the absorbance, as a function of the melting temperature (T _m ) , the temperature where 50% of a duplex has disappeared to give single strands. The higher the T _m , the greater the strengh of the binding of the strands. Duplexes are formed from single-stranded oligodeoxyribonucleotides and the macromolecules of the present invention. The macromolecules of the present invention are synthesized according the description or examples presented herein. Oligodeoxyribonucleotides are synthesized on solid phase with an Applied Biosystems, Inc. 392 DNA/RNA Synthesizer. The oligonucleotide species is purified as their dimethoxytrityl derivatives by reverse- phase chromatography using HPLC (Gilson) . Typically 0.3- 0.5 OD ₂₆₀ oligonucleotide analog is hybridized with 1 equivalent of the other strand and the absorbance (260 n ) hyperchromicity upon duplex to random coil transition is monitored using a Gilford Response II spectrophotometer. The buffers used are 10 mM in phosphate, 0.1 mM in EDTA and either 0.1 M or 1 M in NaCl. The following extinction coefficients are used dA: 15.4 ml/μmol-cm; dT 8.8; dG: 11.7 and dC: 7.3 for both regular oligonucleotides and oligonucleotide analogues. The melting curves are recorded in steps of 0.5 °C/min. The T _m is determined from the maximum of the 1st derivative of the plot A ₂₆₀ vs temperature. Data can be analyzed by the graphic

representation of 1/T _m vs ln[C _oligo ], wherein C _oligo is the total oligonucleotide concentration. Thermodynamic parameters are determined from this analysis.

Nuclease Resistance

A. Evaluation of the resistance of macromolecules of the invention to serum and cytoplasmic nucleases.

The oligonucleotide analogues of the invention were evaluated for their stability in media containing various concentrations of fetal calf serum or adult human serum. Oligonucleotide analogs are incubated for various times, treated with protease K and then analyzed by reverse-phase or ion-exchange HPLC or by gel-electrophoresis on 20% poϊyacrylamide-urea denaturating gels and subsequent autoradiography. Based on the location of the modified linkage and the known length of the oligonucleotide it is possible to determine the effect on nuclease degradation depending on the particular modification of the linkage. For the cytoplasmic nucleases, an HL 60 cell line can be used.

Following the incubation, the oligonucleotide analogs are assessed for degradation as mentioned above for serum nucleolytic degradation. Autoradiography. results are quantitated to compare the regular oligonucleotides and macromolecules of invention.

B. Evaluation of the resistance of macromolecules of the invention to specific endo- and exo- nucleases.

Evaluation of the resistance of oligonucleotide analogues of the invention to specific nucleases (i.e. endonucleases, 3', 5'-exo-, and 5', 3 ' -exonucleases) can be done to estimate precisely the effect of a particular linkage on stability in degradation conditions. The oligonucleotide analogs are incubated in defined reaction buffers specific for various selected nucleases, treated

with proteinase K and then analyzed by reverse-phase or ion-exchange HPLC, or by gel-electrophoresis on 20% polyacrylamide-urea denaturating gels and subsequent autoradiography.