SI9520112A - Amino acid nucleic acids - Google Patents

Abstract

The present invention provides various novel oligonucleotide analogs having one or more properties that make the subject compounds superior to conventional oligonucleotides for use in procedures employing oligonucleotides. The compounds of the invention are oligonucleotide analogs in which the furanose ring of a naturally occurring nucleic acid is replaced with an amino acid or a modified amino alcohol residue. Some embodiments of the novel compounds of the invention are particularly useful for the antisense control of gene expression. The compounds of the invention may also be used as nucleic acid hybridization probes or as primers. Another aspect of the invention is to provide monomeric precursors of the oligonucleotide analogs of the invention. These monomeric precursors may be used to synthesize the subject polynucleotide analogs. Another aspect of the invention is to provide formulations of the subject polynucleotide analogs that are designed for the treatment or prevention of disease conditions. Yet another aspect of the invention is to provide methods for treating or preventing diseases, particularly viral infections and cell growth disorders. The subject disease treatment methods comprise the step of administering an effective amount of the subject polynucleotide analogs for use as antisense inhibitors.

Description

Oligonucleotides that specifically bind to complementary nucleic acids (e.g. sense chain) by binding hydrogen to inhibit gene expression are generally referred to as antisense oligonucleotides. These synthetic oligonucleotides bind to the target (mRNA) and thus inhibit translation of messenger RNA. This antisense principle (Uhlmann, E. et al., Chem. Revievvs, 1990, 90. 543-584; and Stein, CA et al., Cancer Res., 1988, 48. 2659-2688) is used in nature to regulate gene expression. This antisense principle has been used in the laboratory not only to inhibit but also to activate gene expression. Zamecnik and Stephenson were the first to propose the use of synthetic oligonucleotides for therapeutic purposes in 1978 (Stephenson, M. L.; and Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 1978, 75, 280, and 285). The specific inhibition of antisense poinucleotides is based on the specific Watson-Crick base pairing between heterocyclic antisense oligonucleotide and viral nucleic acid bases. The process of binding an oligonucleotide to a complementary nucleic acid is called hybridization. An oligomer having a base sequence complementary to that of a base on an mRNA that has an encoded protein required for disease progression is particularly interesting. By mRNA-specific hybridization, the synthesis of mRNA-encoded proteins may be impaired.

-2Preparation of unmodified oligonucleotides, e.g. Oligonucleotides having a DNA structure have been at the forefront of interest in many research groups over the past decade. Synthesis via phosphoramidites after Caruthers (McBride, LJ; and Caruthers, MH, Tetrahedron Letts., 1983, 24, 245), originally introduced by Letsinger (Letsinger, RL; and Lunsford, WB, J. Amer. Chem. Soc. , 1976, 98. 3655) as the phosphite triester method is currently the most efficient method for the preparation of phosphodiester oligonucleotides. When they are common, e.g. unmodified oligonucleotides used as antisense oligonucleotides have problems such as instability to nucleases and insufficient membrane penetration. In order for the antisense oligonucleotides to be able to inhibit translation, they must reach the interior of the cell unchanged. Useful properties of oligonucleotides for use in antisense inhibition include: (i) stability of oligonucleotides against extra- and intracellular enzymes; (ii) the ability to penetrate the cell membrane; and (iii) the ability to hybridize the target DNA or RNA (Agarwal, KL et al., Nucleic Acids Res., 1979, 6, 3009; Agawal, S. et al., Away from Nati Acad Sci. USA. 1988, 85, 7079) . Thus, it is of interest to provide polynucleotide analogues having better properties when used as antisense or when used as primers (initial oligonucleotides) or hybridization probes.

The modified polynucleotides have already been synthesized in the past and these polynucleotide modifications include methylphosphonates, phosphorothioates, various amidates and sugar particles of nucleic acid species. These substitutions of the spine give increased stability to some degree, but they have the downside of having chiral phosphorus in the bond, which in turn leads to the formation of 2 ⁿ diastereomers, where n is the number of modified diester bonds in the oligomer. The presence of these multiple diastereomers significantly impairs the ability of the modified oligonucleotides to hybridize to the target sequences.

-3Some of these substitutions also retain the ability to maintain a negative charge and the presence of charged groups impairs the ability of the compounds to penetrate cell membranes. There are many other disadvantages associated with these modified bonds, depending on the particular nature of the bond.

Some oligonucleotide analogues having sugar modifications have been synthesized. Previously used sugar modifications (deoxy) of nucleic acid ribose include α-DNA, homo DNA, morpholino and thio nucleosides and peptide nucleic acids (PNA) to provide what is understood as improved structures, especially those structures that have improved cellular uptake . A general scheme for the synthesis of such analogs is to include a primary hydroxyl group of a nucleoside or its nucleotide, either attached to a polymeric carrier or to a sequentially specific 3'-nucleotide with a phosphorus atom, or in a five-valent or trivalent oxidation state . Specific coupling procedures were referred to as phosphite triester (phosphoramidite), phosphorus diester and hydrogen phosphonate processes. For such methods having protected bases (G, A, C, T, U and other heterocycles) together with protected phosphorus atoms, commercially available monomers and polymer-bonded monomers are suitable to allow storage and prevent non-specific reactions during the coupling process .

Nucleic acids containing modified sugars, non-ionic backbones or acyclic polyamides (PNA) have to one degree one or more of the following properties useful for gene modulation: they increase duplex stability (hybridization efficiency), have increased target specificity, nuclease stability, enhanced cellular uptake and involvement in important nucleic acid terminator events (e.g., RNase activity

-4H, catalytic cleavage, stopping hybridization, and others). The use of carbonate diesters has also been suggested. However, these compounds are highly unstable and the carbonate diester bond does not maintain the tetrahedral configuration shown by phosphorus in the phosphodiester. Similarly, carbamate bonds, which are otherwise achiral, give trigonal symmetry and it has been shown that poly dT having these bonds does not strongly hybridize with poly dA (Coull, JM et al., Tetrahedron Letts., 1987, 28, 745 Stirchak, EP et al., J. Org. Chem., 1987, 52, 4202).

Recently, reports of acyclic sugar analogues have appeared in the literature (Augustyns, K. A. et al., Nucleic Acids Res. 1991, IS, 25872593). The incorporation of these acyclic nucleosides into oligonucleotides caused a decrease in Tm, depending on the number of linker-embedded linkers in the oligomers. These oligonucleotides have been found to be enzymatically stable and mate base with a complementary sequence. Given the disadvantages of polynucleotides and known polynucleotide analogues, it is of interest to provide novel polynucleotide analogues for use in antisense inhibition and other techniques using oligomers.

Such efforts in modifying both the sugars and the components of the spine have some disadvantages for use as therapists and in other methods. Therefore, further improvements in these properties are required before effective therapists, diagnostics and research tools become available. Accordingly, there has long been a need for improved oligomer analogues of oligonucleotides, such as pharmaceutical compounds.

The present invention provides novel oligonucleotides and their structural precursors, which have improved resistance to nuclease digestion and which, under physiological conditions, have increased stability and which may be neutral

-5 or positively charged to increase cell permeation. Furthermore, the novel oligonucleotides of the present invention improve the hybridization properties with respect to nucleic acid hybridization targets.

The oligomers of the present invention are generally referred to as oligomers comprising a series of restricted linkers or monomers that are suitable for binding heterocyclic bases to the target nucleic acid in a sequence-specific manner. The limited linkers described herein may, when incorporated into oligomers, have a power greater than a single hydrogen bond, thereby favoring the formation of a bond-competent conformation.

Nucleomonomers of the present invention are generally referred to as particles or residues that replace the furanose ring found in naturally occurring nucleotides with an amino acid or a modified amino alcohol residue. Illustrative monomers and oligomers of the invention are shown in formulas 1 to 41. The incorporation of these monomers described herein into oligonucleotides allows for the synthesis of compounds with improved properties and these improved properties include (i) increased lipophilicity resulting from the removal of charge associated with phosphodiester bonds (Dalge , JM et al., Nucleic Acids Res., 1991, 19, 1805) and (ii) resistance to degradation by enzymes such as nucleases. Oligomers containing such monomers can be completely stable for hybridization to target sequences and better than unmodified nucleoside residues for one or more uses.

Summary of Invention:

The present invention provides various novel oligonucleotide analogues having one or more properties that make the subject compounds better than conventional oligonucleotides for use in processes in which

-6oligonucleotides used. The compounds of the invention are oligonucleotide analogues in which the furanose ring of a naturally occurring nucleic acid is replaced by an amino acid or a modified amino alcohol residue. Some embodiments of the novel compounds of the invention are particularly useful for antisense control of gene expression. The compounds of the invention may also be used as nucleic acid hybridization assays or as primers.

Another aspect of the invention is to provide monomeric precursors to the oligonucleotide analogues of the invention. These monomeric precursors can be used to synthesize the subject poinucleotide analogues.

Another aspect of the invention is to provide formulations of the subject polynucleotide analogues intended to treat or prevent disease states. Another aspect of the invention is to provide methods for treating or preventing diseases, especially viral infections and cell growth disorders. The subject methods of treating the disease include the step of administering an effective amount of the subject poinucleotide analogues for use as antisense inhibitors.

Short ools of pictures:

1 to 25 are illustrations of chemical reaction sequences useful for synthesizing the monomers and oligonucleotides of the present invention. More specifically,

Figure 1 shows the synthesis of the L-serinol-linked thymine monomer phosphoramidite with the -CH ₂ -CO- bond between thymine and serinol.

-7Figure 2 shows the synthesis of the L-serinol-linked thymine monomemic phosphoramidite with the -CH ₂ -CH ₂ - bond between thymine and serinol.

Figures 3 and 4 show the synthesis of the L-serinol-substituted thymine monomer phosphoramidites with the -CH ₂ -CO- bond between thymine and serinol.

Figure 5 shows the synthesis of a five-atom TT dimer long internucleotide bond having a hydroxylamine in the middle of the internucleotide bond, with a -CH ₂ -CO- bond between thymine and serinol.

Figure 6 shows the synthesis of thymine monomemic phosphoramidite in which thymine is linked to N-ethylhydroxylamine by the -CH ₂ -CO- bond.

Figure 7 shows the synthesis of the L-serinol-linked thymine monomemic phosphoramidite in which the NH ₂ group of the L-serine is linked to the 2 hydroxyacetyl group and the hydroxyl group is blocked by the DMT group. This building block is used for the 2'-5 'connection. This figure also shows the synthesis of a thymine monomer in which the NH ₂ group of the L-serine is linked to the 2'-hydroxyethyl group.

Figure 8 shows the synthesis of a T-T dimer having a hydroxamate backbone with a 2′-5 ′ bond. In this dimer, one building block is made of L-aspartic acid and thymine and the other block is made of L-serine and thymine. This dimer has two additional amide bonds in the spine.

Figure 9 shows the synthesis of a T-T dimer having a hydroxamate backbone with a 2′-5 ′ bond. In this dimer, one building block is made of L-aspartic acid and

-8thymine and the second block is made of L-serine and thymine. This dimer in the spine between the monomers lacks an amide bond.

Figure 10 shows the synthesis of L-serinol-β-alanine-linked thymine monomemic phosphoramidite in which β-alanine binds thymine and serinol.

Figure 11 shows the synthesis of L-serinol-alkylamine-linked thymine monomemic phosphoramidite with alkylamine linking thymine and serinol.

Figure 12 shows the synthesis of a T-T dimer having a hydroxamate backbone with a 4′-5 ′ bond. The dimer here is made of two L-aspartic acid units and two thymine units with an acetyl linker between thymine and aspartic acid.

Figure 13 shows the synthesis of a T-T dimer having a hydroxamate backbone with a 4′-5 ′ bond. The dimer here is made of two L-aspartic acid units and two thymine units with an ethyl linker between thymine and aspartic acid.

Figure 14 shows the synthesis of the N-hydroxyamino acid-linked thymine building block.

Figure 15 shows the synthesis of an L-aspartic acid-linked thymine building block with an N-hydroxylamine linker between thymine and aspartic acid.

Figure 16 shows the synthesis of a T-T dimer having a hydroxamate backbone with a 4′-5 ′ bond. The carboxylic acid group is then linked to the thymine building block by an N-hydroxylamine linker.

-9Figure 17 shows the synthesis with thymidinacetic acid of a substituted N-hydroxyamino acid building block 150 and its analogue 149. These monomeric building blocks are useful for nucleic acid formation with a hydroxamate backbone.

Figure 18 shows the synthesis of amino acid building blocks 157 and 158 containing thymidinacetic acid substituted hydroxylamine. These monomers are useful for forming a nucleic acid having an amide backbone with a hydroxylamine functional group.

Figure 19 shows the synthesis of the L-serinol-linked thymidine building block 166, which has a hydroxylamine moiety between thymine and serinol. This building block is useful for preparing nucleic acid with 4′-5 ′ bonds.

Figure 20 shows the synthesis of glutamic acid-glycine-linked thymidine monomer 174. This monomeric building block is useful for producing nucleic acid with an amide backbone and 2′-5 ′ bonds.

Figure 21 shows the synthesis of glycine-glycine-linked thymidine building blocks 181 and 182, which have a hydroxylamine moiety between thymine and glycinol. These two building blocks are useful for preparing nucleic acid with 2′-5 ′ bonds.

Figures 22 to 24 show syntheses of ribose-amino acid-linked thymidine building blocks 191, 199 and 207. These building blocks are useful for the preparation of oligonucleotides having a ribose-amide backbone.

Figure 25 shows the synthesis of oligonucleotide 211 on a solid phase having a ribozamide backbone.

-10Figure 26 shows the synthesis of 1 -O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] - L-propane3-O- (N, N-diisopropyl) -p-cyanoethylphosphoramidite.

Figure 27 shows the synthesis of 1-0- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] - D-propan3-O- (N, N-diisopropyl) -p-cyanoethylphosphoramidite.

Figure 28 shows the synthesis of 2 - [(p- (4,4'-dimethoxytrityl) -O-acetyl) amino] -3-thymynyl-Lpropan-1-O- (N, N-diisopropyl) -p-cyanoethylphosphoramidite.

Figure 29 shows the synthesis of N- (thyminylacetyl) -N - [[(2-isobutyryl) oxy] ethyl] -obenzylhydroxylamine.

Figure 30 shows the synthesis of (2R, 4S) -1- (tert-butyloxycarbonyl) -2- [N ₃ -benzoyl (thymin-1yl)] methyl-4-phthalimido-pyrrolidine.

Detailed description of specific designs:

A. Definitions and abbreviations:

In describing the present invention, the following terms will be used, which will be defined as shown below.

As used herein, "antisense" therapy refers to the administration or in situ production of DNA or RNA oligomers or their analogs that bind specifically to a complementary target nucleic acid sequence. The binding may be complementary to conventional base mating, or the binding may be by other mechanisms, e.g. in the case of DNA duplexing, with specific interactions in the major double-strand

-11helix. Mainly, “antisense refers to a range of techniques commonly used in this work under this description and includes any therapy that relies on specific binding to oligonucleotide sequences. Techniques for antisense gene regulation are well known to a person with ordinary experience in molecular biology. Descriptions of antisense gene regulation can be found, e.g., in U.S. Pat. U.S. Patent 5,107,065, U.S. Pat. U.S. Patent 5,166,195 5,087,617 and Crooke, Annual Revievv Pharmacology Toxicology 1992, 32, 329-376.

The terms "oligomer" or "oligonucleotide" are used interchangeably to include naturally occurring compounds such as DNA and RNA, as well as their synthetic analogs, including the compounds of the invention. Unless otherwise indicated, the terms "oligomer" and "oligonucleotide" refer to both DNA / RNA and their synthetic analogues. The term "oligomer" refers to compounds comprising two or more nucleomonomers which are linked covalently to each other by a phosphodiester bond or by any other alternative bonds. Unless otherwise stated, the term "oligomer" should not be understood to mean a length limit. Thus, a given oligomer may have as little as two covalently linked nucleomonomers (dimers) or may be significantly longer. The oligomers can be binding competent and thus can be base-mated to single-stranded or double-stranded nucleic acid sequences. Oligomers (eg, dimer-hexamers) are also useful as synthons for longer oligomers, as described herein. Oligomers may contain basic sites and pseudonucleosides.

Oligomers include oligonucleotides, oligonucleosides, polydeoxyribonucleotides (containing 2′-deoxy-D-ribose or modified forms thereof), e.g. DNA, polyribonucleotides (containing D-ribose or modified forms thereof), e.g. RNA, and any other type of polynucleotide that is N-glycoside or

-12C-glycoside of purine or pyrimidine base or modified purine or pyrimidine base. An oligomer as used herein is also contemplated to include compounds in which adjacent nucleomonomers are linked via hydroxamate bonds. Elements commonly found in oligomers, such as the furanose ring and / or phosphodiester bond, may be replaced by any corresponding functionally equivalent element. The term "oligomer" is intended to include any structure that serves as a framework or support for bases in which the framework permits binding to the target nucleic acids in a sequentially dependent manner. Oligomers that are currently known may be classified into four groups, which may be designated as having (i) phosphodiester bonds or phosphodiester analogs (phosphorothioate, methyl phosphonate, etc.), (ii) substituent bonds which having non-phosphorus isosters (riboacetal, formacetal, carbamate, etc.), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose or hexose at the site of ribose or deoxyribose; linked through each appropriate alternate bond.

The term "nucleomonomer" as used herein refers to a particle comprising (1) a base covalently linked to (2) another particle. Nucleomonomers include nucleosides, nucleotides or bases linked to amino alcohol. Nucleomonomers may be coupled to form oligomers that bind to the target or complementary nucleic acid base sequences in a sequence-specific manner.

The term "other particle" as used herein refers to a compound linked to a nucleomonomer and includes an amino acid / amino alcohol particle, typically serinol, aspartic acid, glutamic acid, glycine, and those species that comprise modifications of an amino acid particle, e.g. in which

-13 is one or more hydrogen substituted by another functional group (see formulas 24-41) or one carboxylic acid is converted to alcohol, amines, thiols, hydroxylamines and the like. Nucleomonomers as defined herein are also intended to include a base linked to an amino acid or an amino alcohol and / or amino acid / alcohol analog having a free carboxylic / hydroxyl group and / or a free amino group and / or protected forms thereof.

The term "nucleoside" as used herein refers to an amino acid and its amino alcohol derivative, as further described below, which carries purine, pyrimidine, or analogues thereof, as defined below, but does not have a linking moiety such as a phosphodiester analog or modified internucleoside bond. By "5" 'nucleoside is meant a nucleoside that provides a 5' carbon point to the linker. The '5' 'end of the linker binds to the 5' nucleoside. The '3' 'end of the linker binds to the 3' position at the next nucleoside. If a modified nucleoside that does not accurately include 3 'and / or 5' carbon is present, the person skilled in the art understands that this' 3 'and' 5 '' terminology is used to describe the polarity of the chain by analogy with DNA and RNA .

The term "nucleoside" as used herein refers to a base covalently linked to an amino alcohol / amino acid analogue and containing a linker between the base and the amino acid / amino alcohol. The term nucleoside typically includes ribonucleosides, deoxyribonucleosides, or any other nucleoside that is an N-glycoside or a C-glycoside of the base.

The term "nucleotide" as used herein refers to a nucleoside having a phosphate group or a phosphate analog (groups with phosphorus in the same oxidation state as with a phosphate group, eg thiophosphate, amidate).

-14The term "base" as used herein refers to many different nucleoside bases, including purine and pyrimidine heterocycles and heterocyclic analogues and their tautomers. Purines include adenine, guanine, and xanthine, and illustrative purine analogues include 8-oxo-N ₆ methyladenine and 7-deazaxanthin. Pyrimidines include uracil and cytosine and their analogues such as 5-methylcytosine, 5- (1-propynyluracil), 5- (1-propynylcytosine), 5-methyluracil and 4,4-ethanocytosine. "Bases when connected to an appropriate molecular framework, e.g. phosphodiester backbones are capable of entering into the mating ratio of bases occurring in double stranded DNA or other double stranded nucleic acids of similar structure. The bases may also be capable of entering into the mating ratio of the bases in the triple helix.

The term "sugar modification" as used herein refers to any amino acid or amino alcohol moiety other than 2′-deoxyribose.

The term "amino acids / alcohols" as used herein refers to all natural amino acids and alcohols with both the "R" and "S" isomers.

The term "nucleoside bonds" as used herein refers to a bond existing within a monomer.

The term "bond" as used herein refers to a particle used to bind a base to an amino acid / amino alcohol and derivatives thereof.

The term "internucleotide bonds" as used herein refers to a phosphodiester particle (-O-P (O) (O) -O-) or any other functionally equivalent particle that covalently bonds adjacent nucleomonomers.

-15The term "substituent bonds" as used herein refers to any analog of a natural group or to any suitable particle that covariates adjacent nucleomonomers. Alternate bonds include phosphodiester analogs, e.g. such as phosphorothioate and methylphosphonate and phosphorus-free bonds, e.g. such as amides, hydroxamates, hydroxylamine. Alternate bonds include phosphorus-free inventive bonds (2 ', 5' bonds, 3 ', 5' bonds and 4 ', 5' bonds).

The term "crosslinker" as used herein refers to a group or particle in an oligomer that forms a covariate bond with the target nucleic acid. Cross-linking particles include species capable of covalent binding by covalently binding the oligomer to the target nucleic acids either spontaneously (e.g., N ⁴ , N ⁴ -ethancytosine) or via photoactivation (e.g., psoralen) and the like.

The term “blocking groups as used herein refers to a non-hydrogen substituent covalently linked to oligomers or nucleomonomers, or as a protecting group, synthesis linking group, OPO ₃ . ₂ , or other conventional conjugate, such as a solid support, labeling, antibody, monoclonal antibody, or fragments thereof, and the like. As used herein, it is not intended to be interpreted as a "blocking group" as a protecting group by the terminology of the dialect, but is also intended to include, e.g. linking groups such as H-phosphonate or phosphoramidite.

The term "protecting group" as used herein refers to any group capable of protecting the O-atom, S-atom or N-atom to which it is linked from participating in the reaction or in bonding. Such protecting groups for the N-atoms on the base particle in the nucleomonomer and their introduction are

-16 conventionally known in this work. Examples of suitable protecting groups which do not limit these include: diisobutylformamidine, benzoyl, silyl and the like. Suitable protecting groups for O-atoms and Satomes are e.g. DMT, MMT, FMOC or esters. "Protective groups" as used herein include any group capable of deterring the Oatom, S atom or N atom to which it is attached, from participating in the reaction or in its binding. Such protecting groups for the 0-, S- and N-atoms in the nucleomers are described and methods for their introduction are generally known in the art. Protective groups also include any group capable of preventing reactions and binding to carboxylic acids, thiols and the like.

The term " linking group " as used herein refers to any group that is suitable for bonding or substitution bonds between nucleomonomers such as hydrogen phosphonate and phosphoramidite.

The term "conjugate" or "conjugate particle" as used herein refers to any group attached to an oligomer at the terminal end aii within the oligomer itself. The conjugates include solid substrates such as e.g. silica gel, controlled porous glass and polystyrene; indications such as fluorescent, chemiluminescent, radioactive atoms or molecules, enzymatic particles and reporter groups; oligomem transport agents such as polycations, serum proteins and glycoproteins and polymers and the like. Other conjugate particles include O-cholesterol, polyethylene glycol (PEG), amino acids, intercalators, polynucleotide clarifying particles, cross-functional groups, lipids, hydroxamates, alkylating agents and the like.

The term "synthon" as used herein refers to a structural or structural unit within an oligonucleotide analogue of the invention.

-17The term "transfection" as used herein refers to any method that is appropriate for the increased uptake of oligomers into cells.

The term "subject" as used herein refers to a plant or animal, including a mammal, especially a human.

The term "derivatives" and monomemic constituents of their oligomers include those generally known in the art. For example, oligonucleotides may be covalently linked to different particles, such as e.g. intercalators, substances that correlate in particular with the smaller of the two channels on the DNA double helix and other arbitrarily selected conjugates such as labels (radioactive, fluorescent, enzymatic, etc.). These additional particles can be (but need not) be derivatized using a modified spine bond as part of the bond itself. For example, intercalators such as acridine may be coupled via the R-CH ₂ group associated with any available -OH or SH, e.g., to the terminal 5 'position of RNA or DNA, the 2' position of RNA, or OH or SH, wherein, in the 5 position of the pyrimidines, for example, instead of 5 methyl cytosine, a derivatized form containing -CH ₂ CH ₂ CH ₂ OH or -CH ₂ CH ₂ CH ₂ SH in the 5 position was designed. Many different substituents can be linked, including those related to conventional bonds. Accordingly, the displayed OH particles in the oligomer of formula (1) may be replaced by phosphate groups protected by standard protecting groups or activated to prepare additional bonds for other nucleotides or may be coupled to a conjugated substituent. The 5 ′ terminal OH is conventionally phosphorylated; The 2'-OH or OH substituents at the 3 'end can also be substituted. Hydroxyls can also be derivatized into standard protecting groups.

-18The term "phosphodiester analogue" as used herein refers to an analogue with a conventional phosphodiester bond as well as alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which OP (O) is replaced by P (O) S, P (O) NR ₂ , P (O) R, P (O) OR ', where RH or alkyl (1-7C) and R 'is alkyl (1-7C). Not all phosphodiester analogs in the same oligomer are required to be identical, the only requirement is that at least one of these bonds is a modified internucleotide bond as described herein.

The "analog" forms of purines and pyrimidines are those commonly known in the art, many of which are used as chemotherapeutic agents. An illustrative but incomplete list includes: 4-acetylcytosine, 8hydroxy-N ⁶ -methyladenine, aziridinylcytosine, pseudoisocytosine, 5 (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracyl, 5carboxymethylaminomethyl-2-carboxymethylaminomethyl-2-carboxymethylacetyl-2-carboxamide N ₆ -isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3methylcytosine, 5-methylcytosine, N _6- methyladenine, 7-methyladenine, 7-methyladenine 7-methylguanine, 5methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylguanosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N ₆ -isopentenyladenine, uracyl-5-oxiocycetyl acid, oxybutoxosin, pseudouracil, geosine, 2-thiouracil, 4-thiouracil, 5-methyluracil, Nuracyl-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, 2-thiocytosine and 2,6-diocytopenin. A particularly preferred analogue is 5-methylcytosine (abbreviated herein as "Cme").

The term "isosteric" as used herein refers to the spatial properties and properties resulting from the orientation of the internucleoside bond and the fact that these properties are so similar to those of the natural phosphodiester

-19 binds that a modified oligonucleotide containing an isosteric bond will be replaced, replaced, imitated and / or hybridized with a natural oligonucleotide.

The term "ribose-amide" as used herein refers to an internucleotide bond that exists between two nucleobases. The currant-amide internucleotide bond has a combination of ribose / (2′-deoxy) and amino acid functional groups.

Various abbreviations referring to functional groups and compounds are used in this application. These abbreviations are easily understood by a person who is experienced in working in the field of organic chemistry, e.g. “Ph” refers to phenyl, “Me” refers to methyl, “(1-7C)” indicates that a given chain contains from 1 to 7 carbons everywhere, etc.

OdIs Izuma:

The present invention provides novel oligonucleotide analogues containing modified amino acid / amino alcohol bonds between bases and spines (phosphodiester, phosphorothioates and others, as shown in Table 1) also referred to as modified nucleotide bonds. These modifications or their functional equivalent replace the sugar particle lying between the spine and the bases with amino acid derivatives, e.g. as shown in Formula 24. The present invention also provides novel nucleomonomers and methods for incorporating them into oligomers containing nucleomonomers.

The invention provides various nucleomomeric compounds having structures of formulas 1-23.

Base

-21Base

Base

Base

Base

Base

Base

Base

Base

And ^with s, ^A

Base

Base

-22Oligomers of the invention are polymers that comprise one or more of the subject monomemic compounds linked to provide a structural analog of DNA or RNA. The oligomers of the invention comprise two or more nucleomonomers and can actually comprise any number of nucleomonomers, although oligomers with 200 or less nucleomonomers are generally easier to synthesize. Compounds of formulas 1-23 may be linked to each other by 4′-5 ′ bonds, 3′-5 ′ bonds and 2′-5 ′ bonds, as can be seen in formulas 24-41.

The nucleotide bonds in the compounds of the invention are made from the amino acids serine and glycine or derivatives thereof. The oligonucleotides of the invention are stable in vivo. resistant to endogenous nucleases and capable of hybridizing to target nucleotide sequences. The illustrative compounds of the present invention are shown in formulas 24 to 41 and are conformationally more limited relative to the phosphodiester bonds found in unmodified DNA and RNA. This conformational restriction may in part contribute to the increased binding properties of the subject compounds to complementary polynucleotide target sequences; however, the use of the invention for increased binding properties is not dependent on this theory.

In another embodiment, the present invention is directed to modified oligonucleotides or derivatives thereof wherein the furanose particle is a natural oligonucleotide, e.g. DNA or RNA replaced with amino acid / amino

-23 alcohol particles and other modifications comprising substitution at amino acid positions are shown in formulas 25 to 41. The internucleotide bond between adjacent nucleomonomers is a bond between the 4 'and 5' position of adjacent nucleomonomers. In other words, the phosphodiester internucleotide bond or its functional equivalent originates from the 5′-position of one nucleomonomer and associates the 4′-position of the adjacent monomer, as illustrated by the compounds of formulas 24-33:

25A

Base

Base

Z

Base

Base

Base

32Α

32Β

In which each "R" is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CHJk-F; where "x" is 1-7 carbon and is “F” NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each “base” is independently a nucleoside base.

In which each Rf is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each “R ₂ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each “R ₃ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "X" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

-27In which each "R ₄ " is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x 1-7 is carbon and" F "is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S ( O) (O) NH ₂ , ch ₃ , Ph.

In which each “A” is independently (ΟΗ ^ χ, CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where “x 1- 7 carbon.

In which each “B” is independently (CH2) _X , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where “x” 1 -7 carbon.

In which each “X is independently (CH ₂ ) _X , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where“ x ”1 -7 carbon.

In which each "Z" is independently (CH ^, CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" is 1-7 carbon .

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (17C), Me, heteroalkyl (1-7C), aryl (6-7C), - (CH ₂ ) _X F; where “x” is 1-7C and “F” is independently H, OH, SH, OCH ₃ , CN, SCH ₃ , ONH ₂ , ONH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O ) (O) NH ₂ , CH ₃ , Ph.

In which each "V" is independently a phosphodiester analogue, phosphorothioates, methylphosphonates, phosphorodithioates, borphosphonates, selenophosphonates, phosphoramidates, acetamidate, oxyformamido, oxyacetamido, diisopropylsilyl, carbamate, dimethylethenyl sulfoxidate or dimethylene sulfide internucleoside bond selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of the oligomer may vary from dimer to 200-mer

-28or more. Preferred modified internucleotide bonds that include structures for “V” are shown in Table 1.

Additionally, compounds of these formulas may be conjugated to one or more conjugate particles. Suitable conjugate particles include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleavage particles (eg imidazoins), crosslinking particles (eg psoralen), lipids, peptides, alkylating agents, hydroxamates and fluorescent designations. The conjugate particle can independently replace one or more of R, R-ι, R ₂ , R3, R ₄ and R ₅ .

In another embodiment, the present invention provides oligomeric structures as exemplified in formulas 34-36 and derivatives thereof:

Formula 34

In compounds of formulas 34-36, the bonds between adjacent nucleomers are 3 'to 5' bonds.

In which each “R” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each “base” is independently a nucleoside base.

In which each "R-," is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ^ -F; where "x" 1-7 carbon and is "F" NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O ) NH ₂ , CH ₃ , Ph.

In which each “R ₂ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH _3j Ph.

-30V in which each "R ₃ " is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH _3i SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S ( O) (O) NH ₂ , CH _3j Ph.

In which each “R ₄ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where “x” is 1-7 carbon and is “F NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S ( O) (O) NH ₂ , CH _3i Ph.

In which each “A” is independently (CH ₂ ) _X , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se. Where "x" is 1-7.

In which each “B” is independently (CH ₂ ) _X , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH _3> NR ₅ and Se. Where "x" is 1-7.

In which each “X” is independently (CH ₂ ) _X , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR5. Where "x" is 1-7.

In which each Ύ is independently (CH ^, CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR _5. Where "x" is 1-7.

In which each "Z" is independently (CH ₂ ) _X , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ . Where 'x 1-7.

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (17C), Me, heteroalkyl (1-7C), aryl (6-7C), - (CH ₂ ) _X F; where “x” is 1-7C and is “F”

-31 independently of H, OH, SH, OCH ₃ , CN, SCH ₃ , ONH ₂ , ONH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each "V" is independently a phosphodiester analogue, phosphorothioates, methylphosphonates, phosphorodithioates, borphosphonates, selenophosphonates, phosphoramidates and / or two to four atoms long internucleoside bond is selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of the oligomers can vary from dimer to 200mm and above. Preferred modified internucleotide bonds that include structures for “V” are shown in Table I.

In another embodiment of the invention, the present invention provides oligomers having formulas 37 to 41 or variants thereof, oligomers comprising new internucleotide bonds that are 2 ', 5' bonds. These oligonucleotides are stable in vivo. have improved resistance to endogenous nucleases and are capable of hybridizing to target oligonucleotide sequences.

37A

37B

z

Χ-Ν

Base

R, dr \ z Χ-Ν

A

- Base

R, - <

Χ-Ν

2'Baza v

R, - r-Base

Χ-Ν

39A

39B

In z-Base 'Χ-Ν \ Z' Base ^X x - N '

40B

y z-Base 'Χ-Ν \

V and Z '

Base

In which each “R” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x 1-7 is carbon and" F "is NH ₂ , SH, OH, COOH, OCH ₃ , SCH _3i SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O ) (O) NH ₂ , CH _3i Ph.

In which each “base” is independently a nucleoside base.

In which each "R-ι" is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ^ -F; where "x" 1-7 carbon and is "F" NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O ) NH ₂ , CH ₃ , Ph.

In which each “R ₂ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each “R ₃ ” is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x" is 1-7 carbon and "F" is NH ₂ , SH, OH, COOH, OCH ₃ , SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH _3i Ph.

-34V each of which R4 is independently H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _X -F; where "x 1-7 is carbon and" F "is NH ₂ , SH, OH, COOH, OCH ₃₎ SCH ₃ , SPh, NOH, NOH (CH ₃ ), SNH _2l S (O) NH ₂ , S (O ) (O) NH ₂ , CH ₃ , Ph.

In which each “A” is independently (ΟΗ ^ χ, CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se; where “x” 1 -7 carbon.

Wherein each "B" is independently (CH ^, CO, CS, S, S (O), S (O) (O), NH, NOH, NCH _3, NR ₅ and Se, where "x" is 1- 7 carbon.

In which each “X” is independently (CH ₂ ) _X , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ ; where "x" is 1-7 carbon.

In which each “Z” is independently (ΟΗ ^ χ, CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se; where “x 1- 7 carbon.

R ₅ is H, OH. OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (17C), Me, heteroalkyl (1-7C), aryl (6-7C), - (CH ₂ ) _X F; where "x is 1-7C and" F "is independently H, OH, SH, OCH ₃ , CN, SCH ₃ , ONH ₂ , ONH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

In which each &quot; V &quot; is independently a phosphodiester analogue, phosphorothioates, methylphosphonates, phosphorodithioates, borphosphonates, selenophosphonates, phosphoramidates and / or two to four atoms long intemucoside bond is selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of the oligomer may vary from dimer to 200 mm or more. Preferred modified internucleotide bonds that include structures for “V” are shown in Table I.

-35 In other embodiments of the invention, the present invention is directed to an oligomer of the following formulas (Formula 42) and its monomer component (Formulas 85-90).

In which

X is selected from the group consisting of (CH ₂ ) _n , where n = 1-3, CO (CH2) _n , where n = 0-2, and (CH ₂ ) _n SO ₂ , where n = 1-2,

Y is selected from the group consisting of CH ₂ , CO, COOH, CS and SO ₂ ,

Y 'is selected from the group consisting of CH ₂ , CO, COOH, CS and SO ₂ ,

Z is selected from the group consisting of O, S, NH and CH ₂ ,

R is selected from the group consisting of CH ₂ OH, CH ₂ NH ₂ , CH ₂ NHCHO, CONH ₂ and COOH,

B is a nucleoside base.

In which

X is selected from the group consisting of (CH ^ n, where n = 1-3, COfCH ^ n, where n = 0-2, and (CH2) _n SO ₂ , where n = 1-2,

V is selected from the group consisting of CH ₂ , CO, COOH, CS and SO ₂ ,

Y 'is selected from the group consisting of CH ₂ , CO, COOH, CS and SO ₂ ,

Z is selected from the group consisting of O, S, NH and CH ₂ ,

R is selected from the group consisting of CH ₂ OH, CH ₂ NH ₂ , CH ₂ NHCHO, CONH ₂ and COOH,

B is a nucleoside base.

In other embodiments, the invention provides methods for treating diseases mediated by the presence of a nucleotide sequence comprising administering an effective amount of the above modified oligonucleotides capable of specifically binding the nucleotide sequence to its deactivation to a subject in need of such treatment.

-37V The oligonucleotides of the invention are at least one of the phosphodiester groups included within the "Vs" of formulas 24-41, substituted by the modified internucleoside bonds described herein. It is desirable that multiple phosphodiester bonds in an unmodified oligonucleotide substituted with a modified internucleoside bond may be reused in this structure or, if desired, different types of modified internucleotide bonds may be used in a single oligonucleotide. In a preferred embodiment of the oligonucleotides in question, these alternative bonds are non-chiral, increasing the ability of the oligonucleotide to hybridize to the desired target; however, useful compounds of the invention include those embodiments in which chiral forms are used.

-38 Preferred modified internucleotide bonds that include structures for “V” are shown in Table 1.

Table 1

-o-s-S (0) -S (0) (0) -Se-si-C (0) —C (S) -

-NH-NOH-NCHj-NR ₅ -CH ₂ -O-CHj-CH ₂ -O-O-CHj-CKj-CH ₂ -CH ₃ -O-ch ₂ -o-ch ₂ -o-ch ₂ -oS -CHj-CH ₂ -SS-CH ₂ -CH ₂ -CH ₂ -CH ₂ -S-CHj-S-CH, -s-ch ₂ -so-ch ₂ -s ~

-S-CH3-OS (O) -CH _; -CH ₂ -S (O) -S (O) -CH ₂ -CH ₂ -CH ₂ -CH ₂ -S (O) -CHj-S (O) -CHj-S (O) -CH ₂ -S ( O) -O-CHj-S (O) -S (O) -CH -, - 0-S {0) (O) -CHj-CH ₂ -S (O) (0) -S (O) (O) ) -CH ₂ -CH ₂ -CH ₂ -CH ₂ -S (O) (O) -CH ₂ -S (O) (O) -CHj-S (0) (O) -CH ₂ -S (O) (0) -O-CHj-S (O) (O) - -S (0) (O) -CHj-OssS (O) -S (O) -39-S (O) (O) -S (O) ) - (O) -Se-CH ₂ -CHj-Se-Se-CHj-CHj-CHj-CHj-Se-CHj-Se-CH ₂ -Se-CH ₂ -Se-O-CHj-Se-Se-CHj -O-Se (O) -CH ₂ -CH ₂ -Se (O) -Se (O) -CHj-CHj-CH ₂ -CH ₂ -Se (O) -CH ₂ -Se (O) -CH ₂ - Se (O) -CH ₂ -Se (O) -O-CH ₂ -Se (O) -Se (O) -CH ₂ -O-Se (O) (O) -CH, -CH ₂ -Se (O ) (O) -Se (O) (O) -CH, -CHj-CH ₂ -CH ₂ -Se (O) (O) -CH ₂ -Se (O) (O) -CHj-Se (O) ( O) -CH ₂ -Se (O) (0) -Se-Se-Se (0) -Se (O, -Se (0) (O) -Se (O) - (0) -O-CH ₂ - Se (O) [0) -Se (0) (0) -CH ₂ -0-5-CH ₂ -Se-Se-CH ₂ -SS (0) -CH ₂ -Se (O) -Se (O) -CH ₂ -S (O) -

-S (O) (O) -CHj-Se (O) (O) - -Se (O) (O) -CH ₂ -S (O) (0) -SSS (O) -S (O) -S {0, (O) -S (O) (0, -Se-Se-Se (O) -Se (O) -Se (O) (O) -Se (O) (0) -N (R _s ) -CH ₂ -CH ₂ -N (R _s ) -N (R _s ) -CH ₂ -CH ₂ -CH ₁ -CH ₂ -N (R ₅ ) -CH ₂ -N (R _s ) -CH ₂ -N ( R _S ) -OON (R ₅ ) -N (Rj) -O-CH ₂ -CH ₂ -ON (Rj) -CH ₂ -N (R _s ) -O-0-N (Rj) -CHj-40- O-CH ₂ -N (R _S ) -N (R _s ) -CH ₂ -ON {Rj) -SSN (R ₅ ) -N (R ₅ ) -S-CH ₂ -CH ₂ -SN (R ₅ ) -CH ₂ -N (R _s ) -SSN (Rj) -CH ₂ -S-CH ₂ -N (R _s ) -N (Rj) -CH ₂ -SN (R _s ) -S (O) -S ( O) -N (R _s ) -N (R ₅ ) -S (O) -CHj-CH ₂ -S (O) -N (Rs) -CH ₂ -N (R _s ) -S (O) -S (O) -N (R ₅ ) -CH ₂ -S (O) -CH ₂ -N (R _s ) -N (Rj) -CH ₂ -S (O) -N (R 1) -S (O) ( O) -S (O) (O) -N (Rj) -N (Rj) -s (O) (O) -CH, -CHj-S (O) (O) -N (R _s ) -CH ₂ -N (R ₅ ) -S (O) (O) -S {0) (Or-N (R _S ) -CH ₂ -S (O) (O) -CH ₂ -N (R _s ) -N ( Rj) -CH ₂ -S (O) (O) -0-N (R ₅ ) -SSN (Rj) -O ~ -0-N (R _s ) -S (O) -S (O) -N ( R ₅ ) -OON (R ₅ ) -S (O) (O) “S (O) (0) -N (R ₅ ) -0-osoOS (O) -OOS (O) (O) -ON (R) _s ) -SN (R _s ) -N (R _s ) -S (O) -N (R _s , -N (R _s ) -S (O) (O) -N (RJ-CHj-SO-CH ₂ -S (O) -O-CHZ-S (O) (0) -0-CH ₂ -C (O) -O-CH ₂ -C (S) -0-CH ₂ -N (R ₅₎ -C (O) -O-CH ₂ -N ( Rj) -C (S) -ON (R _s ) -C {O) -O-CH ₂ -N (R ₅ ) -C (S) -O-CH ₂ -OC (O) -N (R _S ) -O-0-C (S) -N (R ₅ ) -O-OC (O) -N (R ₅ ) -CH ₂ -O-C {S) -N (R _s ) -CH ₂ -41- OC (O) -CHj-N (R ₅ ) -OC (S) -CHj-N (Rj) -OC (O) -CHi-ON (R _s ) -OC (S) -CH ₂ -ON (R ₅ ) -OC (O) -N {R _S ) -O-CH _: -OC (S) -N (R ₅ ) -O-CH ₂ -ON (R ₅ ) -C (O) -O-CH ₂ - ON {R _s ) -C (S) -O-CH ₂ -CH, -OC (O) -N (R ₅ ) -0-CH ₂ -OC (S) -N (R _s ) -O-CH ₂ - OC (O) -N (R _s ) -CH ₂ -CH ₂ -OC (S) -N (R _s ) -CH ₂ -CH ₂ -OC (O) -CH ₂ -N {R ₅ ) -CH ₂ -OC (S) -CH ₂ -N (Rj) -CH ₂ -OC (O) -N (Rj) -CH ₂ -OC (S) -N (R _s ) -CH ₂ -OC (O) -N (R ₅ ) -O-CH ₂ -OC (S) -N {R ₅ ) -O-CH ₂ -O-N {R _s ) -C (O) -O-CH ₂ -ON (R ₅ ) - C (S) -O-CH ₂ -N (R ₅ ) -C (O) -S-CH ₂ -N (Rs) -C (S) -SN (R 1) -C. (O) -S-CH . "-N (Rj) -C (S) -S-CH, -SC (O) -N (R _s ) -OOC (S) -N (R ₅ ) -SSC (O) -N (Rj) - CHj-SC (S) -N (Rj) -CH ₂ -SC (O) -CH ₂ -N (R ₅ ) -SC (S) -CH ₂ -N (RJ-SC (O) -CH ₂ -ON (R ₅ , -OC (S) -CH ₂ -SN (Rj) -OC (O) -N (R _s ) -S-CH ₂ -SC (S) -N (R ₅ ) -O-CHj-SN (R ₅ ) -C (O) -O-CHj-ON (R _s ) -C {S) -S-CH ₂ -CHj-SC (O) -N (R ₅ ) “O-CH ₂ -OC ( S) -N (R ₅₎ -S-CH ₂ -SC (O) N (R _s) -CH ₂ -CH ₂ -SC (S) N (R _s) -CHj-CH ₂ -SC (O ) -CH ₂ -N (R _s.) ) -CH ₂ -SC (S) -CH ₂ -N (R _s ) -CH ₂ -SC (O) -N (R _s ) -CHj-SC (S) -N (R ₅ ) -CH ₂ -SC (O) -N (R ₅ ) -O-CH ₂ -SC (S) -N (R ₅ ) -O-CH ₂ -SN (R _s ) -C (O) -O-CH ₂ -SN (R ₅ ) -C (S) -O-CH ₂ -OC (O) -N {R _s ) -S "

-CHj-OC (S) -N (R ₅ ) -S-CH ₂ -ON (R _s ) -C (O) -S-42-CH ₂ -ON (R _s ) -C (S) -SN ( R}) ^_ N (Rj) -N {R _s ) -N (R _s ) -CH ₂ -CH ₂ -N (R _s ) -N (R ₅ ) -N = C (NHj) -N {R ₅ ) -N (R ₅ ) -N = C (NH ₂ ) -S (O) -CH ₂ -O-O-CH ₂ -S (O) -S-CH (R _s ) -OO-CH (R 1) -So-ch ₂ -ch «ch-S-CHj-CH = CH-S — CHj — C — C—

-N (R ₅ ) -CH ₂ -N (R ₅ ) -N (R _s ) -C {O) -N (R ₅ ) -N (Rs) -C (S) -N (R ₅ ) -N (R ₅ ) -C (O) -SN (R _s ) -C (S) -SN (R ' ₅ ) -C (S) -ON (R _S ) -C (O) -OOC (O) -N (R $) - OC (S) -N {R _s ) -SC (O) -N (R _s ) -SC (S) -N (R 1) R ₅ is H, OH, OMe, CN, NH, NOH , ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (ΙΣΟ), Me, heteroalkyl (1-7C), aryl (6-7C), - (CH ₂ ) _X F; where “x” is 1-7C and “F” is independently H, OH, SH, OCH ₃ , CN, SCH ₃ , ONH ₂ , ONH (CH ₃ ), SNH ₂ , S (O) NH ₂ , S (O ) (O) NH ₂ , CH ₃ , Ph. Additionally, the conjugate of one or more particles may be bonded to form an oligomeric conjugate. Suitable conjugate particles include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleavage particles (eg imdazole), crosslinking particles (eg psoralen), lipids, peptides, alkylating agents, hydroxamates and fluorescent designations.

Particularly preferred 4′-5 ′ bonds include phosphodiester, phosphorothioates, methylphosphonates, carboxamide, thiocarboxamide, hydroxamate, sulfonamide, hydroxylamine and carbamate. The same modifications are also preferred for 2'-5 'and 3'-5' bonds.

-43Oligomers of the invention are not limited to oligomers with a homogeneous bond type and these alternately or randomly distributed alternate bonds, including 2 ', 5' bonds are included. Because the oligomers of the invention can be so synthesized that one nucleomomeric residue is synthesized at a time, each individual bond and / or substituent bond and the nature of each individual "base" substituent can be selected independently to produce oligonucleotides having the desired sequence.

The oligomers of the invention may contain any desired number of alternative bonds. These alternate bonds may be identical or different, based on the embodiments selected for "V", including other alternate bonds not covered by the invention. As the oligomers are prepared sequentially, any pattern of bonds or types of substitution bonds, bases and sugar modifications may be used.

In preferred embodiments of the invention, the alternate bonds of the invention are arranged in a regular pattern. For example, one alternate bond is followed by two phosphodiester bonds, followed by one alternate bond of the invention, etc. Additional embodiments include e.g. alternating bonds, such as an alternate bond followed by a phosphodiester analogue (e.g., thioate, etc.), followed by an alternative bond of the invention, this bond followed by a phosphodiester analogue, etc., for example, the oligomer of the invention may comprise one alternation of two types of alternative ties. The oligomers of the invention comprising more than one type of bond may have any number of regular patterns formed by alternations between different types of bonds present between the subunits of the oligomer.

The sugar modifications may be made in one or more nucleomomeric residues in the oligomers of the invention; however, 4′-5 ′, 3′-5 ′ and 2′5 ′ nucleotide bonds between amino acid residues are preferred when it comes to

-44to include such modifications. In such a case, a further abbreviation may be used to represent the sequence of bases of the oligonucleotide analogue. For example, for standard DNA (or RNA), the sequences are usually indicated by the sequences of the bases themselves, such as, for example, ATG CGC TGA. It is generally predetermined whether this represents an RNA or DNA sequence. The corresponding recording system is used herein to represent oligonucleotide analogues with a given base sequence.

Additional nucleomomeric modifications:

In addition to the alternate bonds of the invention, the oligomers of the invention may also comprise various modifications. Additional modifications include oligomers, where (i) one or more nucleomomeric residues are modified at 2 ', 3', 4 'and 5' positions, (ii) one or more covalent crosslinking particles are included, (iii) other alternatives are included (iv) other base analogs, such as 8-oxo-N ⁶ -methyladenine, are included, and (v) conjugates such as intercalator agents or polylysine which increase the binding affinity to the target nucleic acid are included sequences or that increase the association of the oligomer with cells.

The sequence-specific binding properties of the oligomers of the invention for single-stranded and duplex targets are compatible with further modifications in the oligomer. These further modifications may also confer other useful properties, such as stability to nuclease cleavage (e.g. in the field of the oligomer of the invention having phosphodiester bonds) or enhancing their ability to penetrate cell membranes and the like.

The oligomers of the invention may comprise one or more substituent bonds such as sulfide or sulfone bonds (Benner, S.A., International Publication No. WO

-4589/12060), sulfamate bonds (International Publication No. WO 91/15500), carbamate or other substitute bonds in morpholino-linked oligomers (Stirchak, EP et al., Nucleic Acids Res. 1989, 17, 6129-6141; Summerton, J., et al., International Publication No. 216 860) and Related Links.

Thus, exemplary embodiments of the oligomers of the invention include oligomers having (1) at least one alternative bond and one amino acid attached to the adjacent monomer and (2) one or more alternative bonds not subject to this invention selected from the group consisting of phosphorothioate, methylphosphonate and thionomethylphosphonate and / or (3) one or more phosphodiester bonds and / or (4) purine or pyrimidine analogues that increase binding affinity for complementary target sequences. Other illustrative oligomers would include (1) an oligomer having alternate bonds of the invention at the 3 'and / or 5' ends and phosphorothioate bonds elsewhere in the oligomer; (2) oligomers having alternative linkages of the invention and standard purine or pyrimidine bases (e.g., adenine, guanine, cytosine, thymine or uracil); (3) oligomers having alternative linkages of the invention and one or more bases that increase the binding affinity or permeation competence of the oligomer (e.g., 5-methylcytosine, 5 '(1-propynyl) uracil, 5- (1-propynyl) cytosine). Also included are oligomers containing nucleomomeric residues linked via hydroxamates.

Synthesis of oligomers:

The oligomers of the invention may be formed using nucleomonomers of the invention alone or in combination with conventional nucleomonomers and synthesized using standard solid phase (or in solution) oligomeric synthesis techniques now commercially available. In general, the oligomers of the invention can be synthesized by a method comprising the following steps: synthesizing a nucleomonomer or oligomer synthon having a protective

-46 group and base and linker group that can bind to a nucleomonomer or oligomer; coupling a nucleomonomer or oligomer synthon to a receiving nucleomonomer or to a receiving oligomer; removal of the protecting group; and repeating the cycle for as long as is necessary for the desired oligomer to be synthesized.

Oiigomers of the present invention may be of any length, including those longer than 40, 50, 100, 200, or 500 nucleomonomers. In general, preferred oiigomers contain 2-30 nucleomomers. Lengths greater than or equal to about 8 to 20 nucleomonomers may be useful for therapeutic or diagnostic uses, and oiigomers of these lengths have corresponding base sequences provided. Short oligomers containing 2, 3, 4 or 5 nucleomonomers are specifically included in the present invention and can be used as synthons.

Oiigomers having a random sequence and containing about 6, 7 or 8 nucleomomers may be used as primers for use in cloning or amplification protocols using random sequence primers, provided that the oligomer contains 1 or 2 residues at 3 ' an end that may serve as an example for polymerases or reverse transcriptases or that does not otherwise interfere with polymerase activity.

In addition to the bonds described herein first, the oiigomers of the invention may comprise conventional phosphodiester bonds or may contain other substituent bonds, such as phosphoramidate bonds in addition to those of the invention. These alternate bonds include, but are not limited to, embodiments of which have a particle of formula -OP (O) (S) -O- ("phosphorothioate"'), -OP (O) (NR ₂ ¹¹ ) -X2, -OP (O) (R ¹¹ ) -O-, -OP (S) (R ¹¹ ) -O- ("thioalkylphosphonate"), -P (O) (OR ⁹ ) -X ² , -OC (O) -X ² , or -OC (O) (NR ₂ ¹¹ ) -X 2 ~ where R ^{11 is} H (or salt) or alkyl (1-12C, including methyl and

-47ethyl) and R ^{9 is} alkyl (1-9C) and the bond is attached to adjacent nucleomonomers by -O- or -S- carbon-linked nucleomonomers and X ^{2 is} O or S. Phosphorothioate and phosphodiester bonds are well known. Particularly preferred alternative bonds for use in the oligomers of the present invention include phosphodiester, phosphorothioate, methylphosphonate, and thionomethylphosphonate replacement bonds. The phosphorothioate and methylphosphonate replacement bonds that give the oligomer additional stability must be identical, especially preferred oligomers of the invention contain one or more phosphorothioate or methylphosphonate replacement bonds.

The oligomers of the invention and their segments can be synthesized using methods well known to one of ordinary skill in the art. Synthesis methods known in the art and described herein may be used to synthesize oligomers containing alternative bonds of the invention, as well as other or alternative bonds known in the art, using suitably protected nucleomonomers. Methods for the synthesis of oligomers having phosphorus-containing bonds are found, for example, in Froehler, B., et al., Nucleic Acids Res., 1986, 14, 5399-5467; Nucleic Acids Res., 1988, 16, 4831-4839; Nucleosides & Nucleotides, 1987, 6, 287291; Froehler, B., Tetrahedron Letts., 1986, 27, 5575-5578; Caruthers, M.H. in Oligodeoxynucleotides Antisense Inhibitions of Gene Expression, 1989, J.S. Cohen, editor, CRC Press, Boca Raton, p7-24; Reese, C.B. et al., Tetrahedron Letts., 1985, 26, 2245-2248. The synthesis of methylphosphonate-linked oligomers via methyl phosphonamidite chemistry has already been described (Agravval, S. et al., Tetrahedron Letts., 1987, 28, 3539-3542; Klem, RE, et al., International Publication No. WO 92/07864 ).

The oligomers having the bonds of the present invention are also properly synthesized by the preparation of dimeric or trimeric compounds with liquid phase chemistry, followed by

-48 Conversion of synthon to a derivative incorporated in oligomers either by solid or liquid chemistry. Typical synthons are 5 'DMT or MMT blocked 3' phosphonate or phosphoramidate derivatives prepared by standard methods (see: Gait, M.J. ed., Oligonucleotide Synthesis; A Practical Approach 1984, IRL Press, Oxford).

The synthons included in the scope of the present invention include dimers, trimers, tetramers, hexamers and a longer oligomer made by solid or liquid phase synthesis. Trimmers and longer synthons can have more than one type of bond. Synthons may include any base as described above or 2 ', 3', 4 'and 5' groups such as OH, DMTO, MMTO, O-allyl, phosphate, phosphonate or amidite as described above.

Ribose-amide oligonucleotides can be synthesized using standard conditions of solid phase peptide synthesis (Fmoc chemistry), (see Figure 26).

Blocking groups for the synthesis of compounds of the Invention:

1. Liaison groups.

Suitable linking groups are, for example, the H-phosphonate, methylphosphonamidite or phosphoramidite group.

Phosphoramidites that may be used include β-cyanoethylphosphoramidites (preferably).

Methylphosphonamidites, alkylphosphonamidites (including ethylphosphonamidites and propylphosphonamidites) may also be used. Illustrative phosphoramidites are shown in Figures 1 to 21.

-49Equivalent "linking groups" at the 2 ', 3', 4 'or 5' position for the synthesis of oligomers via the phosphoramidite chemistry of triesters, referred to herein as 'amidite' chemistry, include N-diisopropylamino-p-cyanoethoxyphosphine, N, N-diisopropylamino -methoxyphosphine, Ν, Ν-diethylamino-cyanoethoxyphosphine and (N-morpholino) -methoxyphosphine linking group (Moore, MF, et al., J Org Chem., 1985, 50, 2019-2025; Uznanski, AW, et al., Tetrahedron Letts., 1987, 28, 3401-3404; Bjergarde, K., et al., Nucl Acids Res., 1991, 19, 5843-5850; Dahi, O., Sulfur Reports, 1991, H, 167-192). Related linking groups such as Ν, Ν-diisopropylamino-methyl-phosphine or N, Ndiethylamino-methyl-phosphine can also be used to prepare methylphosphonates. Methylphosphonate oligomers can be properly synthesized using linker groups such as N, N-diisopropylaminomethylphosphoramidite. The synthesis of the nucleo-monomeric amidites of the invention can be carried out by conventional methods (e.g., Gryaznov, S. M., et al., Nucl Acids Res., 1992, 20, 1879-1882; Vinayak, R., et al., Nucl Acids Res., 1992 , 20, 1265-1269; Sinha, ND, et al., Nucl Acids Res., 1984, 12, 4539-4557; and other references cited herein).

2. Protective groups.

Protective groups such as, for example, diisobutylformamidine, benzoyl, isobutyryl, FMOC, dialkylformamidine, dialkylacetamidine, or other groups known in the art can be used to protect the exocyclic nitrogen of cytosine, adenine or guanine heterocycles. Alternatively, cytidine may be directly incorporated into the oligomers using the described methods without the protecting group on exocyclic nitrogen (Gryaznov, SM et al., J Amer Chem Soc. 1991, 113. 5876-5877; Gryaznov, SM et al. Nucl Acids Res., 1992, 20, 1879-1882; Kung, P.-P. et al., Tetrahedron Letts., 1992, 33, 5869-5872).

-50Convenient protecting groups are DMT (dimethoxy trityl), Bz (benzoyl), Bu (isobutyryl), phenoxyacetyl, MMT (monomethoxytrityl) or FMOC at the 5 'end and / or hydrogen phosphonate, methyl phosphoramidite, methyl phosphonamidite, β-cyanoethylyl, β-cyanoethyl, (t-butyldimethylsilyl) or TBDPS (tbutyldiphenylsilyl) at the 3'-end.

Preferred protecting groups are Bz (benzoyl), DMT (dimethoxytrityl), MMT (monomethoxytrityl) or FMOC at the 5 'end or position and / or TBS, hydrogen phosphonate, methylphosphoramidite, methylphosphonamidite, βcyanoethylphosphoramidite at the 3′-end. However, it is thought that the position of the protecting groups may be reversed when necessary (e.g., phosphoramidite at 5 'position and DMT at 3' position). In general, the nucleomonomers and oligomers of the invention can be derivatized into such "protecting groups" as exemplified in the corresponding formulas by methods known in the art.

Koniugati:

The present invention also provides "conjugates" of the oligomers of the invention. The "conjugates" of conventional oligomers are known to one of ordinary skill in the art. For example, the oligomers of the invention may be covalently linked to different particles, such as, for example, intercalators and compounds that specifically correlate with the smaller double-stranded DNA channel. Other particles for conjugation to the subject oligomers include labels (e.g., radioactive, fluorescent, enzymatic) or particles that facilitate cellular association by using fissile linkers and the like. The corresponding radioactive indications are ³² P, ³⁵ S, ³ H, ¹³¹ 1 and ¹⁴ C; and appropriate fluorescent designations include fluorescence, resorufin, rhodamine, BODIPY (molecular probes) and Texas red; suitable enzymes include alkaline phosphatase and

-51hren peroxidase. Other compounds that can be used as covalently linked particles, including biotin, antibodies or antibody fragments, asialoglycoprotein, transferrin and HIV Tat protein, may also be covalently linked to the oligomers of the invention.

These additional particles can be derivatized with the aid of each corresponding particle. For example, intercalators such as acridine or psoralen may be coupled to the oligomers of the invention by any suitable -OH or SH particle, e.g., at the oligomer end 5'-position, 2'-RNA positions, or OH, NH ₂ , COOH or SH included in the 5-position of the pyrimidines. A derivatized form having, for example, -CH ₂ CH ₂ CH ₂ OH or -CH ₂ CH ₂ CH ₂ SH in the 5-position of the pyrimidines is suitable. Conjugates, including polylysine or lysine, can be synthesized as described and can further increase the binding affinity of the oligomer to its target nucleic acid sequence (Lemaitre, M. et al., Natl Acad Sci. USA, 1987, 84, 648- 652; Lemaitre, M. et al., Nucleosides and Nucleotides, 1987, 6, 311-315).

Many different substituents can be bonded, including those bonded or bonded. In oligomers, the -OH particles may be replaced by phosphate groups protected by standard protecting groups or linking groups to prepare additional bonds for other nucleomonomers or may be linked to a conjugated substituent. The 5′-terminal OH can be phosphorylated; 2′-OH or OH substituents at the 3′-end can also be phosphorylated. Hydroxyls may also be derivatized into standard protecting groups.

The oligomers of the invention may be covalently derivatized into particles that facilitate cell assembly by the use of fission linkers. Suitable conjugates also include solid substrates for the synthesis of oligomers and for facilitating detection

-52nucleic acid sequences. Solid substrates include, but are not limited to, silica, controlled-size glass, polystyrene, and magnetic glass beads.

Sugar modifications:

Derivatives may be made by substitution on sugars. Preferred oligomer derivatives of the invention are the 2′-0-allyl or 3′-allyl group, which appear to increase permeation ability and stability for degradation by nuclease but do not appear to reduce the affinity of the oligomer for single-stranded or duplex targets. Especially in ribosamide backbone oligonucleotides, different functional groups may be introduced at the Γ, 2 ′, 3 ′, 4 ′ and 5 ′ positions of the ribose particle to enhance the pharmacokinetic properties of the corresponding oligonucleotides.

Alternate Links:

The oligomers of the invention may also have one or more "substituent bonds" in addition to the 2'5 ', 3'-5' and 4'-5 'bonds disclosed herein, which are generally understood in the art. These "substitute linkages" include phosphorothioate, metilfosfonatno, tionometilfosfonatno, fosforoditioatno, alkilfosfonatne, morpholino sulfamidno, boranofosfatno (-OP (OCH ₃₎ (BH ₃₎ -O-), siloxane (-O-Si (X ⁴⁾ (X ⁴⁾ -O-; X ⁴ is 1-6C alkyl or phenyl) and phosphoramidate (methoxyethylamine (-OP (OCH ₂ GH ₂ OCH ₃ ) (O) -O-) and the like) and have been synthesized as described in generally available literature including with the following references (Sood, A., et al., J. Am. Chem. Soc., 1990, 112, 9000-9001; WO 91/08213; WO 90/15065; WO 91/15500; Stirchak, EP, s et al., Nucleic Acids Res., 1989, 17. 6129-6141; US patent 5,034,506; US patent 5,142,047; Hewitt, JM et al., Nucleosides & Nucleotides, 1992, H, 1661-1666; Summerton, J. et al. , international

-53post no. 216 860). Alternate bonds that may be used in the oligomers disclosed herein also include sulfonamide (-O-SO ₂ -NH-), sulfide (-CH ₂ -S-CH ₂ -), sulfonate (-O-SO ₂ -CH ₂ -), carbamate (OC (O) -NH-, NH-C (O) -O-), dimethylhydrazino (-CH ₂ -NCH ₃ -), sulfamate (-OS (O) (O) -N-; -NS (O) (O) -N-), 3'-amino (-NH-CH ₂ -), N-methylhydroxylamino (-CH ₂ -NCH ₃ -O-) and 2 ', 5' bonds (such as eg 2 ', 5' carbamate (2 '-N (H) -C (O) -O- 5'), 5 ', 2' carbamate (2'-OC (O) -N (H) -5 '), 5', 2 'methylcarbamate (2'-OC (O) -N (CH ₃ ) -5') and 5 ', 2' thioformacetal (2'-O-CH ₂ -S-5 '). suitable substitutions include amide bonds described by Buchardt, O. et al. (International Publication No. WO 92/20702) and those described by Cook, PD et al. (international publication). No. WO 92/20822) and De Mesmaeker, A. et al. (International Publication No. WO 92/20823) and as described in PCT / US92 / 04294.

Except where specifically indicated, alternative bonds such as the formacetal bond are -O- CH ₂ -O- bonded to either the 4 ', 3', 2 'carbon nucleomonomer on the left and the 5' carbon nucleomonomer on the right. Accordingly, the positions of 4 ', 3', 2 'or 5' of carbon may be modified when any structure other than ribose, deoxyribose or arabinose is attached to a neighboring nucleomonomer. Such structures include xylose, hexose, morpholino ring, carbocyclic ring (e.g. cyclopentane) and the like.

The use of carbamate, carbonate, sulfide, sulfoxide, sulfonate, Nhydroxylamine and dimethylhydrazino bonds in synthons or oligomers has already been described (Vaseur, JJ. Et al., J Amer Chem Soc., 1992, 114, 4006-4007; WO 89/12060 ; Musicki, B. et al., J Org Chem., 1990, 55, 4231-4233; Reynolds, RC et al., J. Org. Chem., 1992, 57, 2983-2985; Mertes, MP, et al. ., J. Med. Chem., 1969, 12, 154-157; Mungall, WS, et al., J. Org.

-54Chem., 1977, 42, 703-706; Stirchak, E.P., et al., J. Org. Chem., 1987, 52, 4202-4206; Wang, H., et al., Tetrahedron Letts., 1991, 32, 7385-7388; international application no. PCT US91 / 03680). The substitution bond (s) can be used in oligomers for many purposes, such as further facilitating binding by complementary target nucleic acid sequences and / or enhancing oligomer stability against nucleases.

Bases:

Suitable bases for use as nucleoside bases in the compounds of the invention include not only naturally occurring purine and pyrimidine bases but also analogues of these heterocyclic bases and their tautomers. Such analogues include alkylated purines or pyrimidines, acylated purines or pyrimidines or other heterocycles. Such &quot; purine analogues &quot; and &quot; pyrimidine analogs or purine or pyrimidine analogues are those commonly known in the art, some of which are used as chemotherapeutic agents. An illustrative but incomplete list includes N ⁴ N ⁴ -ethancytosine, 7deazaxanthosine, 7-deazaguanosine, 8-oxo-N ⁶ -methyladenine, 4-acetylcytosine, 5 (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracyl, 5carboxymethyl thiouracil, 5-carboxymethylaminomethyl uracil, inosine, N ⁶ -isopentenyl-adenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine, N ⁶ methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-methoxyaminomethyl-2- thiouracil, 4-thiouracil, 5- (1-propynyl) -4-thiouracil, 5- (1-propynyl) -2thiouracil, 5- (1-propynyl) -2-thiocytosine, 2-thiocytosine and 2,6-diaminopurine. In addition to these base analogs, pyrimidine analogs, including 6azacytosine, 6-azathimidine and 5-trifluoromethyluracil, have been described in Cook, DP, et al., International Publication no. WO 92/02258 (incorporated herein by reference) may be suitably included in the oligomers of the invention.

-55The incorporation of 4-thiouridine and 2-thiothymidine into oligomers has been described (Nikiforov, TT, et al., Tetrahedron Letts. 1992, 33, 2379-2382; Clivio, P., et al., Tetrahedron Letts., 1992 33 : 65-68; Nikiforov, TT, et al., Tetrahedron Letts., 1991 32: 2505-2508; Xu, Υ.-Ζ., et al., Tetrahedron Letts., 1991 32: 2817-2820; Clivio, P ., et al., Tetrahedron Letts., 1992 33: 69-72; Connolly, BA, et al., Nucl. Acids Res., 1989 17: 4957-4974). Preferred bases include the following bases: adenine, guanine, thymine, uracil, cytosine, 5methylcytosine, 5- (1-propynyl) uracil, cytosine, 5-methylcytosine, 5- (1-propynyl) uracil, 5 (1-propynyl) cytosine, 8-oxo-N ^6- methyladenine, 7-deaza-7-methylguanine, 7-deaza-7methyladenine and 7-deazaxanthosine.

Covariate coupling particle:

Some of the oligomers of the invention include a particle capable of at least one covariate bond between the oligomer and the duplex. By providing many such crosslinking particles, multiple covalent bonds can also be formed. The covalent bond is preferred to the base residue in the target chain, but may also be formed by other parts of the target, including saccharide or phosphodiester. The reaction nature of the particle performing the crosslinking is determined by the nature of the target in the duplex. Preferred crosslinking moieties include acylating and alkylating agents, and in particular those agents positioned relative to the moiety, which gives sequence specificity in such a way as to allow reaction with the target location in the chain.

It is understood that the heterocycle is not required to be purine or pyrimidine; it is also really not necessary that the pseudo-base to which the reactive group is linked is at all heterocycle. All methods of connecting a reactive group are satisfactory as long as the installation is correct.

-56Polarity of oligomers:

In their most general form, the symbol 3'-5 'indicates the region of the oligomer in which the bonds are consistently formed between the 5'-hydroxyl of the amino acid residue on the left by the 3'- (or 2'- for oligomers having 2'-5' bonds or 4 'for oligomers having 4'-5' bonds) by the hydroxyl of the amino acid residue of the nucleomonomer on the right (e.g., the region of the same polarity), thus leaving the 5'-hydroxyl of the rightmost nucleomomeric amino acid residue free for additional conjugation. Analogously, 5'-3 'shows the region of a counter-directed oligomer in which the bonds are formed between the 3'-hydroxyl of the amino acid residue of the left nucleomonomer and the 5'-hydroxyl of the amino acid residue of the nucleomomer on the right, leaving the 3'-hydroxyl of the most right nucleomonomer residue free conjugation. The same goes for the 5 '- - 4' oligomer range.

Pharmaceutically acceptable salts:

The invention also provides various salts of all the compounds disclosed herein, including pharmaceutically acceptable salts for administration to animals or humans. Pharmaceutically acceptable salts and materials for forming such salts are well known in the art. Pharmaceutically acceptable salts are preferably metal or ammonium salts of the oligomers of the invention and include alkali or alkaline earth metal salts, e.g., sodium, potassium, magnesium or calcium salts; or more preferably ammonium salts which crystallize easily and are obtained from ammonia or organic amines such as mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl) -amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl) alkylammonium bases , e.g. methylamine, diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, tris- (hydroxymethyl) -aminomethane or benzyltrimethylammonium hydroxide. Oiigomers of the invention may form additional acid salts,

-57 preferably, therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrophilic, e.g., hydrochloric or hydrobromic acid; sulfur, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, e.g., formic, acetic, propionic, succinic, glycolic, lactic, malic, tartaric, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-amine, benzoic, 4-amine , 4-hydroxybenzoic, salicylic, 4aminosalicylic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, sulfanyl or cyclohexylsulfamic acid and the like.

Utility and benefits:

Because the oligomers of the invention are capable of characterizing single or double stranded target nucleic acid binding activities to form duplexes, triplexes or other forms of stable bonds with naturally occurring polynucleotides and their structural analogues, the oligomers of the invention can be used in most processes using conventional oligomers. Thus, the oligomers of the invention can be used, for example, polynucleotide hybridization probes, primers for polymerase chain reaction, and similar cyclic amplification reactions, primers for sequencing, and the like. The oligomers of the invention can also be used in the diagnosis and therapy of diseases. The therapeutic uses of the oligomers of the invention include specific inhibition of gene expression (or inhibition of translation of RNA sequences encoded by these genes), which are associated with either the establishment or maintenance of a pathological condition through the use of antisense oligomers. The oligomers of the invention can be used to mediate the antisense inhibition of many genetic targets, Illustrative genes or RNA molecules encoded by these genes, which can be targeted by an antisense method using oligomers include those encoding enzymes, hormones, serum proteins, transmembrane

-58proteins, adhesion molecules (LFA-1, ΘΡΐνΐΙΙ ₃ , ELAM-1, VACM-1, ICAM-1, E-selection and the like), receptor molecules, including cytokinin receptors, cytokines (IL-1, IL-2, IL-3, IL-4, IL-6 and the like), oncogenes, growth factors and interleukins. Target genes or RNA molecules can be associated with any pathological condition, such as those associated with inflammatory conditions, cardiovascular disorders, immune responses, cancer, viral infections, bacterial infections, yeast infections, parasite infections and the like.

The oligomers of the present invention are suitable for use in both in vivo and ex vivo applications.

Indications for ex vivo administration include the treatment of cells such as bone marrow or peripheral blood cells in conditions such as e.g. leukemia (chronic myelogenous leukemia, acute lymphocytic leukemia) or viral infection. Target genes or RNA molecules encoded by these genes that serve as targets for cancer treatments include oncogenes such as ras, k-ras, bcl-2, c-myb, ber, c-myc, c-abl, or overexpressed sequences of such such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2, and translocations such as bcr-abl. Viral gene sequences or RNA molecules encoded by these genes, such as the polymerase or reverse transcriptase genes of herpes viruses such as CMV, HSV-1, HSV-2, retroviruses such as HTLV-1, HIV-1, HIV-2 or other DNA or RNA viruses such as HBV, HPV, VZV, influenza virus, adenoviruses, flaviviruses, rhinoviruses and the like are also suitable targets. Administration of specific binding oligomers may be used in conjunction with other therapeutic treatments. Other therapeutic uses for the oligomers of the invention include (1) modulating inflammatory responses by modulating gene expression, such as, for example, IL-1 receptor, IL-1, ICAM-1 or E-selection, which play a role in mediating inflammation and (2) modulation of cell proliferation under conditions such as

-59 artery blockage (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as non-muscle myosin, myc, fox, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c-myb. Other suitable proliferation factors or signal transduction factors, such as TGFx, IL-6, gINF, protein kinase C, tyrosine kinases (such as p210, p190) may be targeted for the treatment of psoriasis or other conditions. Likewise, EGF receptor, THFα or MHC alleles may be targeted in autoimmune diseases.

The uptake of the oligomers of the invention into cells can be enhanced by any suitable method, including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation, or using cationically anionic and / or neutral lipid mixtures or liposomes according to the methods described (International Publication No. WO 90 / 14074, WO 91/16024, WO 91/17424, U.S. Patent 4,897,355). Oligomers can be inserted into cells by complexation with cationic lipids such as DOTMA (which may or may not form liposomes), the complex of which is then in contact with the cells. Suitable cationic lipids include N- (2,3-di (9- (Z) -octadecenyloxy)) - prop-1-yl-N, N, N-trimethylammonium (DOTMA) and its salts, 1-O-oleyl-2-O -oleyl-3dimethylaminopropyl-p-hydroxyethylammonium and its salts and 2,2-bis (oleioxy) -3 (trimethylammonio) propane and its salts, but are not limited to these compounds.

Increased uptake of the oligomers of the invention may also be mediated by the use of (i) viruses such as Sendai virus (Bartzatt, R., Biotechnol App! Biochem., 1989, H, 133-135) or adenovirus (VVagner, E. et al. , Off Natl Acad Sci. USA, 1992, 89, 6099-6013); (ii) polyamine or polycationic conjugates using compounds such as polylysine, protamine or Na, N ₁₂ -bis (ethyl) spermine (Vagner, E. et al., Natl Acad Sci. USA, 1991, 88, 4255-4259 Females,

M. et al., Off. Natl. Acad. Sci. USA, 1990, 87, 3655-3659; Chank, B.K. s

-60sod., Biochem Biophys Res Commun., 1988, 157, 264-270; U.S. patent 5,138,045); (iii) lipopolyamine complexes using compounds such as lipospermine (Behr, J.-P. et al., Natl Acad Sci. USA, 1989, 86, 69826986; Loeffler, JP et al., J. Neurochem., 1990 , 54, 1812-1815); (iv) anionic, neutral or pH-sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D. et al., Biochem Biophys ACTA, 1992, 1103 185-197; Cheddar, G. et al., Arch Biochem Biophys, 1992, 294. 188-192; Yoshimura, T., et al., Biochem Int., 1990, 20, 697706); (v) conjugates with compounds such as transferrin or biotin; or (vi) conjugates with proteins (including albumin or antibodies), glycoproteins or polymers (including polyethylene glycol), which increase the pharmacokinetic properties of the oligomers in the subject. As used herein, transfection refers to any method suitable for the uptake of oligomers into cells. Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocols is collectively referred to herein as a "permeation enhancer". The uptake of oligomers into cells may occur via cotransfection with other nucleic acids, such as (i) expression DNA fragments having a protein (s) or protein fragment encoded, or (ii) RNA molecules that are translatable and having a protein (s) encoded, or protein fragment.

The oligomers of the invention can thus be incorporated into any suitable formulation that enhances the uptake of oligomers into cells. Suitable pharmaceutical formulations also include those commonly used for administration where compounds are introduced into cells or tissues by topical administration. Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical preparations containing oligomers.

-61Oligomers of the invention may be suitably used as reagents for research or production purposes, where inhibition of gene expression is desirable. Currently, very few reagents are available that effectively and specifically inhibit target gene expression by each mechanism. Oligomers previously cited to inhibit target gene expression often have nonspecific effects and; or do not reduce target gene expression to very low levels (less than about 40% of uninhibited targets).

Thus, the oligomers described herein generate a reagent that can be used in methods of inhibiting the expression of a selected protein or proteins in a subject or in cells in which proteins are encoded by DNA sequences and proteins are translated from RNA sequences, comprising the following steps: insertion the oligomer of the invention into cells; and allowing the oligomer to form tripiex with DNA or RNA or duplexing with DNA or RNA thereby inhibiting protein or protein expression. The methods and compound of the present invention are suitable for modulating gene expression in both prokaryotic and eukaryotic cells, such as bacterial cells, giva parasite cells, yeast cells and mammalian cells.

RNase H "competent or RNase H" incompetent "oligomers can be quickly designed using alternative substitution bonds. RNase H competent oligomers may comprise one or more RNase H competent regions made from linked, RNase H competent nucleomonomers. Oligomers that have modifications such as 2'-substitutions (2'-O-allyl and the like) or certain charge-free bonds (methylphosphonate, phosphoramidate and the like) are usually incompetent as a substrate recognized and / or acted upon by RNase H . RNase H competence can facilitate antisense oligomer function by degrading target RNA in the RNA oligomer duplex (Dagle, J.M.

-62s et al., Nucl Acids Res., 1990, 18, 4751-4757; Walder, J.A. et al., International Publication no. WO 89/05358). The enzyme cleaves RNA in RNA-DNA duplexes.

In order to retain RNase H competence, the oligomer requires the RNase H competent domain of three or more competent adjacent nucleomonomers located within the oligomer (Ouartin, R.S., et al., Nucl Acids Res., 1989, 17, 7253-7262). The model of oligomers resistant to nuclease metabolism will have the final binding and modifications of sugars and / or bases to exert nuclease resistance. Thus, the oligomers may be designed to have modified nucleomomeric residues at one or both, 5 'and / or 3' ends, while the internal RNase H has a competent domain. Illustrative oligomers that retain RNase H competence would generally have the same polarity and would comprise about 2 to about 12 nucleomonomers at the 5'-end and 3'-end, stabilizing the oligomer against nuclease degradation and about three to about 26 nucleomonomers, acting as RNase H competent domain, between RNase H incompetent 3 'and 5'- ends. Variations on such an oligomer would include (1) shorter RNase H competent region comprising 1 to 2 RNase H competent bonds or substituent bonds, (2) longer RNase H non-competent region comprising up to 15, 20 or more replacement bonds or nucleomonomers, (3 ) longer RNase H competent region, comprising up to 30, 40 or more bonds, (4) oligomers with only one RNase H incompetent region at the 3 'end or 5' end.

Oligomers having as few as about 8 nucleomonomers can be used to perform inhibition of target protein (s) expression by forming duplex or triplex structures with target nucleic acid sequences. However, linear oligomers will be used for inhibition

-63Expression of the target protein via duplex or triplex formations, preferably having from about 10 to about 20 nucleomomeric residues.

The oligomers having alternative linkages of the invention may be properly rounded as described (International Publication No. WO 92/19732; Kool, ET J Am Chem Soc. 1991, 113, 6265-6266; Prakash, G. et al. , J Am Chem Soc., 1992, 114, 3523-3527). Such oligomers are suitable for binding to single-stranded or double-stranded nucleic acid targets. Circular oligomers can be of different sizes. Such oligomers in the size range of about 2250 nucleomonomers can be more readily prepared. Circular oligomers can have in the loop region about three to about six nucleomomeric residues separating the binding regions of the oligomer as described (Prakash, G. in the same publication). The oligomers may be enzymatically rounded by terminal phosphate by ligase or by chemical means via bonds using 5′- and 3′terminal sugars and / or bases.

Oligomers can be used to modulate target gene expression by inhibiting the interaction of nucleic acid binding proteins responsible for modulating transcription (Maher, L.J., et al., Science, 1989, 245. 725730) or translation. Oligomers are therefore suitable as sequence-specific agents that compete with nucleic acid binding proteins (including ribosomes, RNA polymerases, DNA polymerases, translation initiation factors, transcription factors that either increase or decrease transcription, protein-hormone transcription factors and the like) . Suitably designed oligomers can thus be used to enhance the synthesis of the target protein by mechanisms such as binding to or near the regulatory site, that transcription factors serve to arrest expression or by inhibiting the expression of the selected repressor protein itself.

-64Oligomers of the invention comprising additional modifications that increase the binding affinity may be designed to have secondary or tertiary structures, such as pseudo-nodes or pseudo-half-nodes (Ecker, DJ et al., Science, 1992, 257, 958- 961). Such structures may have a more stable secondary or tertiary structure than the corresponding unmodified oligomers. The increased stability of such structures would rely on the increased binding affinity between the complementarity regions in a single oligomer or the complementarity regions between two or more oligomers forming a given structure. Such structures can be used to mimic structures such as the HIV TAR structure, in order to interfere with the binding of the HIV Tat protein (TAR-binding protein). A similar approach can be used with other transcription or translation factors that recognize higher nucleic acid structures such as stems, loops, loops, knots and the like. Alternatively, the oligomers of the invention may be used to (1) break or (2) attach to such structures as a method of (1) obstructing or (2) enhancing protein binding to nucleic acid structures.

In addition to their use in antisense or triple helix therapies, the oligomers of the invention may also be used as therapeutic or diagnostic agents that act by directly displacing a single chain in duplex nucleic acid. Chain displacement in natural duplexes such as chromosomal DNA or duplex viral DNA, RNA, or a DNA / RNA hybrid is possible for oligomers with high binding affinity to their complementary sequence and is not good enough for efficient displacement of DNA or RNA strands in the duplex. The therapeutic efficacy of Dluping-functioning oligomers would result from a higher binding affinity to the complementary sequence, resulting in the modulation of normal biological function associated with Dluping.

-65nucleic acid target. Different types of target nucleic acids include (i) gene sequences, including exons, introns, exon / intron junctions, promoter / promoter regions and 5 'or 3' untranslated regions, (ii) nucleic acid regions which function to utilize secondary structures (e.g., HIV TAR stem-loop or tRNA molecule element), (iii) nucleic acids that serve structural or other functions, such as telomeres, centromeres or replication sources (virus, bacterium, etc.), and (iv) any other duplex region . It is clear that oiigomers can be synthesized by discrete functional regions in which one oligomer region binds to the target by D-luping, while the adjacent region binds the target molecule, for example, by forming a triple helix or by binding to a protein. Alternatively, the D-loop forming oligomer can bind to each chain in the duplex by changing the chain to which the oligomer binds (e.g., by having one oligomer region that can bind to one chain and another region to bind to the complementary chain). The control elements that dictate the binding mode (eg, triple helix or DOZANKA) are the oligomer sequence and the inherited affinity embedded in the oligomer. The rules for recognizing bases for Watson-Crick duplex binding are different from those for Hoogsteen controlled triplex binding. Therefore. the oligomer base sequence can serve to dictate the type of binding rules that the oligomer will use. D-loop structures are naturally formed by enzyme-mediated processes (Harris, LD. Et al., J Biol Chem., 1987, 262, 9285-9292) or are linked to regions where DNA replication occurs (Jacobs, HT et al., Nucl Acids Res., 1989, 17, 8949-8966). D-loops resulting from the binding of oligomers can result from a one- or two-step process. Direct relocation of the target chain will result in a D-loop with a single binding event. However, D-lapping can also occur by forming a triple

-66screws, which facilitates the relocation of the chain, the event leading to the formation of Dzanka.

Ribozymes having alternative linkages of the invention may be designed to form species with altered characteristics. Ribozymes cleaving single-stranded RNA or DNA (Robertson, D.L. et al., Nature, 1990, 344, 467-468) have already been described. Therapeutic uses of ribozymes have been postulated (Sarver, N., et al., Science, 1990, 247, 1222-1225; International Publication No. WO 91/04319). The secondary or tertiary structure required for ribozyme function may be affected by the formation of suitable oligomer sequences. For example, ribozymes having nuclease stable target regions containing alternative linkages of the invention may have greater affinity for target sequences while maintaining the specificity of base pairing. Due to the greater affinity and / or nuclease stability of the substitution bonds of the invention, shorter recognition regions may be formed in the ribozyme (advantage in fabrication), which may lead to more favorable substrate metabolism (advantage in ribozyme action).

In therapeutic applications, the oligomers of the invention may be used in a manner suitable for treating various conditions by inhibiting the expression of the corresponding target genes. For such therapy, oligomers may be formulated for a variety of routes of administration, including systemic, topical or topical administration. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, Merk Publishing Co., Easton, PA, latest edition. Typically, the oligomeric active ingredient is combined with a carrier such as a diluent or excipient, which may include fillers, extenders, binders, wetting agents, disintegrating agents, surfactants or lubricants, depending on the nature of the route of administration and dosage forms. Typical dosage forms include tablets, powders, liquid preparations, including

-67 Suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injection, including liposomal preparations.

Injection, including intramuscular, intravenous, intraperitoneal and subcutaneous injection, is preferred for systemic administration. For injection, the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringers' solution. Additionally, the oligomers may be formulated in solid form and re-dissolved or suspended immediately before use. Freeze-dried forms are also included. The doses that can be used for administration to the system are preferably in the range of about 0.01 mg / Kg to 50 mg / Kg given once or twice daily. However, different dosage schedules may be used depending on (i) the ability of each oligomer to inhibit the activity of its target DNA or RNA, (ii) the difficulty or extent of the pathological disease condition associated with a given target gene, or (iii) the pharmacokinetic behavior of a given oligomer.

Systemic administration may also be transmucosal or transdermal, or the compounds may be administered orally. For transmucosal or transdermal administration, the formulations also use penetrators suitable for the barrier to be punctured. Such penetrators are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. Additionally, detergents may be used to facilitate permeation. Transmucosal administration may be e.g. using nasal sprays or suppositories. For oral administration, oligomers are formulated into conventional oral administration forms such as capsules, tablets and tonics.

For topical administration, the oligomers of the invention are formulated into ointments, balms, gels or creams as is commonly known in the art. Formulations of oligomers

-68 The invention for visual indications, such as viral infections, would be based on standard preparations known in the art.

In addition to use in therapy, the oligomers of the invention can be used as diagnostic reagents to detect the presence or absence of target nucleic acid sequences to which they specifically bind. The increased binding affinity of the oligomers of the invention is an advantage for their use as primers and probes. Diagnostic tests can be performed by assisted hybridization, either by forming a double or triple helix, which is then detected in the usual ways. For example, oligomers may be labeled using radioactive, fluorescent or chromogenic indications, and then the presence of a tag attached to a solid support is detected. Alternatively, antibodies can detect the presence of double or triple helix that specifically recognize these forms. Methods for performing assays using such oligomers as probes are generally known.

The use of the oligomers with alternative linkages of the invention as diagnostic agents with the formation of the triple helix is more advantageous because triple helices are formed under mild conditions and can thus be performed without exposing the test specimens to too harsh conditions. Diagnostic tests based on RNA detection to identify bacterial, fungal, or porcine sequences often require isolation of RNA from laboratory-grown samples or organisms, which is difficult and time consuming because RNA is extremely sensitive to ubiquitous nucleases.

Oligomer probes may also include additional modifications, such as modified sugars and / or substitute bonds, that make the oligomer particularly nuclease stable and would thus be useful for tests performed in the presence of cellular or tissue extracts that normally contain nuclease

-69activity. Oligomers that have terminal modifications often retain their ability to bind to complementary sequences without losing specificity (Uhlmann et al., Chemical Reviews, 1990, 90, 543584). As explained above, the assays of the present invention may also contain linkers that allow specific binding on the alternating DNA strand by including a linker allowing such binding (Froehler, B.C. et al., Biochemistry, 1992, 31, 1603-1609); Horne et al., J Am Chem Soc., 1990, 112, 2435-2437).

The incorporation of the base analogs of the present invention into samples that also contain covalent crosslinking agents has the potential to increase sensitivity and reduce background in diagnostic or detection tests. Additionally, the use of cross-linking agents will permit new test modifications, such as (1) use of cross-linking to increase sample differentiation, (2) incorporate denaturing wash rate, reduce background, and (3) perform hybridization and cross-linking at or near the melting point of the hybrid to reduce the secondary structure in the target and increase the specificity of the sample. Modifications of hybridization conditions have already been described (Gamper et al., Nucleic Acids Res., 1986, 14, 9943).

The oligomers of the invention are suitable for use in diagnostic tests using methods in which either the oligomer or the nucleic acid to be detected is covalently attached to a solid support as described (U.S. Patent No. 4,775,619). The oligomers of the invention are also suitable for use in diagnostic tests based on polymerase chain reaction techniques for amplifying target sequences by the methods described (European Patent Publication No. 0 393 744). The oligomers of the invention having a 3 'end that can serve as an example are compatible with the polymerases used in polymerase chain reaction methods such as Tag or

-70Vent ™ (New England Biolabs) polymerase. The oligomers of the invention can thus be used as primers in PCR protocols.

The oligomers of the invention are useful as primers that are discrete sequences or as primers with random sequences. The primers with random sequences can generally be about 6, 7 or 8 nucleomers long. Such primers can be used in various nucleic acid amplification protocols (PCR, ligase chain reaction, etc.) or in cloning protocols. The substitute bonds of the invention generally do not interfere with the oligomer's ability to act as an example. The oligomers of the invention having 2'-modifications at sites other than the 3 'terminal residue, and other modifications that make the RNase H oligomer incompetent or otherwise nuclease stable, may preferably be used as probes or primers for RNA or DNA sequences in cellular extracts or other solutions containing nuclease. Thus, oligomers can be used in nucleic acid amplification protocols in a sample by mixing the oligomer with a sample containing the target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplification of the target nucleic acid by PCR, LCR or other suitable methods.

Oligomers derivatized into chelators such as EDTA, DTPA or acetic acid 1,2diaminocyclohexane analogues can be used in various in vitro diagnostic tests (U.S. Patent Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, the oligomers of the invention may be derivatized by cross-linking agents such as 5- (3-iodoacetamidoprop-1-yl) 2'-deoxyuridine or 5- (3- (4bromobutyramido) prop-1-yl) -2'-deoxyuridine and used in different test methods or kits, as described (International Publication No. WO 90/14353).

-71Additionally, for the aforementioned uses, the ability of oligomers to inhibit gene expression can be verified by appropriate method in in vitro systems, by measuring expression levels in subject cells or in recombinant systems (Graessmann, M et al., Nucleic Acids Res., 1991 , 19, 53-59).

The following examples are given for a better explanation of the invention described above. Examples are given to illustrate the invention and should not be construed as limiting the invention.

EXAMPLES

Synthesis of nucleomonomic slnton synthesis and oligomers:

The oligomers of the invention can be synthesized using reactions known in the art for the synthesis of oligonucleotide derivatives. Glei e.g. .. Fiandor, J. and Yam, S.Y., Tetrahedron Letts., 1990, 31, 597-600; Mattson, R.J. et al., J Org Chem., 1990, 55, 2552-2554; Chung, C.K. et al., J Org Chem., 1989, 54, 2767-2769.

As can be seen from the variety of substituent bonds, especially those listed in Table 1, the substituent bonds of the invention can be varied by having one or more nitrogen, sulfur and / or oxygen atoms in their structure. The positions of these atoms in the alternate bonds can vary from the "5" 'end, to the' middle ', to the' 2 '' or '3' 'and' 4 '' ends. This section shows a series of representative synthesis reaction images that provide pathways for different locations and combinations of nitrogen and oxygen atoms within the substituent bonds.

-72The syntheses illustrated in Figures 1-25 may be modified as is known to those skilled in the art of oligonucleotide chemistry. For example, although base protection is not always exemplified in synthesis images, such protection may be desirable and may be performed using reagents and techniques known in the art. See, e.g., Protective Groups and Organic Synthesis (Theodora W. Greene, John Wiley and Sons, 1981). Similarly, although the use of protecting groups is demonstrated, in some cases it is not always necessary to block the reactants in order to synthesize the illustrative oligomers of the invention.

Example 1

The first five steps shown in Figure 1 relate to the preparation of isobutyl protected amino acid alcohol serinol. The sixth and next steps in Figure 1 are directed to the synthesis of serinol-substituted thymine phosphoramidite building block.

In step 1 of Figure 1, the amino group of the amino acid serine protected by reaction of 1 with diethyl tert-butyl dicarbonate to obtain compound 2. Other equivalent protecting groups may also be used. In the next step, the β-hydroxyl group of compound 2 is blocked with dihydropyran to form a fully protected amino acid 3. Amino acid 3 then reacts with the diboran-dimethyl sulfide complex to form alcohol 4, which, when exposed to isobutyl chloride, yields 5. This reduction reaction it can also be performed using isobutyl chloroformate and sodium borohydride (see: K. Ramasamy, R. K. Olsen, and T. Emery, Synthesis, 1982, 42). Reaction 5 with trifluoroacetic acid for 30 minutes followed by washing with NaHCO ₃ gave 6.

-73Time acetic acid 7 was prepared as described in the literature (see: L. Kosynkina, W. Wang, and T. C. Liang, Tetrahedron Letts., 1994, 35, 5173). Coupling 7 to 6 under mixed anhydride condition provided 8. Dimethoxytritylation 8 with DMTCI gave compound 9 which after hydrolysis with the base gave 10. Phosphylation 10 provided the formation of a serinol-bound thymine building block 11 under standard condition. can be added to the growing oligomer using conventional chemistry. Any DNA synthesis chemistry, such as phosphoramidate or phosphonate chemistry, can be used to bind monomers or dimers in a manner analogous to that shown above.

Example 2

In reaction Figure 2, thymine acetaldehyde 13 was produced by treating thymine with bromoacetaldehyde dimethylactal, followed by hydrolysis of compound 12 with aqueous TFA. Aldehyde 13 and amine 6 were then combined and the corresponding intermediate was converted to phosphoramidite, building block 17, in an analogous manner as in the rates used in Figure 1.

Example 3

In reaction Figure 3, the starting material is a β-substituted amino acid 18. Substituted amino acid can be transformed into phosphoramidite by building a block in the steps used in Figures 1 and 2

27.

Example 4

-74In Figure 4, starting material is amino alcohol 21 oxidized with CrOs / pyridine mixture to produce aldehyde 28. The aldehyde, in reaction with alkyl halide, in the presence of base, should give compound 29. Amino alcohol 29 can then be transformed to building block 35 in an analogous manner as in the stages of Figures 1 and 2.

Example 5

Turning to Figure 5, the first four stages are essentially the same as those used in Figure 1, and aspartic acid is used instead of serine. Aspartic acid methyl ester 36 gave a fully protected alcohol 40 which, on selective deprotection of acetic acid, provided 41. Oxidation 41 with a CrO ^ pyridine mixture gave the corresponding aldehyde 42. Reductive amination of aldehyde 42 with o-benzylhydroxyl amine in the presence of sodium triacetoxide, triacetoxide sodium had to give 43 (see: T. Kolasa and MJ Miller, J. Org. Chem. 1990, 55, 1711). Alcohol 39 is then converted to aldehyde 46, using essentially the same reaction conditions as above, but with an allyl protecting group for the hydroxyl group of compound 39. Coupling of aldehyde 46 and hydroxylamine 43, in the presence of sodium triacetoxyborohydride, followed by amino removal of protecting groups should allow for the formation of bisamine 48. By following the steps used in Figure 1, the bisamine 48 could then be converted to dimer 53.

Example 6

In Figure 6, the coupling of alcohol 54 with O-benzylhydroxylamine 55, sub

Mitsunobu reaction conditions (see: O. Mitsunobu, Synthesis, 1981,

-751) provides compound 56. Intermediate 56, after hydrogenation followed by acetylation, should give 57. Exposure 57 to TFA unblocks the “TBDMSi” protecting group and gives 58. Coupling 58 to 7, followed by dimethoxytritylation, could provide 60. The final building block 62, should be made of 60 after hydrolysis of the base followed by phospho- fylation.

Example 7

In Figure 7, serinol 4 is converted to halide 59 and alkylated with thymine to form 63. The protecting groups in 63 are removed, bound to DMT protected hydroxyacetic acid and phosphitylated to form 66.

Example 8

In Figure 8, alcohol 64 is coupled to N-hydroxylaminopropanoic acid 69 to form 70. Alkylation of thymine with halide 73 yields 74 which, after removal of the protection and coupling with 76, followed by hydrolysis, can give 78. Condensation 78 with 70, which followed by phosphitillation, should yield a hydroxamate dimer 80.

Example 9

In Figure 9, the N-hydroxylamino propanoic aldehyde 81 is used to bind alcohol 64. The dimer 88 is prepared from 83 and 86 by performing the steps used in Figure 8.

Example 10

-76In Figure 10, alkylation (see T. T. Kolasa and M. J. Miller, J. Org. Chem., 1990, 55, 4246) of? -Bromo-p-aminopropanoic acid methyl ester 89 with thymine would give 90. Intermediate 90 after hydrolysis with sodium hydroxides give acid 91, which is linked to 6 to provide 92. Compound 92 is then converted to phosphoramidite, building block 95 using the steps described in Figure 1.

Example 11

In Figure 11, thymine is alkylated with alkylamine halide 96 (see: R. K. Olsen,

K. Ramasamy and T. Emery, J. Org. Chem., 1984, 49, 3527 and Islam et al., J. Med. Chem., 1994, 37, 293-304 for the preparation of aminoalkyl halide) to give 97. Exposure of compound 97 to TFA followed by alkylation would give 100. Building block 103 is obtained from 100 by dimethoxytritylation and hydrolysis followed by phosphitylation .

Example 12

12 is an alternative route to dimer 111 having a hydroxamate backbone and is made of N-hydroxylamine 43 and aldehyde 107, the latter of which is prepared from aspartic acid.

Example 13

In Figure 13, dimer 115 is prepared from intermediates 108 and 13, following the same reaction rates as described in Figure 2.

Example 14

-77In Figure 14, N-hydroxylthymine is prepared (see: Kim, CU, et al., Tetrahedron Letts., 1992, 33, 25-28) and linked to N-hydroxyphthalimide to form 117 which, upon exposure to hydrazine and ethanol had to give 118. Treatment with DMT-protected glycerol epoxide 119, provides 120. Intermediate 120 was then converted to phosphoramidite 121. using a standard procedure. In the second synthesis, compound 118, under reductive amination conditions, is linked to amino acid aldehyde 122 to form 123. Protection of the secondary amino group by FMOCCI followed by hydrolysis should give 125.

Example 15

In Figure 15, 1,2-dihydroxypropanoic acid 126 is linked to Nhydroxylamine thymine 118 to form 127, which is then transformed into phosphoramidite synth 129. Under standard conditions, Compound 118 is also linked to adipic acid and transformed to nucleic acid building block 133.

Example 16

In Figure 16, the first building block 136 is synthesized from 118 and 134 in a similar manner to that described in Figure 1. Linking 139 to 118 provided 140. Processing 137 to 118 should provide 138 which, after condensing with 140, gives a dimer 141.

Example 17

-78In Figure 17, the aldehyde 142 and glycine benzyl ester are linked to form 143. Treatment 143 with 7 should provide 145 which, after exposure to acetic acid, gives 148. Mitsunobu alkylation of compound 148 with Boc-NH-Oacetylhydroxy, amine should give 147 , and building block 150 may be obtained after hydrogenation of this compound. Similarly, coupling 143 to 13 and following the same reactions as above should give sinton

149.

Example 18

In Figure 18, reductive amination of aldehyde 142 and Boc-NH-Obenzylhydroxylamine gave 151. Hydrogenation 151 followed by alkylation 152 with glycolic acid 153 (BC Borer and DC Balogh, Tetrahedron Letts. 1991, 32, 1039) should yield 154. Treatment with TFA will remove the Boc protecting group, which, after coupling, will result in 155. The hydroxyl protecting group of compound 155 could be selectively removed with acetic acid to form 156. Compound 156 will then be converted to building block 157 using standard reaction conditions. Similarly, building block 158 will be made by connecting 154 with 13 and tracking the reaction rates used to prepare 157.

Example 19

In Figure 19, alkylation of thymine-N-hydroxylamine 160 with alcohol 162 will give 163. Compound 163 can be transformed into phosphoramidite, building block 166, by following the steps used in Figure 1.

Example 20

-79 In Figure 20, the first intermediate 169 is synthesized from glutamic acid using standard reaction conditions. Alkylation of thymine with 169 would give 170, which after treatment with TFA should give 171. Intermediate 171 could be linked to Boc-glycine to provide 173 which, upon hydrolysis, could give monomeric sintone 174. Similarly, intermediate 172 could be prepared by coupling 118 and Boc-aminoacetic aldehyde, followed by hydrolysis of benzyl ester.

Example 21

In Figure 21, intermediate 177 is prepared from Boc-NH-O-benzylhydroxylamine and 175 using standard reaction conditions. Hydrogenation of compound 177, followed by coupling with N-hydroxytymine 116 would give 178. Removal of the THP protecting group followed by dimethoxytrityl and phosphitillation should give a synthon block 181. Similarly, 182 could be prepared by following all the above reactions. and using THP-hydroxyacetic aldehyde instead of THP-hydroxyacetic acid.

Example 22

In Figure 22, building block 191 may be prepared using known starting material 183 and following the reaction conditions presented in the lower portion of Figure 22.

Example 23

-80In Figure 23, synthesis of building block 199 can be performed using starting material 183 and following the reaction conditions presented in the lower portion of Figure 23.

Example 24

In Figure 24, the starting material 200 is transformed to the building block 207, following the reaction conditions presented in the lower part of Figure 24.

Example 25

The compounds used and made in this example are shown in Figure 1.

Serine (1): Thymine (37.8 g, 300 mmol) was dissolved in a solution of potassium hydroxide (64.5 g, 1150 mmol) in 200 ml of water. While this solution was heated in a water bath at 40 ° C, a solution of bromoacetic acid (62.5 g, 450 mmol) in 100 ml of water was added over a period of more than 1 hour. At this temperature, the reaction mixture was stirred for 1 h. It was then allowed to cool to room temperature and adjusted to pH 5.5 with concentrated HCl. The solution was then refrigerated in the refrigerator for 2 h. All precipitate (unreacted thymine) was removed by filtration. The solution was then adjusted to pH 2 with concentrated HCl and placed in the freezer for 2 h. The white precipitate was collected by filtration and dried in a vacuum oven at 40 ° C for 6 h. We got 44g (88%).

N-Boc-L-serine methyl ester (2): L-Serine methyl ester (15.6 g, 100 mmol) was suspended at THF / DMF (100 ml each) at room temperature.

-81mixers. Triethylamine (11.13 g, 110 mmol) was added to this stirred mixture, followed by the addition of di-tert-butyl bicarbonate (24.0 g, 110 mmol) and stirring was continued at room temperature for a further 30 minutes. Water (20 ml) was added and the solution was stirred at room temperature for 8 h. The solution was evaporated to dryness. The residue was suspended in ethyl acetate (250 ml) and treated with potassium hydrogen sulfate (0.25 N solution, 100 ml). The product was immediately extracted with ethyl acetate solution. The organic extract was washed with water (100 ml), brine (100 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic solvent provided 26g (90%) of the oily residue.

N-Boc-L-serine (OTHP) methyl ester (3): Compound 2 (15 g, 68.49 mmol) was dissolved in dry CH ₂ Cl ₂ (100 ml) and treated with 3,4dihydro-2H-pyran at room temperature. (8.4 g, 100 mmol) and a catalytic amount of p-toluene sulfonic acid (100 mg). The reaction mixture was allowed to stir at room temperature for 12 h and evaporated to dryness. The residue was dissolved in ethyl acetate (200 ml), washed with 5% NaHCO ₃ solution (100 ml), water (50 ml) and brine (50 ml). The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The residue was clean enough for the next level and used as such. We gained 15g (72%).

N-Boc-L-serinol (OTHP) (4): Serine (OTHP) methyl ester (10 g, 33 mmol) was dissolved in dry THF (100 ml) and cooled to 0 ° C in an argon ice bath. To this cold stirred solution, a borane-methyl sulfide complex (2 M solution in THF, 100 ml, 200 mmol) was added over a period of 1 h at 0 ° C. After the addition of borane, the reaction mixture was warmed to room temperature and heated at 40 ° C for 6 h. The reaction mixture was cooled to 0 ° C, neutralized with water and acetic acid to pH 6-7 and extracted with ether (3x100 ml). The ether extract was washed with

-82 water (2x100 ml) and brine (100 ml), dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as an oil. Purification by flash column silica gel using hexane -> acetone mixture as eluent gave 8g (88%) of the pure product.

N-Boc-L-serine (OTHP) Olb (5): TEA (3.54 g, 35 mmol) was added to a stirred solution of compound 4 (8 g, 29.09 mmol) in dry CH ₂ Cl ₂ (100 ml) at 0 ° C. ) and isobutyl chloride (3.71 g, 35 mmol) was added over a 30 minute time interval. The reaction mixture was then stirred at room temperature for 4 hours and evaporated to dryness. The residue was dissolved in EtOAc (200 ml), washed with 5% NaHCO ₃ solution (50 ml), water (50 ml) and brine (50 ml). The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as an oil. After purification with a silica gel flash column using hexane -> acetone mixture as eluent, 7.9g (79%) of the pure product was obtained from the oil.

L-Serinol (Olb) (6): Dissolve compound § (10 g, 28.98 mmol) in CH ₂ OI ₂ (100 ml) and then stirred at room temperature for 1 h with TFA (50 ml), and evaporated to dryness. The residue was dissolved in methanol (50 ml) and evaporated again. The residue was dissolved in CH ₂ Cl ₂ (200 ml), washed with saturated NaHCO ₃ solution (2x200 ml), water (100 ml) and brine (50 ml). The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give 4.5g (96%) of the product as an oil.

N- {Tfminylacetyl) -L-serinol (Olb) (8): Thymine acetic acid 7 (7.3 g, 40 mmol) and N-methylmorpholine (4.4 ml, 40 mmol) were dissolved in 100 ml DMF.

The solution was allowed to cool to -20 ° C in an argon atmosphere. Isobutyl chloroformate (5.2 ml, 40 ml) was completely added to this cold stirred solution

-83mmol). After 15 minutes, a solution of 6 (6.44 g, 40 mmol) in 30 ml of DMF (cooled to the same temperature) was added. The reaction mixture was stirred at -20 ° C for 30 minutes, warmed to room temperature, and stirred for 1 h. The reaction mixture was evaporated to dryness and the residue was dissolved in CH ₂ Cl ₂ (200 ml). The organic solution was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as a foam. The crude product was purified by flash column of silica gel using CH ₂ Cl ₂ -> acetone as eluent to give 12 g (92%) of the pure product.

4,4'-Dimethoxytrityl-N- (thyminylacetyl) -L-serinol (Olb) (9): Compound 8 (10 g, 30.58 mmol) was evaporated together with dry pyridine (3x50 ml) and dissolved in dry pyridine (100 ml ). TEA (3.54 g, 35 mmol) was added to this solution, followed by addition of DMTCI (11.83 g, 35 mmol) at room temperature under argon. The reaction mixture was stirred for 12 h, quenched with methanol (20 ml) and stirred for a further 30 minutes. The solution was evaporated to dryness and dissolved in CH ₂ Cl ₂ (200 ml). The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as a foam. The crude product was purified by flash silica gel column using CH ₂ Cl ₂ -> acetone as eluent to give 17 g (88%) of pure product.

1-0- (4,4'-Dimethoxytrityl) -2- [amino (thyminylacetyl)] - L-propane-1,3-diol (10):

Compound 9 (10 g, 15.89 mmol) was dissolved in methanol (20 ml). To this solution, a 1N NaOH solution (20 ml, 20 mmol) was added at 0 ° C. The reaction mixture was stirred for 1 h and quenched with acetic acid to pH 7. The solution was extracted with EtOAc (2x100 ml). Organic extract

-84 We were washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The EtOAc layer was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give a crude foam product. The crude product was purified by flash silica gel column using CH ₂ Cl ₂ -> acetone as eluent to give 8.2 g (92%) of the pure product.

1'-O- (4,4'-Dimethoxytrityl) -2- [amino (thyminylacetyl) -L-propane-3'-O- (N, Ndiisopropyl) -p-cyanoethylphosphoramidite (11): Compound 10 (8.00 g. 14.31 mmol) was evaporated along with dry pyridine (3x50 ml) and dried under vacuum over solid NaOH overnight. The dried material was dissolved in dry CH ₂ Cl ₂ (100 ml) and cooled to 0 ° C under argon. To this cold solution was added N, N-diisopropylethylamine (5.23 g, 25 mmol), followed by addition of 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (4.72 g, 20.00 mmol) under argon. The reaction mixture was stirred at 0 ° C for 1 h and at room temperature for 1 h. The reaction mixture was diluted with CH ₂ Cl ₂ (100 ml). The CH ₂ Cl ₂ solution was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ Cl ₂ layer was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as a foam. The crude product was purified by flash silica gel column using CH ₂ Cl ₂ -> acetone containing 0.1% TEA as eluent to give 10 g (x%) of pure product. The foam was vacuum dried over solid NaOH overnight. The foam was dissolved in CH ₂ Cl ₂ (15 ml) and was dropped into a stirred solution of dry hexane (2000 ml) under argon for 1 h. After the addition of the CH ₂ Cl ₂ solution, the resulting precipitate was stirred for another 1 hour and filtered, washed with dry hexane (200 ml) and dried overnight over solid NaOH. Yield: 9.5g (87%).

Example 26 (See Figure 26)

-85N- (Tert-butyloxycarbonyl) -O-benzyl-L-serine (2): O-Benzyl-L-serine 1 (10 g, 51.28 mmol) was suspended at room temperature in THF / H ₂ O (8: 2 , 100 ml) to the mixture. Triethylamine (6.06 g, 60 mmol) was added to this stirred mixture, followed by the addition of di-tert-butyl dicarbonate (13.08 g, 60 mmol) and stirring was continued at room temperature overnight. The homogeneous solution was evaporated to dryness and the residue was dissolved in ethyl acetate (300 ml). The organic extract was washed with 0.5N potassium hydrogen sulfate solution (100 ml), water (100 ml) and brine (50 ml). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated to dryness to give 14 g (93%) of an oily residue.

N-CTert-butyloxycarbonyl-O-benzyl-L-serinol (3): N- (Tert-butyloxycarbonyl) O-benzyl-L-serine 2 (6.0 g, 20.34 mmol) was dissolved in dry THF and cooled in argon atmosphere. at -20 ° C. To this cold stirred solution was added TEA (2.32g, 23 mmol) and isobutyl chloroformate (3.13 g, 23 mmol). Stirring was continued at -20 ° C for 30 minutes in an argon atmosphere. The reaction mixture was then filtered immediately under an argon blanket and the precipitate was washed with dry THF (50 ml). The combined filtrate was slowly added to a cold (0 ° C) solution of NaBH ₄ (7.4 g, 200 mmol) in THF / water (80:20, 200 ml) over a 10 minute time interval. After this addition, the reaction mixture was stirred for 2 h at 0 ° C and the pH adjusted to 7 with acetic acid. The solution was evaporated to dryness and partitioned between ethyl acetate / water (300: 150 ml) and extracted into ethyl acetate. The organic extract was washed with brine (100 ml), dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure product was combined and evaporated to dryness to give 4.7 g (82%) of the pure product as an oil. ¹ HNMR (CDCI ₃ ): δ

-861.41 (s, 9H, Boc), 3.60 - 3.70 (m, 4H), 3.82 (d, 2H), 4.53 (s, 2H, OCH ₂ Ph), 5.20 (bs, 1H, NH), and 7.30 - 7.40 ( m, 5H, Ph).

N-tert-butyloxycarbonyl-O-benzyl-L-serinol-O-1b (4): To a dried solution of N- (tert-butyloxycarbonyl) -O-benzyl-L-serinol 3 (4.3 g, 14.3 mmol) in dry pyridine (50 ml) was added TEA (2.02 g, 20 mmol) at room temperature. Isobutyric anhydride (3.16 g, 20 mmol) was added to this stirred solution and stirring was continued under argon atmosphere overnight. The reaction mixture was evaporated to dryness, the residue was partitioned between EtOAc (100 ml) and NaHCO ₃ (5% solution, 100 ml) and extracted into EtOAc. The organic extract was washed with water (100 ml), brine (50 ml) and dried over anhydrous Na ₂ SO ₄ . The dried solution was evaporated to dryness to give a crude residue. The residue was purified by flash chromatography over silica gel using hexane-> EtOAc as eluent. The pure fractions were combined and evaporated to give an oily product of 4.5 g (84%). ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.39 (s, 9H, Boc), 2.46 (m, 1H, IbCH), 3.40 (m, 2H), 3.92 (m, 2H). 4.12 (m, 1H), 4.46 (s, 2H, OCH ₂ Ph), 6.84 (d, 1H, NH) and 7.24 - 7.40 (m, 5H, Ph).

N- (Thinylacetyl) -O-benzyl-L-serinol-O-1b (6): N- (Tert-butyloxycarbonyl-Obenzyl-L-serinol-O-1b 4 (4.3 g, 12.25 mmol) was kept at room temperature 30 minutes was allowed to stir in trifluoroacetic acid (20 ml) and CH ₂ Cl ₂ (20 ml). the reaction mixture was evaporated to dryness, dissolved in dry CH ₃ OH (10 ml) and again evaporated to dryness. the residue was under vacuum for 12 h dried The solid residue was used as such for further reaction without characterization.

Thymine acetic acid 5 (2.76 g, 15 mmol) was dissolved in dry DMF (75 ml) and cooled to -20 ° C in argon. We are in to this cold stirred solution

-87 N-methylmorpholine (1.72 g, 17 mmol) was added followed by the addition of isobutyl chloroformate (2.31 g, 17 mmol). After stirring for 15 minutes, the solution of the above TFA salt in dry DMF (50 ml) was neutralized with Nmethylmorpholine (1.72 g, 17 mmol) and added at once to a cold stirred solution of acetic acid thymine. The reaction mixture was stirred at 20 ° C for 1h, warmed to room temperature and stirred overnight. The solution was evaporated to dryness and the residue was dissolved in CH ₂ Cl ₂ (250 ml) and water (100 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ Cl ₂ extract was dried and evaporated to dryness to give the crude product. The crude product was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> acetone as eluent. The necessary fractions were collected and evaporated to give 4.8 g (94%) of pure product. The pure product was crystallized from CH ₂ Cl ₂ / hexane mixture. Melting point: 122-124 ° C. ¹ HNMR (CDCI ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.44 (m, 1H, IbCH), 3.42 (m, 2H), 4.06 (m, 2H) , 4.18 (m, 1H), 4.30 (s, 2H), 4.46 (s, 2H, OCH ₂ Ph), 7.24 -7.40 (m, 6H, C ₆ H and Ph), 8.22 (d, 1H, NH) and 11.22 (s, 1H, NH).

N- (Thinylacetyl) -L-serinol-O-1b (7): N- (Thinylacetyl) -O-benzyl-L-serinol-O-1b 6 (2.08 g, 5 mmol) was dissolved in ethanol (50 ml). . To this solution, Pd (OH) ₂ (0.6 g) and cyclohexane (5 ml) were added at room temperature. The reaction mixture was heated at 70 ° C for 12 h. The catalyst was filtered off and washed with methanol (20 ml). The filtrate was evaporated to dryness to give a white solid. The white solid was dissolved in a minimal amount of MeOH and cooled to room temperature. The product crystallized as a fine powder. Melting point: 196-198 ° C. Yield: 1.48 g (91%). ¹ HNMR (Me ₂ SO-d ₆ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.42 (m, 1H, IbCH), 3.40 (m, 2H), 3.94 ( m,

-882Η), 4.06 (m, 1H), 4.28 (s, 2H), 4.90 (t, 1H, OH), 7.20 (s, 1H, C ₆ H), 8.12 (d, 1H, NH) and 11.22 (s , 1H, NH).

N, N-Dimethoxytrityl-N-trimethylacetyl-0-L-serinol-O-1b (8): N- (Thyminylacetyl) -Lserinol-O-1b 7 (1.48 g, 4.5 mmol) was dissolved in dry pyridine (50 ml) in the atmosphere argon. TEA (0.51 g, 5 mmol) and N, N-dimethylamino pyridine (0.10 g) were added to this stirred solution. After 10 min, 4,4'-dimethoxytrityl chloride (1.69 g, 5 mmol) was added and stirring was continued at room temperature under argon overnight. The reaction mixture was quenched with MeOH (10 ml), stirred for 10 min and evaporated to dryness. The residue was dissolved in EtOAc (200 ml), washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure fractions were collected and evaporated to give 2.5 g (88%) of foam. ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.40 (m, 1H, IbCH), 3.38 (m, 2H), 3.72 (s, 6H. 2.0CH ₃ ), 4.12 (m, 2H), 4.20 (m, 1H), 4.32 (d, 2H), 6.84 (m, 4H, Ph), 7.20 - 7.40 (m, 12 H, C ₆ H and Ph) , 8.30 (d, 1H, NH) and 11.28 (s, 1H, NH).

-0- (4,4'-Dimethoxytrityl) -2- [amino (thymnylacetyl)] - L-propane-1,3-diol (9):

4,4'-Dimethoxytrityl-N- (thyminylacetyl) -L-serinol-O-1b 8 (3.4 g, 5.41 mmol) was dissolved in MeOH (30 ml) and cooled to 0 ° C in an ice bath. To this cold stirred solution was added 2N NaOH (10 ml, 20 mmol) and stirring continued at 0 ° C for 30 min. The solution was adjusted to pH 7 with acetic acid and evaporated to dryness. The residue was partitioned between water (50 ml) and CH ₂ Cl ₂ (150 ml) and extracted into CH ₂ Cl ₂ . The aqueous layer was re-extracted with CH ₂ Cl ₂ (50 ml). The combined organic extract was washed with brine

-89 water (50 ml), dried and evaporated to dryness. The residue was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> acetone as eluent. Yield: 3.0 g (99%). ¹ HNMR (CDCl ₃ ): 1.72 (s, 3H, CH ₃ ), 3.0 (m, 2H), 3.42 (m, 2H), 3.72 (s, 6H, 2.0CH ₃ ), 3.94 (m, 1H), 4.32 (d, 2H), 4.68 (m, 1H, OH), 6.84 (m, 4H, Ph), 7.20 - 7.40 (m, 12H, C ₆ H and Ph), 8.06 (d, 1H, NH) and 11.28 ( bs, 1H, NH).

1- O- (4,4'-Dimethoxytrityl) -2- [amino (tminylacetyl)] - L-propane-3-O- (N, Ndiisopropyl) -p-cyanoethylphosphoramidite (10): 1- 0- (4, 4'-Dimethoxytrityl) -2 [amino (thyminylacetyl)] - L-propane-1,3-diol 9 (3.1 g, 5.55 mmol) was dried in vacuo overnight over solid NaOH and dissolved in dry CH ₂ Cl ₂ (100 ml). The solution was cooled to 0 ° C in an argon atmosphere. To this cold stirred solution was added N, N-diisopropylethylamine (1.29 g, 10 mmol) and then

2- cyanoethyl-N, N-diisopropyl-chlorophosphoramidite (1.96 g, 8.3 mmol). The reaction mixture was stirred at 0 ° C for 1 h and at room temperature for 2 h. The reaction was diluted with CH ₂ CH ₂ (100 ml) and the organic layer was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ CI ₂ extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ ~> EtOAc mixture with 0.1% TEA as eluent. The pure fractions were collected together and evaporated to give a foam. The foam was dried in vacuo overnight over solid NaOH. The dried foam was dissolved in dry CH ₂ Cl ₂ (20 ml) and added to a stirred solution of dry hexane (2000 ml) under an argon atmosphere for 1 h. After the addition, the resulting precipitate was stirred for a further 1 h and filtered, washed with dry hexane (100 ml) and the solid in vacuo dried for 4 h over solid NaOH. Yield: 3.5 g (83%).

Example 27

-90 (See Figure 27)

N- (Tert-butyloxycarbonyl) -O-benzyl-D-serine (12): O-Benzyl-D-serine U (5 g, 25.64 mmol) was suspended at THF / H ₂ O (8: 2, at room temperature). 70 ml) to the mixture. To this stirred mixture was added triethylamine (4.04 g, 40 mmol) followed by di-tert-butyl dicarbonate (6.54 g, 30 mmol) and stirring was continued overnight at room temperature. The homogeneous solution was evaporated to dryness and the residue was dissolved in ethyl acetate (150 ml). The organic extract was washed with 0.5N potassium hydrogen sulfate solution (100 ml), water (100 ml) and brine (50 ml). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated to dryness to give 7.56 g (100%) of an oily residue.

N- (Tert-Butyloxycarbonyl) - O-benzyl-D-serinol (13): N- (Tert-Butyloxycarbonyl) -O-benzyl-D-serine 10 (7.56 g, 25.63 mmol) was dissolved in dry THF and argon atmosphere. cooled to -20 ° C. To this cold stirred solution was added TEA (3.03 g, 30 mmol) and isobutyl chloroformate (4.08 g, 30 mmol). At -20 ° C, stirring was continued for 30 min in an argon atmosphere. The reaction mixture was immediately filtered under an argon blanket, and the precipitate was washed with dry THF (50 ml). The combined filtrate was slowly added to a cold (0 ° C) solution of NaBH ₄ (7.4 g, 200 mmol) in THF / water (80:20, 200 ml) over a 10 min interval. After the addition, the reaction mixture was stirred at 0 ° C for 2 h and the pH adjusted to 7 with acetic acid. The solution was evaporated to dryness, partitioned between ethyl acetate / water mixture (300: 150 ml) and extracted into ethyl acetate. The organic extract was washed with brine (100 ml), dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure product was collected together and evaporated to

-91 dry to give 6.68 g (92%) of the pure product as an oil. ¹ HNMR (CDCl ₃ ): δ 1.41 (s, 9H, Boc), 3.60 - 3.70 (m, 4H), 3.82 (d, 2H), 4.53 (s, 2H, OCH ₂ Ph), 5.20 (bs, 1H. NH) and 7.30 - 7.40 (m, 5H, Ph).

N- (Tert-butyloxycarbonyl) -O-benzyl-D-serinol-O-1b (14): To a dried solution of N- (tert-butyloxycarbonyl) -O-benzyl-D-serinol 13 (6.6 g, 23.5 mmol) in TEA (3.03 g, 30 mmol) was added at room temperature to dry pyridine (50 ml). Isobutyric anhydride (4.74 g, 30 mmol) was added to this stirred solution and stirring was continued overnight under argon. The reaction mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and NaHCO ₃ (5% solution, 100 ml) and extracted into EtOAc. The organic extract was washed with water (100 ml), brine (50 ml) and dried over anhydrous Na ₂ SO ₄ . The dried solution was evaporated to dryness to give a crude residue. The residue was purified by flash chromatography over silica gel using hexane -> EtOAc as eluent. The pure fractions were combined and evaporated to give an oily product of 8.0 g (97%). ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.39 (s, 9H, Boc), 2.46 (m, 1H, IbCH), 3.40 (m, 2H), 3.92 (m, 2H). 4.12 (m, 1H), 4.46 (s, 2H, OCH ₂ Ph), 6.84 (d, 1H, NH) and 7.24 - 7.40 (m, 5H, Ph).

N- (Thinylacetyl) -O-benzyl-D-serinol-O-1b (15): N- (Tert-butyloxycarbonyl) -Obenzyl-D-serinol-O-1b 14 (5.0 g, 14.25 mmol) was kept at room temperature. allowed to stir in trifluoro acetic acid (20 ml) and CH ₂ Cl ₂ (20 ml) for 30 min. The reaction mixture was evaporated to dryness, dissolved in dry CH ₃ OH (10 ml) and evaporated again to dryness. The residue was dissolved in CH ₂ Cl ₂ (150 ml), the pH was adjusted to 7 with 5% NaHCO ₃ solution and extracted into CH ₂ Cl ₂ . The organic layer was washed with water (50 ml) and brine (50 ml). The CH ₂ Cl ₂ extract was dried and evaporated to dryness. So

-92 The residue obtained was dried under solid KOH in vacuo for 12 h. The dried residue was used as such for further reaction without characterization.

Thymine acetic acid 5 (2.57 g, 14 mmol) was dissolved in dry DMF (50 ml) and cooled to -20 ° C in argon. To this cold stirred solution was added N-methylmorpholine (1.52 g, 15 mmol) followed by isobutyl chloroformate (2.04 g, 15 mmol). After stirring for 15 minutes, a solution of the above amine in dry DMF (50 ml) was added at once to a cold stirred solution of acetic acid thymine. The reaction mixture was stirred at -20 ° C for 1 h, warmed to room temperature and stirred overnight. The solution was evaporated to dryness and the residue was dissolved in CH ₂ Cl ₂ (250 ml) and water (100 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ Cl ₂ extract was dried and evaporated to dryness to give the crude product. The crude product was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> acetone mixture as eluent. The necessary fractions were collected and evaporated to give 2.8 g (54%) of pure product. ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.44 (m, 1H, IbCH), 3.42 (m, 2H), 4.06 (m, 2H) , 4.18 (m, 1H), 4.30 (s, 2H), 4.46 (s, 2H, OCH ₂ Ph), 7.24-7.40 (m, 6H, C ₆ H and Ph), 8.22 (d, 1H, NH) and 11.22 (s, 1H, NH).

The title compound was also prepared using the method described for the preparation of the L isomer. The reagents used are: thymine acetic acid (2.2 g, 12 mmol); isobutyl chloroformate (1.77 g, 13 mmol); N-methylmorpholine (1.52 g, 15 mmol); TFA salt (3.65 g, 10 mmol); N-methylmorpholine (1.5 g, 15 mmol) and dry DMF (100 ml). Yield: 3.5 g (84%).

-93N- (Thinylacetyl) -D-serinol-O-1b (16): N- (Thinylacetyl) -O-benzyl-D-serinol-O-1b 15 (3.5 g, 8.39 mmol) was dissolved in ethanol (50 ml ). To this solution was added Pd (OH) ₂ (1.00 g) and cyclohexane (10 ml) at room temperature. The reaction mixture was heated at 70 ° C for 12 h. The catalyst was filtered off and washed with methanol (20 ml). The filtrate was evaporated to dryness to give a white solid. Yield: 2.7 g (98%). ¹ HNMR (Me ₂ SO-d ₆ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.42 (m, 1H, IbCH), 3.40 (m, 2H), 3.94 ( m, 2H), 4.06 (m, 1H), 4.28 (s, 2H), 4.90 (t, 1H, OH), 7.20 (s, 1H, C ₆ H), 8.12 (d, 1H, NH) and 11.22 ( s, 1H, NH).

4,4'-Οίιτΐ6ΐοΙ (8ηι1ΙΗ-Ν- (ϋιηΙηΗ3θβϋΙ) -0-8θΐ1ηοΙ-0- ^ (17): N- (Thinylacetyl) -Dserinol-O-lb 16 (2.7 g, 8.26 mmol) was dissolved in an argon atmosphere in dry pyridine (50 ml) TEA (1.01 g, 10 mmol) was added to this stirred solution, followed by 4,4'-dimethoxytrityl chloride (3.38 g, 10 mmol) and continued at room temperature in argon overnight. The reaction mixture was quenched with MeOH (10 ml), stirred for 10 min and evaporated to dryness, the residue was dissolved in EtOAc (250 ml), washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml) The organic extract was dried over anhydrous Na ₂ SO ₄ and the residue was evaporated to dryness The crude product was purified by flash column chromatography using CH ₂ Cl ₂ -> EtOAc as eluent The pure fractions were collected and evaporated to give 5.0 g (96%) of foam. ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.40 (m, 1H, IbCH), 3.38 (m, 2H), 3.72 (s, 6H, 2.OCH3), 4. 12 (m, 2H), 4.20 (m, 1H), 4.32 (d, 2H), 6.84 (m, 4H, Ph),

7.20 - 7.40 (m, 12H, C ₆ H and Ph), 8.30 (d, 1H, NH) and 11.28 (s, 1H, NH).

-0- (4,4'-Dimethoxyphthyl) -2- [amino (thyminylacetyl)] - D-propane-1,3-diol (18):

4,4′-Dimethoxytrityl-N- (thyminylacetyl) -D-serinol-O-lb 17 (5.0 g, 7.95 mmol) was dissolved in MeOH (30 ml) and cooled to 0 ° C in an ice bath. To this cold

-94 To the stirred solution was added 2N NaOH (10 ml, 20 mmol) and stirred at 0 ° C for 30 min. The solution was adjusted to pH 7 with acetic acid and evaporated to dryness. The residue was partitioned between water (50 ml) and CH ₂ Cl ₂ (250 ml) and extracted into CH ₂ Cl ₂ . The aqueous layer was re-extracted with CH ₂ Cl ₂ (50 ml). The combined organic extract was washed with brine (50 ml), dried and evaporated to dryness. The residue was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> acetone mixture as eluent. Yield: 4.0 g (90%). ¹ HNMR (CDCl ₃ ): 1.72 (s, 3H, CH ₃ ), 3.0 (m, 2H), 3.42 (m, 2H), 3.72 (s, 6H, 2.0CH ₃ ), 3.94 (m, 1H), 4.32 (d, 2H), 4.68 (m, 1H, OH), 6.84 (m, 4H, Ph),

7.20 - 7.40 (m, 12H, C ₆ H and Ph), 8.06 (d, 1H, NH) and 11.28 (bs, 1H, NH).

1-0- (4,4'-Dimethoxytrityl) -2- [amino (thyminylacetyl)] - D-propane-3-O- (N, N-lisopropyl) -p-cyanoethylphosphoramidite (19): 1-O- (4, 4'-Dimethoxytrityl) -2 [amino (thyminylacetyl)] - D-propane-1,3-diol 18 (2.79 g, 5.0 mmol) was dried in vacuo overnight over solid NaOH and dissolved in dry CH ₂ Cl ₂ (100 ml). The solution was cooled to 0 ° C in an argon atmosphere. To this cold stirred solution was added N, N-diisopropylethylamine (1.29 g, 10 mmol) followed by 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (1.96 g, 8.3 mmol). The reaction mixture was stirred at 0 ° C for 1 h and at room temperature for 2 h. The reaction was diluted with CH ₂ Cl ₂ (100 ml) and the organic layer was washed with 5%

NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ CI ₂ extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc mixture with 0.1% TEA as eluent. The pure fractions were collected together and evaporated to give a foam. The foam was vacuum dried over solid NaOH overnight. The dried foam was dissolved in dry CH ₂ Cl ₂ (20 ml) and added to argon in a stirring solution for one hour in a dry solution of dry hexane (2000 ml). After the addition

-95 The resulting precipitate was stirred for a further 1 h and filtered, washed with dry hexane (100 ml) and the solid dried in vacuo for 4 h over solid NaOH. Yield: 3.3 g, (87%).

Example 28 (Figure 28)

1- O-Benzyl-2 - [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -Lpropanol (21): To a dry solution of N ₂ -benzoylthymine 20 (5.75 g, 25 mmol) in dry THF (200 mi) was stirred at room temperature under argon was added triphenyl phosphine (10:48 g, 40 mmol) and N _a -tert-butyloxycarbonyl-ss-benzyloxy-Lserinol 3 (5.3 g, 18.86 mmol). After 15 min, diethyl azodicarboxylate (6.96 g, 40 mmol) was added slowly over a 30 min interval. The reaction mixture was covered with aluminum foil and allowed to stir at room temperature in argon for 24 h. The solvent was evaporated to dryness and the residue was dissolved in EtOAc (300 ml). The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (100 ml) and dried over anhydrous Na ₂ SO ₄ . The dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography over silica gel using hexane -> EtOAc mixtures as eluent. The fraction with the desired product was collected and evaporated to give an oil a pale pink color. Yield: 8.0 g (86%). ¹ HNMR (CDCl ₃ ): 1.41 (s, 9H, Boc), 1.72 (s, 3H, CH ₃ ), 3.56 (m, 2H), 4.20 (m, 2H), 4.32 (m, 1H), 4.52 (d , 2H, OCH ₂ Ph), 5.20 (d, 1H, NH), 7.06 (s, 1H, C ₆ H), and 7.20 - 7.60 (m, 10H, Ph).

2- [(Τθπ; -5υϋΙοΚ8ίΚ3ΓόοηΙΙ) 3ηΊΐηο] -3- [Ν ₃ -56ηζοίΙ (ΝιηίηΙΙ) -1.-ρΓορ3η-1-οΙ (22):

1-O-Benzyl-2 - [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L-propanol 21 (4.93 g, 10 mmol) was dissolved in MeOH (100 ml) and treated with §. Pd / C

-96 (10%, 1 g). The reaction mixture was hydrogenated at 3.44 bar of hydrogen for 12 h. The catalyst was filtered, washed with MeOH (50 ml) and the filtrate evaporated to dryness. The residue was crystallized from acetone / hexane to give 3.70 g (92%) of pure product. Melting point: 156-159 ° C. ¹ HNMR (CDCl ₃ ): 1.42 (s, 9H, Boc), 1.94 (s, 3H, CH ₃ ), 3.64 (m, 4H), 3.84 (m, 1H), 4.14 (m, 1H), 5.22 (d , 1H, NH), 7.18 (s, 1H, C ₆ H), 7.48 (t, 2H, Ph), 7.62 (t, 1H, Ph) and 7.98 (d, 2H, Ph).

1-O-Isobutyryl-2 - [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L · propanol (23): 2 - [(Tert-butyloxycarbonyl) amino] -3- [N _3- Benzoyl (thymynyl) -Lpropan-1-ol 22 (1.60 g, 3.97 mmol) was dissolved in dry pyridine (30 ml) and the solution was allowed to stir in argon at room temperature. TEA (0.51 g, 5 mmol) and isobutyric anhydride (0.79 g, 5 mmol) were added to this stirred solution. The reaction mixture was stirred at room temperature for 12 h and evaporated to dryness. The residue was dissolved in EtOAc (150 ml) and washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The organic extract was dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure fractions were collected together and evaporated to give

1.6 g (85%) of the foam. The pure product was crystallized from acetone / hexane. Melting point: 165-167 ° C. ¹ HNMR (CDCl ₃ ): 1.16 (d, 6H, 1bCH ₃ ), 1.42 (s, 9H, Boc), 1.94 (s, 3H, CH ₃ ), 2.52 (m, 1H), 3.64 (m, 4H). 3.84 (m, 1H), 4.14 (m, 1H), 5.22 (d, 1H, NH), 7.18 (s, 1H, C ₆ H), 7.48 (t, 2H, Ph), 7.62 (t, 1H, Ph) ) and 7.98 (d, 2H, Ph).

1-0-Isobutyryl-2 - [(p-hydroxyacetyl) amino] -3- [N3-benzoyl (thyminyl) -L-propanol (24): 1-0-Isobutyryl-2 - [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -Lpropanol 23 (1.6 g, 3.38 mmol) was allowed to stir in a mixture of TFA (5 ml) and CH ₂ Cl ₂ (10 ml) at room temperature for 30 min and then evaporated to

-97dry. The residue was dissolved in dry MeOH (10 ml) and evaporated again. The resulting residue was dried in vacuo overnight over solid NaOH. The dried material was used as such for the next reaction.

To a stirred solution of glycolic acid (0.53 g, 7 mmol) in dry DMF (50 ml) was added 1-hydroxybenzotriazole (0.67 g, 5 mmol) and 1-ethyl-3- (3dimethylaminopropyl) -carbodiimide hydrochloride (EDC) (1.91 g , 10 mmol). After stirring for 15 min, TEA (1.01 g, 10 mmol) and the above TFA salt in DMF (20 ml) were added at room temperature. The reaction mixture was stirred for 12 h and evaporated to dryness. The residue was partitioned between CH ₂ Cl ₂ (150 ml) and water (100 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with brine (50 ml), dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> acetone mixture as eluent. The fractions with the desired product were collected and evaporated to give 1.35 g (92%) of foam.

¹ HNMR (CDCl ₃ ): 1.16 (d, 6H, 1bCH ₃ ), 1.94 (s, 3H, CH ₃ ), 2.52 (m, 1H),

3.20 (bs, 1H), 3.80 - 4.30 (m, 6H), 4.56 (m, 1H), 7.14 (d, 2H, C ₆ H and

NH), 7.50 (t, 2H, Ph), 7.64 (t, 1H, Ph) and 7.94 (d, 2H, Ph).

1-0- Isobutyryl-2 - [(p- (4,4'-dimethoxytrityl) -0-acetyl) amino] -3- [N ₃ benzoyl (thyminyl) -L-propanol (25): 1 -O- Isobutyryl-2 - [(p-hydroxyacetyl) amino] -3 [N ₃ -benzoyl (thyminyl) -L-propanol 24 (1.2 g, 2.78 mmol) was dissolved in dry pyridine (50 ml) and allowed to stir at room temperature. argon atmosphere. To this stirred solution was added TEA (0.35 g, 3.5 mmol) and 4,4'-dimethoxytrityl chloride (1.18 g, 3.5 mmol). The reaction mixture was stirred at room temperature for 12 h, quenched with MeOH (10 ml) and evaporated to dryness. The residue was dissolved in EtOAc (150 ml), washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The organic extract was dried over Na ₂ SO ₄ and evaporated to dryness. Remainder

The -98 was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were collected and evaporated to yield

1.7 g (83%) of pure product. ¹ HNMR (CDCI ₃ ): 1.16 (d, 6H, 1bCH ₃ ), 1.94 (s, 3H, CH ₃ ), 2.52 (m, 1H), 3.74 (s, 6H, 2.0CH ₃ ), 3.80 - 4.30 (m , 6H), 4.56 (m, 1H), 6.82 (d, 4H, Ph), 7.14 (d, 2H, C ₆ H and NH) and 7.26 - 8.00 (m, 14H, Ph).

2 - [(p- (4,4'-Dimethoxytrityl) -O-acetyl) amino] -3-timinyl-L-propanol (26): 1-Oxobutyryl-2 - [(p- (4,4'-dimethoxytrityl) ) -O-acetyl) amino] -3- [N ₃ -benzoyl (thyminyl) -propanol 25 (1.55 g, 2.05 mmol) was dissolved in MeOH (20 ml) and cooled to 0 ° C in an ice bath. To this cold stirred solution was added 2N NaOH (5 ml, 10 mmol) and stirring continued at 0 ° C for 30 min. The solution was adjusted to pH 7 with acetic acid and evaporated to dryness. The residue was partitioned between water (50 ml) and CH ₂ Cl ₂ (150 ml) and extracted into CH ₂ Cl ₂ . The aqueous layer was re-extracted with CH ₂ Cl ₂ (50 ml). The combined organic extract was washed with brine (50 ml), dried and evaporated to dryness. The residue was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ ~> acetone as eluent. Yield: 1.0 g (99%). ¹ HNMR (CDCl ₃ ): 1.94 (s, 3H, CH ₃ ), 3.74 (s, 6H, 2.OCH3), 3.80 - 4.30 (m, 6H), 4.56 (m, 1H), 6.82 (d, 4H. Ph), 7.14 (d, 2H, C ₆ H and NH) and 7.26 - 8.00 (m, 14H, Ph).

2 - [(β- (4,4'-0ΙιηβΙοΙ (8ηιΉΙΙ) -0-80βΙΗ) 3ΐηΙηο] -3-ϋπιΙηίΙ-Ι.-ρΐΌρ8η-1-Ο- (Ν, Νdiisopropyl) -p-cyanoethyl-phosphoramidite (27 ): 2 - [(p- (4,4'-Dimethoxytrityl) -Oacetyl) amino] -3-timinyl-L-propanol 26 (1.00 g, 2.09 mmol) was dried in vacuo overnight over solid NaOH and dissolved in dry CH ₂ Cl ₂ (50 ml) The solution was cooled to 0 DEG C. under argon, to which N, N-diisopropylethylamine (0.54 g, 4.2 mmol) was added to this cold stirred solution.

2-cyanoethyl-N, N-diisopropyl-chlorophosphoramidite (0.73 g, 3.1 mmol). Reactionary

-99 the mixture was stirred at O ° C for 1 h and at room temperature for 2 h. The reaction was diluted with CH ₂ Cl ₂ (100 ml) and the organic layer was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ CI ₂ extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc mixture with 0.1% TEA as eluent. The pure fractions were collected together and evaporated to give a foam. The foam was dried in vacuo overnight over solid NaOH. The dried foam was dissolved in dry CH ₂ Cl ₂ (10 ml) and dripped in argon, at a time interval of 30 min, into a stirred solution of dry hexane (800 ml). After the addition, the resulting precipitate was stirred for an additional 30 min and filtered, washed with dry hexane (100 ml) and the solid dried in vacuo over solid NaOH for 4 h. Yield: 1.3 g (82%).

Example 29 (Figure 29)

N ^a -tert-butyloxycarbonyl-O-benzylhydroxylamine (28): O-benzyl hydroxylamine hydrochloride (15.9 g, 100 mmol) was suspended in a mixture of THF (150 ml) and water (50 ml). To this stirred solution was added TEA (15.15 g, 150 mmol) followed by di-tert-butyldicarbonate (23.98 g, 110 mmol). The reaction mixture was stirred at room temperature for 12 h and evaporated to dryness. The residue was partitioned between EtOAc (250 ml) and water (200 ml) and extracted into EtOAc. The EtOAc extract was washed with potassium hydrogen sulfate (100 ml) and brine (100 ml), dried and evaporated to dryness to give 15 g (91%) of a clear oil.

1-Chloro-2- (tetrahydropyranyl) oxy-ethane (29): 1-Chloro ethanol (8.06 g, 100 mmol) was dissolved in dry CH ₂ Cl ₂ (100 ml) and cooled to 0 ° C in ice

-100baths in argon atmosphere. To this stirred solution was added dihydropyran (12.6 g, 150 mmol) followed by pyridine-p-toluene-4-sulfonate (1.25 g, 5 mmol) and stirring overnight. The reaction mixture was evaporated to dryness and dissolved in EtOAc (200ml). The EtOAc extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (100 ml). The organic extract was dried and evaporated to dryness. The crude material was purified by flash chromatography over silica gel using hexane -> CH ₂ Cl ₂ as eluent. The pure fractions were combined and evaporated to give 11 g (67%) of the pure product.

N-Tert-Butyloxycarbonyl-N - [(tetrahydropyranyl) oxy] ethyl-Obenzylhydroxylamine (30): To a stirred solution of N-tert-butyloxycarbonyl-Obenzylhydroxylamine 28 (5.79 g, 25.96 mmol) in dry DMF (15 ml) was slowly added to dry DMF (15 ml). min at a time interval under argon at 0 ° C, NaH (60%, 1.2 g, 30 mmol) was added. The reaction was allowed to stir at 0 ° C for 30 min and at room temperature for 1 h. 1-Chloro-2- (tetrahydropyranyl) oxy-ethane 29 (4.95 g, 30 mmol) was added and the reaction mixture was heated at 80 ° C for 12 h. The reaction was cooled and evaporated to dryness. The residue was suspended in water (50 ml), the pH of the solution was adjusted to 7 and extracted into EtOAc (150 ml). The EtOAc extract was washed with water and brine, dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using hexane -> CH ₂ Cl ₂ as eluent. The necessary fractions were collected and evaporated to give 6.0 g (66%) of the oily product. ¹ HNMR (CDCl ₃ ): δ 1.48 (s, 9H, Boc), 1.49 - 1.84 (m, 6H, 3.CH ₂ ), 3.48 3.70 (m, 4H, 2.CH ₂ ), 3.86 (m, 2H. CH ₂ ), 4.60 (t, 1H, CH), 4.84 (s, 2H, CH ₂ Ph) and 7.32 - 7.42 (m, 5H, Ph).

N-Tert-butyloxycarbonyl-N - [(2-hydroxyl) ethyl] -O-benzylhydroxylamine (31):

A stirred solution of N-tert-butyloxycarbonyl-N - [(tetrahydropyranyl) oxy] ethyl-O-101benzylhydroxylamine 30 (3.51 g, 10 mmol) in THF: water: AcOH (1: 1: 1, 100 ml) was kept at 70 ° C. heated for 3 h. The reaction was cooled to 0 ° C and the pH adjusted to 7 with solid NaHCO ₃ . The reaction mixture was extracted with EtOAc (2x75 ml). The combined organic extract was washed with water (100 ml) and brine (100 ml), dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure fractions were collected and evaporated to give 2.5 g (94%) of foam. ¹ HNMR (CDCl ₃ ): δ 1.48 (s, 9H, Boc), 3.60 (t, 2H, CH ₂ ), 3.74 (m, 2H, CH 2, 4.84 (s, 2H, CH ₂ Ph) and 7.32 - 7.42 (m, 5H, Ph).

N-Tert-butyloxycarbonyl-N - [[(2-isobutyryl) oxy] ethyl] -O-benzylhydroxylamine (32): To a stirred solution of N-tert-butyloxycarbonyl-N - [(2-hydroxy) ethyl] -Obenzylhydroxylamine 31 ( 4.2 g, 16.6 mmol) in dry pyridine (50 ml) was added TEA (2.02 g, 20 mmol) and then isobutyric anhydride (3.16 g, 20 mmol) at room temperature under argon. The reaction mixture was stirred at room temperature for 12 h and evaporated to dryness. The residue was dissolved in EtOAc (200 ml), washed with 5% NaHCO ₃ solution (100 ml), water and brine (50 ml). The organic extract was dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ as eluent. The pure fractions were collected and evaporated to give 4.5 g (80%) of the pure compound. ¹ HNMR (CDCl ₃ ): δ 1.04 (d, 6H, 1bCH ₃ ), 1.48 (s, 9H, Boc), 2.44 (m, 1H, IbCH), 3.60 (t, 2H, CH2, 3.74 (m, 2H , CH ₂ ), 4.84 (s, 2H, CH ₂ Ph) and 7.32 - 7.42 (m, 5H, Ph).

N- (Thinylacetyl) -N - [[(2-isobutyryl) oxyl] ethyl] -O-benzylhydroxylamine (33): Nert-butyloxycarbonyl-N - [[(2-isobutyryl) oxy] ethyl] -0-benzylhydroxylamine 32 ( 5.0 g, 14.84 mmol) was dissolved in CH ₂ Cl ₂ (10 ml) and stirred for 30 min in TFA (12 ml). The reaction mixture was evaporated to dryness and dissolved in dry methanol (10 ml). The mixture was re-evaporated to

-102 dry and vacuum dried over solid NaOH overnight. The dried material was used as such for the next reaction without characterization.

Thymine acetic acid 5 (3.13 g, 17 mmol) was dissolved in dry DMF (75 ml) and cooled to -20 ° C in argon. To this cold stirred solution was added N-methylmorpholine (2.02 g, 20 mmol) followed by isobutyl chloroformate (2.72 g, 20 mmol). After stirring for 15 min, a solution of the above TFA salt in dry DMF (50 ml) was neutralized with N-methylmorpholine (2.02 g, 20 mmol) and immediately added to a cold stirred solution of acetic acid thymine. The reaction mixture was stirred at -20 ° C for 1 h, warmed to room temperature and stirred overnight. The solution was evaporated to dryness and the residue was dissolved in CH ₂ Cl ₂ (250 ml) and water (100 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (50 ml). The CH ₂ Cl ₂ extract was dried and evaporated to dryness to give the crude product. The crude product was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> acetone as eluent. The necessary fractions were collected and evaporated to give 4.0 g (70%) of pure product. The pure product was crystallized from CH ₂ Cl ₂ / hexane. Melting point: 185-188 ° C. ¹ HNMR (Me ₂ SO-d ₆ ); δ 1.00 (d, 6H, 1bCH ₃ ), 1.74 (s, 3H, CH ₃ ), 2.44 (m, 1H, IbCH), 3.92 (m, 2H), 4.18 (t, 2H), 4.68 (bs, 2H) , 4.98 (s, 2H), 7.34 (s, 1H, C ₆ H), 7.40 - 7.50 (m, 5H, Ph) and 11.32 (bs, 1H, NH).

Example 30 (Figure 30) (2R, 4R) -2-Carbomethoxy-4-hydroxypyrrolidine (35): Dry methanol (40 ml) was poured into a 250 ml round bottom flask equipped with a magnetic stirrer and a return condenser and cooled to icy

-103Bars in argon atmosphere. Acetyl chloride (4.32 g, 55 mmol) was added to this stirred solution followed by cis-4-hydroxy-D-proline 34 (5.00 g, 38.17 mmol). The resulting solution was heated at reflux for 7-8 min and cooled to room temperature. The solution was diluted with ether and the resulting white solid was collected by suction, washed with ether and dried in vacuo over solid NaOH. Yield: 6.9 g (100%). ¹ HNMR (CDCl ₃ ): 2.09 (2 dd, 1H), 2.34 (m, 1H), 3.49 - 3.73 (m, 3H), 3.79 (s, 3H, CH ₃ ), 4.34 (m, 2H).

(2R, 4R) -1- (Tert-Birtyloxycarbonyl-2-carbomethoxy-4-hydroxypyrrolidine (361:

TEA (10.1 g, 100 mmol) was added to a stirred solution of (2R, 4R) -2-carbomethoxy-4-hydroxypyrrolidine 35 (6.9 g, 38.12 mmol) in THF / water (8: 2, 150 ml), and then at di-tert-butyldicarbonate (10.9 g, 50 mmol) was added at room temperature. The reaction was stirred at room temperature for 6 h and evaporated to dryness. The residue was dissolved in EtOAc (200 ml) and washed with 0.5N potassium hydrogen sulfate (50 ml), water (100 ml) and brine (50 ml). The organic extract was dried over Na ₂ SO ₄ and evaporated to dryness to give 7.8 g (84%) of the oily product. The oily product gave a colorless solid, drying: melting point: 75-77 ° C. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 2.09 (2 dd, 1H), 2.34 (m, 1H), 3.49 - 3.73 (m, 3H), 3.79 (s, 3H, CH ₃ ). 4.34 (m, 2H).

(2R, 4R) -1- (Tert-Butyloxycarbonyl) -2-hydroxymethyl-4-hydroxypyrrolidine (371:

(2R, 4R) -1- (Tert-butyloxycarbonyl) -2-carbomethoxy-4-hydroxypyrrolidine 36 (7.0 g, 28.6 mmol) was dissolved in dry THF (100 ml) and cooled in an ice-salt bath under argon. To this cold solution, lithium borohydride (1.88 g, 85.8 mmol) was added in small doses over a 15 min time interval. After the addition of lithium borohydride, the reaction mixture was allowed to stir at 0 ° C for 1 h and then at room temperature for 15 h under argon. The solution was cooled to 0 ° C and diluted with water (50 ml) and adjusted

-104pH with AcOH at 6. The reaction was evaporated to dryness and dissolved in EtOAc (200 ml), washed with water (100 ml) and brine (100 ml). The EtOAc extract was dried and evaporated to dryness. The residue was purified by flash column chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The pure fractions were collected and evaporated to dryness to give 5.00 g (81%) of a clear oil. As the oil stood, a colorless solid formed from it. Melting point: 95-97 ° C. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 - 3.62 (m, 3H), 4.00 (m, 2H), 4.28 (bs. 1H), 4.44 (m, 1H).

(2R, 4R) -1- (Tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl-4hydroxypyrrolidine (38): (2R, 4R) -1- (Tert-butyloxycarbonyl) -2-hydroxymethyl-4hydroxypyrrolidine 37 (4.4 g, 20.28 mmol) was dissolved in dry pyridine (50 ml) and allowed to stir under argon. To this stirred solution was added TEA (2.53 g, 25 mmol) followed by 4,4'-dimethoxytrityl chloride (7.45 g, 22 mmol). The reaction mixture was stirred at room temperature for 12 h and quenched with MeOH (10 ml). The solution was evaporated to dryness and dissolved in EtOAc (200 ml). The EtOAc layer was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine. The organic extract was dried over anhydrous Na ₂ SO ₄ and evaporated to dryness. The residue was purified by flash chromatography over silica gel using hexane -> EtOAc as eluent. The necessary fractions were combined and evaporated to give 8.09 g (100%) of orange foam. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 - 3.62 (m, 3H), 3.74 (s, 6H, 2.OCH3). 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 6.82 (d, 4H, Ph) and 7.20 - 8.00 (m, 9H, Ph).

(2R, 4R) -1- (Tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxylmethyl-4 - [(ptoluenesulfonyl) oxy] pyrrolidine (39): (2R, 4R) -1- (Tert- butyloxycarbonyl) -2- (4,4-105 dimethoxytrityl) oxymethyl-4-hydroxypyrrolidine 38 (8.09 g, 20.27 mmol) was dissolved in dry pyridine / CH ₂ Cl ₂ mixture (2: 1, 200 ml) and cooled in an ice bath in argon atmosphere. TEA (3.03 g, 30 mmol) and then p-toluenesulfonyl chloride (5.7 g, 30 mmol) were added to this cold solution. The reaction mixture was allowed to stir at 0 ° C for 3 h and below 30 ° C for 8 h. The reaction mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and 5% NaHCO ₃ solution (100 ml) and extracted into EtOAc. The EtOAc extract was washed with water (100 ml) and brine (100 ml), dried and evaporated to dryness. The crude product was purified by flash chromatography over silica gel using hexane EtOAc as eluent. The pure fractions were combined and evaporated to give 12.2 g (89%) of an orange oil. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 2.40 (s, 3H, CH ₃ ), 3.40 -3.62 (m, 3H), 3.74 (s, 6H, 2.0CH ₃ ), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 6.82 (d, 4H, Ph) and 7.26 - 8.00 (m, 13 H, Ph) ).

(2R, 4S) -1- (Tert-butyloxycarbonyl) -2- (4,4'-dlmethoxytrityl) oxymethyl-4-azidopyrrolidin (40): (2R, 4R) -1- (Tert-butyloxycarbonyl) -2- ( 4,4'-Dimethoxytrityl) oxymethyl4 - [(p-toluenesulfonyl) oxy-pyrrolidine 39 (5.1 g, 7.58 mmol) was dissolved in dimethylformamide (50 ml) and diluted with water (5 ml). Sodium azide (0.65 g, 10 mmol) was added to this stirred solution and heated at 80 ° C for 8 h. We cooled it and evaporated to dryness. The residue was partitioned between CH ₂ Cl ₂ (200 mi) and water (100 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with brine (50 ml), dried over Na ₂ SO ₄ and evaporated to dryness. The crude product was purified by flash chromatography over silica gel using hexane -> EtOAc as eluent. The pure fractions were combined and evaporated to give 3.8 g (92%) of a clear oil. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 3.62 (m, 3H), 3.74 (s, 6H, 2.0CH ₃ ), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 6.82 (d, 4H, Ph) and 7.26 - 7.80 (m, 9H, Ph).

-106 (2R, 4S) -1- (Tert-Butyloxycarbonyl) -2-hydroxymethyl-4-amino-pyrrolidine (41):

(2R, 4S) -1- (Tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl-4-azido-pyrrolidine 40 (2.72 g, 5 mmol) in methanol (75 ml) was hydrogenated in the presence of 10 % palladium on charcoal (0.3 g) at room temperature and 5 bar pressure. After 12 h, the catalyst was filtered off, washed with methanol (20 ml) and the solvent removed in vacuo. Yield 1.0 g (93%). ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 - 3.62 (m, 3H), 4.00 (m, 2H), 4.28 (bs. 1H) and 4.44 (m, 1H).

(2R, 4S) -1- (Tert-butyloxycarbonyl) -2-hydroxymethyl-4-phthalimido-pyrrolidine (42): (2R, 4S) -1- (Tert-butyloxycarbonyl) -2-hydroxymethyl-4-amino-pyrrolidine 41 (1.00 g, 4.63 mmol) was dissolved in dry methanol (20 ml) and treated with N-ethoxycarbonyl phthalimide (1.09 g, 5 mmol) at room temperature. The reaction mixture was stirred for 6 h and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were collected and evaporated to yield

1.5 g (94%) of pure compound as foam. ¹ HNMR (CDCl ₃ ): 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 - 3.62 (m, 3H), 4.00 (m, 2H), 4.28 (bs. 1H), 4.44 (m, 1H) and 7.3-6.6 (m, 4H, Ph).

(2R, 4S) -1- (Tert-butyloxycarbonyl) -2- [N ₃ -benzoyl (tlmin-1-yl)] methyl-4-phthalimido-pyrrolidine (43): To a stirred solution of N ₃ -benzoylthymine 20 (1.15 g. 5 mmol) in dry THF (70 ml) was added in argon at room temperature triphenyl phosphine (2.62 g, 10 mmol) and (2R, 4S) -1- (tert-butyloxycarbonyl) -2-hydroxymethyl-4-phthalimido-pyrrolidine (1.4 g, 4.05 mmol). After 15 min, diethyl azodicarboxylate (1.74 g, 10 mmol) was added slowly over a 10 min interval. The reaction mixture was covered with aluminum foil and allowed to stir at room temperature in argon for 24 h. The solvent was evaporated to

-107 dry and the residue was dissolved in EtOAc (150 ml). The organic extract was washed with 5% NaHCO ₃ solution (100 ml), water (100 ml) and brine (100 ml) and dried over anhydrous Na ₂ SO ₄ . The dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography over silica gel using hexane -> EtOAc as eluent. The fractions with the required product were combined and evaporated to give an oil a pale pink color. Yield: 2.0 g, (89%). ¹ HNMR (CDCl ₃ ): 1.41 (s, 9H, Boc), 1.72 (s, 3H, CH ₃ ), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40 - 3.62 (m, 3H), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 7.06 (s, 1H, C ₆ H) and 7.20 - 7.60 (m, 9H, Ph).

Example 31

Oligonucleotide Synthesis: Oligonucleotides having modified amino acid nucleic acid backbones were synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry. β-Cyanoethyl phosphoramidites, synthesis reagents and CPG polystyrene columns were purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a tetraethylthiuram disulfide / acetonitrile mixture and a standard ABI phosphorothioate program was used for the gradual titration of the phosphate bonds. After cleavage from the controlled-sized glass column, the protecting groups were removed by treating the oligonucleotides with concentrated ammonium hydroxide at 55 ° C for 8 h. Oligonucleotides (with DMT protecting group) were purified by HPLC using a reversed-phase semipreparative C ₃ column (ABI) and a linear gradient of 5% acetonitrile in 0.1 M triethylammonium acetate (buffer A) and acetonitrile (buffer B). The DMT protecting group was cleaved by treatment with 80% acetic acid and the product precipitated in ethanol. The purity of the products was checked with

-108HPLC, using an analytical C18 column (Beckman). Amino acid nucleic acid monomers were incorporated at the 3′-end and 5′-end and in the middle of the DNA sequence with 100% binding efficiency. Without any problem, we also prepared a homo polymer that has 16 amino acids modified thymine.

Example 32

Hybridization assay: The ability of the amino acid-modified oligonucleotides of the invention to hybridize to their complementary RNA and DNA sequences was determined by thermal melting analysis. The RNA complement was synthesized on a Genset Corporation synthesizer (La Jolla, CA) and purified by denaturing urea PAGE. Natural antisense oligonucleotides or those containing functionalized at specific sites were added to either RNA or DNA complement at stoichiometric concentrations required for the formation of hybrid duplexes. The dependence of the absorbance hyperchromicity (260 nm) on the temperature at the transition of the duplex to a random ball was recorded using a Varian Cary 1E UV-Visible spectrophotometer. Measurements were taken in 10 mM Na-phosphate buffer, pH 7.4, 0.1 mM EDTA and NaCl to give ionic strength of either 0.1 M or 1.0 M. The data were analyzed by graphically representing 1 / Tm against ln [Ct], where [ Ct] total oligonucleotide concentration. Thermodynamic parameters were determined from this analysis. Based on the information obtained regarding the stability of the resulting duplex or hetero-duplex, we evaluated the placement of modified pyrimidine in oligonucleotides for its effects on helix stability. Modifications that drastically alter the stability of the hybrid show reductions or increases in free energy (delta G), so we can decide on their usefulness in antisense oligonucleotides.

-109Hybridization studies were performed with oligonucleotides containing the amino acid nucleic acid backbone at the 3′-end as well as at the 5′-end. Preparatory studies have shown that modified oligonucleotides form a duplex, with their complementary RNA and DNA sequences, as unmodified oligonucleotides.

Example 33

Nuclease resistance. The natural, phosphorothioate and modified oligonucleotides of the invention were evaluated for their resistance to serum nucleases by incubation of oligonucleotides in medium containing different concentrations of fetal calf or adult human serum. The labeled oligonucleotides were incubated for a long time, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamide urea denaturing gels and subsequent autoradiography or phospho-imaging. The autoradiograms were quantified by laser densitometry. Based on the location of the modifications and the known length of the oligonucleotide, it is possible to determine the effect of a particular modification on nuclease degradation. HL60 cell line was used for cytoplasmic nuclease. The post-mitochondrial supernatant was prepared by differential centrifugation, and the labeled oligonucleotides in this supernatant were incubated for various periods of time. By following the incubation, the oligonucleotides were evaluated for degradation as described above for serum nucleolytic degradation. The results of autoradiography were quantified to compare unmodified, e.g. phosphorothioate and modified oligonucleotides.

Preparatory studies on amino acid-modified oligonucleotides have shown that they are resistant to the phosphodiesterase of snake venom.

-110Inclusion with reference

All cited patents, patent applications and publications are incorporated herein by reference.

Equivalents

The above specification is considered satisfactory to enable one skilled in the art to carry out the invention. Of course, within the scope of the following claims for implementation of the invention, various modifications of the embodiments described above are readily apparent to those skilled in the art of molecular biology, organic chemistry, or related fields.

Claims (8)

REQUIREMENTS 1. A compound of compound 1 in any of Groups I - V: W-L Compound 1 Group I, where: X is CHR ₂ OH, where R _{2 is} H, lower alkyl amine or lower alkyl imidazole; Y is a Base- (CH ₂ ) _n -, where the Base is a halogenated variable nucleoside base, where n is 1 to 7; A is carbonyl; ZH or OR is ₃ , where R _{3 is} H, lower alkyl, lower alkyl amine or lower alkyl imidazole; W is CHR ₄ OH, where R _{4 is} H or lower alkyl amine or lower alkyl imidazole; N is N (nitrogen); and L is zero; Group II, where: X is Base- (CH ₂ ) _n -, where Baza is a halogenated variable nucleoside base, where n is 1 to 7; Y is CHR ₂ OH, where R _{2 is} H, lower alkyl amine or lower alkyl imidazole; A is carbonyl or CH ₂ ; ZH or OR is ₃ , where R _{3 is} H, lower alkyl, lower alkyl amine or lower alkyl imidazole; -112 is W CHR4OH, where R _{4 is} H or lower alkyl amine or lower alkyl imidazole; N is N (nitrogen); and L is zero; Group III, where: X is CHR2OH, where R _{2 is} H, lower alkyl amine or lower alkyl imidazole; Y is a Base- (CH ₂ ) _n -, where the Base is a halogenated variable nucleoside base, where n is 1 to 7; A is CH ₂ ; Z is OH or OR ₃ and R _{3 is} H, lower alkyl, lower alkyl amine or lower alkyl imidazole; W is CHR ₄ OH, where R _{4 is} H or lower alkyl amine or lower alkyl imidazole; N is N (nitrogen); and L is zero; Group IV, where: X is Base- (CH ₂ ) _n -, where Baza is a halogenated variable nucleoside base, where n is 1 to 7; Y is COOH or CHR ₂ OH, where R _{2 is} H, lower alkyl amine or lower alkyl imidazole; A is carbonyl or CH ₂ ; Z is CH ₂ ; W is CH ₂ ; is NN (nitrogen); and L is CHNHR ₅ , where R _{5 is} H, OH or OR ₃ , and R _{3 is} H, lower alkyl, lower alkyl amine or lower alkyl imidazole; -113Group V, where: X is CH ₂ OH, CH ₂ NH ₂ , CONH ₂ or COOH; Y is zero; Z is CH ₂ or CHO-L _r B; WO, S or CH ₂ ; N is CH; and L and A are independently COOH, CHCOOH, CHCH ₂ COOH, NH ₂ , CHNH ₂ , L 1 -NH-L ₂ -B or CH-L _r NH-L ₂ -B; where BH is a nucleoside base, and Li and L _{2 are} independently (CH ₂ ) _n , where n = 1-3, or (CH ₂ ) _n CO, where n = 0-2. A compound according to claim 1 polymerized into an oligonucleotide, characterized in that internucleotide bonds are formed between W and X in Group I, W and Y in Group II, W and Z in Group III, L and Y in Group IV, and L and A in Group V. An oligonucleotide according to claim 2, characterized in that the internucleotide bond comprises a phosphodiester. Oligonucleotide according to claim 2, characterized in that the internucleotide bond comprises a phosphorothioate. An oligonucleotide according to claim 2, characterized in that the internucleotide bond comprises phosphoramidate. An oligonucleotide according to claim 2, characterized in that the internucleotide bond comprises hydroxamate. -1147. An oligonucleotide according to claim 2, characterized in that the oligonucleotide can be distinguished in a plurality of monomers wherein at least one of the monomers comprises a 2'-deoxyribose nucleoside and further characterized to comprise at least two different internucleotide bonds from the group consisting of phosphodiester, phosphorothioate , phosphoramidate and hydroxamate. Oligonucleotide according to claim 7, characterized in that the oligonucleotide 2'deoxyribose nucleoside has a pentafuranosyl ring wherein the oxygen in the ring is replaced by one of S, CH ₂ or NR ₆ , wherein R _{6 is} acetyl, lower alkyl, carbonyl, carbonyl lower alkyl amine, or carbonyl lower alkyl imidazole. An oligonucleotide according to claim 2, characterized in that at least one monomer corresponds to Group V of claim 1, and a maximum of fifty monomers is selected from 2′-deoxyribonucleoside, ribonucleoside and 2′-methylribonucleoside.