RU2154638C2 - Nucleic acids modified with amino acids - Google Patents

Abstract

FIELD: organic chemistry, biochemistry. SUBSTANCE: invention describes the novel derivatives of amino acids or aminoalcohols of the formula (I)

in any of groups I, II where N means nitrogen. Group I: X means CHR₂OH where R₂ means H, (lower alkyl)-amine or (lower alkyl)-imidazole; Y means Base-(CH₂)_n- where Base means nonhalogenated variable nucleoside base where n = 1-7; A means carbonyl; Z means H or OR₃ where R₃ means H, lower alkyl, (lower alkyl)-amine or (lower alkyl)-imidazole; W means CHR₄OH where R₄ means H, (lower alkyl)-amine or (lower alkyl)- -imidazole. Group II: A means carbonyl; X means Base-(CH₂)_n- where Base means nonhalogenated variable nucleoside base where n = 1-3; Y means -CH₂OH; W means -CH₂OH; Z means H. Compounds are analogs of oligonucleotides where furanose ring occurring in natural nucleic acid is substituted with amino acid or modified aminoalcohol residues. In some variants new compounds are useful for nonsense control of gene expression. Compounds can be used also as probes for hybridization of nucleic acids or as primers. EFFECT: new derivatives indicated above, valuable properties. 8 cl, 3 tbl, 34 dwg, 33 ex

Description

Field of Invention
The invention relates to the field of analogues of polynucleotides in which there are no furanose rings.

State of the art
Oligonucleotides that bind in a sequence-specific manner to complementary nucleic acids (sense strands) via hydrogen bonds so that gene expression is inhibited are commonly called antisense oligonucleotides. These synthetic oligonucleotides bind the target (mRNA) and thus inhibit the translation of the RNA messenger. Such an antisense principle (Uhlmann, E. et al., Chem. Reviews, 1990, 90, 543-584; and Stein, C. A. et al., Cancer Res., 1988, 48, 2659-2688) are used in nature to regulate gene expression. This antisense principle was used in laboratory conditions not only for inhibition, but also for activation of gene expression. For the first time in 1978, the Observer and Stephenson proposed the use of synthetic oligonucleotides for therapeutic purposes (Stephenson, ML; and Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 1978, 75, 280 and 285). Specific inhibition by antisense polynucleotides is based on the specific Watson-Crick base pairing between heterocyclic bases of the antisense oligonucleotide and the viral nucleic acid. The process of binding oligonucleotides to complementary nucleic acids is called hybridization. In particular, an oligomer having a base sequence complementary to the base sequence of an mRNA encoding a protein necessary for the development of the disease is of interest. Through specific hybridization with such mRNA, the synthesis of the protein encoded by this mRNA can be impaired.

The preparation of unmodified oligonucleotides, that is, oligonucleotides having a DNA structure, has been the focus of attention of many groups of scientists in the last decade. Synthesis using phosphoramidites according to Carusers (McBride, LJ; and Caruthers, MH, Tetrahedron Letts., 1983, 24, 245), originally proposed by Letsinger (Letsinger, RL; and Lunsford, WB, J. Amer. Chen.Soc., 1976 , 98, 3655) as a phosphitriether method, is currently the most effective method for producing phosphodiester oligonucleotides. When normal (i.e., unmodified) oligonucleotides are used as antisense oligonucleotides, there is a problem of their instability due to the action of nucleases and insufficient penetration through the membrane. In order for antisense oligonucleotides to be able to inhibit translation, they must reach the cell interior unchanged. Properties useful for oligonucleotides used for antisense inhibition include:
(1) the resistance of oligonucleotides to the action of extra- and intracellular enzymes;
(2) the ability to pass through the cell membrane; and
(3) the ability to hybridize with a DNA or RNA target (Agarwal, KL et al., Nucleic Acid Res., 1979, 6, 3009; Agawal, S. et al., Proc Natl Acad Sci. USA. 1988, 85, 7079) . Thus, it is necessary to obtain analogues of polynucleotides that have the above properties, for use as antisense or for use as primers or hybridization probes.

Modified polynucleotides have already been synthesized; such polynucleotide modifications include methylphosphonates, phosphorothioates, various amidates and sugar moieties of nucleic acid species. These substitutions in the skeleton give to some extent enhanced stability, but have the disadvantage of obtaining chiral phosphorus in the bond, which leads to the formation of 2 ⁿ diastereomers, where n is the number of modified diester bonds in the oligomer. The presence of such multiple diastereomers significantly impairs the ability of the modified oligonucleotide to hybridize with the target sequences. Some of these substitutions also leave the ability to maintain a negative charge, and the presence of charged groups reduces the ability of these compounds to penetrate cell membranes. There are many other disadvantages associated with such modifications of the bonds, depending on the specific nature of the connection.

 Some oligonucleotide analogues containing sugar modification have been synthesized. Modifications of sugar (deoxy) ribose nucleic acids designed to provide what is understood by improved structures, especially structures that penetrate better into cells, include α-DNA, homo-DNA, morpholino and thionucleosides and peptide nucleic acids (PNA) ) The general synthesis scheme for the preparation of such analogues was to include the primary hydroxyl group of the nucleoside or its nucleotide bound either to the polymer carrier or to the sequence-specific 3'-nucleotide via a phosphorus atom in the pentavalent or trivalent oxidation state. Specific binding methods are represented by phosphite-triester (phosphoramidite), phosphodiester and hydrophosphonate methods. Commercially available monomers and polymer-associated monomers are suitable for such methods due to the presence of protected bases (G, A, C, T, U and other heterocycles) together with protected phosphorus atoms, which allows them to be stored and prevents non-specific reactions during the process binding.

 Types of nucleic acids containing modified sugars, non-ionic skeletons or acyclic polyamides (APA) have, to some extent, one or more of the following properties useful for gene modification: enhanced duplex stability (hybridization efficiency), increased target specificity, and resistance to the action of nucleases, improved penetration into the cell and assistance in important terminating events of nucleic acids (for example, the activity of RNase H, catalytic cleavage, stopping hybridization and others). It has also been proposed to use carbonate diesters. However, these compounds are highly unstable, and such a carbonate-diester bond does not support the tetrahedral configuration established by phosphorus in the phosphodiester. Similarly, carbamate bonds, being achiral, impart trigonal symmetry, and it has been shown that poly dT having such a bond does not hybridize strictly with dA (Coull, J.M. et al. Tetrahedron Letts., 1987, 28, 745; Stirchak , Ep et al., J. Org. Chem., 1987, 52, 4202).

 In later literature, information appears on acyclic sugar analogues (Augustyns, K. A. et al., Nucleic Acids Res., 1991, 19, 2587-2593). The inclusion of these acyclic nucleosides in the oligonucleotides entails a gap in Tm depending on the number of linkers incorporated into these oligomers. It was found that these oligonucleotides are resistant to the action of enzymes and mate with the bases of the complementary sequence. Given the shortcomings of polynucleotides and known polynucleotide analogs, it is of interest to create new polynucleotide analogs for use in antisense inhibition and other methods using oligomers.

 Such attempts with the modification of both sugar and skeletal components have several disadvantages when used as therapeutic agents in other methods. Consequently, more serious improvements in these properties are required before it can be used as effective therapeutic, diagnostic, and research agents. According to this, there has long been a need to improve oligomeric analogues of oligonucleotide oligomers as pharmaceutical compounds.

 The present invention provides new oligonucleotides and their structural precursors that are more resistant to the action of nucleases, have increased stability under physiological conditions and which can be neutral or positively charged, which could facilitate penetration into the cell. Further, the novel oligonucleotides of the invention have improved hybridization properties with respect to hybridizable target nucleic acids.

 The oligomers of the present invention are generally characterized in that they contain a series of built-in linkers or monomers suitable for binding the heterocyclic bases to the target nucleic acid in a sequence-specific manner. When the built-in linkers described herein are incorporated into oligomers, they may have greater strength than a single hydrogen bond, thereby facilitating the formation of a competent conformation upon binding.

 The nucleomonomers of the present invention are mainly characterized as groups or residues that replace the furanose ring, which is present in the natural nucleotides, with amino acid or modified amino alcohol residues. Exemplary monomers and oligomers of the invention are shown in formulas 1-41. The inclusion of these monomers described herein in the composition of the oligonucleotides allows the synthesis of compounds with improved properties; these improved properties include (1) increased lipophilicity due to the removal of charge due to phosphodiester bonds (Dalge, J.M. et al., Nucleic Acids Res., 1991, 19, 1805), and (2) resistance to degradation enzymes such as nucleases. Oligomers containing these monomers can be perfectly stable for hybridization with target sequences and are superior to unmodified nucleoside residues in one or more applications.

Summary of invention
The present invention provides various new oligonucleotide analogs having one or more properties that provide superiority of the compounds of interest to traditional oligonucleotides when used in methods employing oligonucleotides. The compounds of the invention are analogous to oligonucleotides in which the furanose ring present in natural nucleic acids is replaced by an amino acid or a modified amino alcohol residue. In some implementations, the new compounds of the invention are particularly useful for antisense control of gene expression. The compounds of the invention can also be used as probes for hybridization with nucleic acids or as primers.

 In another aspect, the invention provides monomeric precursors of the oligonucleotide analogs of the invention. These monomeric precursors can be used to synthesize the polynucleotide analogues of interest to us.

 In another aspect, the present invention provides preparations of the polynucleotide analogs of interest to us that are designed to treat or prevent painful conditions. In yet another aspect, the present invention provides methods for treating or preventing diseases, in particular viral infections and cell growth disorders. The methods of treating diseases of interest to us include the stage at which an effective amount of polynucleotide analogues of interest to us is administered for use as antisense inhibitors.

Brief Description of the Drawings
In FIG. 1-25 shows the sequence of chemical reactions used for the synthesis of monomers and oligonucleotides according to the invention. More specifically:
In FIG. 1 shows the synthesis of L-serinol-linked thymine monomeric phosphoramidite with a -CH ₂ -CO-bond between thymine and serinol.

In FIG. Figure 2 shows the synthesis of L-serinol-linked thymine monomeric phosphoramidite with a -CH ₂ -CH ₂ bond between thymine and serinol.

In FIG. Figures 3 and 4 show the synthesis of thymine monomeric phosphoramidite bound with substituted L-serinol with the -CH ₂ -CO bond between thymine and serinol.

In FIG. 5 shows the synthesis of a TT dimer with an internucleotide bond of 5 atoms having hydroxylamine in the middle of the internucleotide bond, with a —CH ₂ —CO bond between thymine and serinol.

In FIG. Figure 6 shows the synthesis of thymine monomeric phosphoramidite in which thymine is attached to N-ethylhydroxylamine via an -CH ₂ -CO bond.

In FIG. 7 shows the synthesis of L-serinol-linked thymine monomeric phosphoramidite in which the NH ₂ L-serine group is attached to a 2-hydroxyacetyl group and the hydroxy function is blocked by the DMT group. This building block is used for 2'-5 'connection. The drawing also shows the synthesis of a thymine monomer in which the NH ₂ group of L-serine is linked to a 2'-hydroxyethyl function.

 In FIG. Figure 8 shows the synthesis of a TT dimer having a hydroxamate backbone with a 2'-5 'bond. In this dimer, one building block consists of L-aspartic acid and thymine, and the other consists of L-serine and thymine. This dimer also has two additional amide bonds in the skeleton.

 In FIG. 9 shows the synthesis of a TT dimer having a hydroxamate backbone with a 2'-5 'bond. In this dimer, one building block consists of L-aspartic acid and thymine, and the other consists of L-serine and thymine. There is no amide bond in the skeleton in this dimer.

 In FIG. 10 shows the synthesis of L-serinol-β-alanine-bound thymine monomeric phosphoramidite, in which β-alanine binds thymine and serinol.

 In FIG. 11 shows the synthesis of L-serynol-alkylamine-associated thymine monomeric phosphoramidite in which an alkylamine binds thymine and serinol.

 In FIG. 12 shows the synthesis of a TT dimer having a hydroxamate backbone with a 4'-5 'bond. In this case, the dimer consists of two units of L-aspartic acid and two thymine units with an acetyl linker between thymine and aspartic acid.

 In FIG. 13 shows the synthesis of a TT dimer having a hydroxamate backbone with a 4'-5 'bond. In this case, the dimer consists of two units of L-aspartic acid and two thymine units with an ethyl linker between thymine and aspartic acid.

 In FIG. 14 shows the synthesis of a thymine building block bound to N-hydroxamic acid.

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

 In FIG. 16 shows the synthesis of a TT dimer having a hydroxamate backbone with a 4'-5 'bond. In this case, the carboxylic acid group is linked to the thymine building block via an N-hydroxylamine linker.

 In FIG. Figure 17 shows the synthesis of thymidine acetic acid substituted with N-hydroxylamine acid building block 150 and its analogue 149. These monomeric building blocks are useful for producing nucleic acid with hydroxamate skeletons.

 In FIG. Figure 18 shows the synthesis of thymidine acetic acid substituted with hydroxylamine containing amino acid building blocks 157 and 158. These monomers are useful for producing a nucleic acid having an amide skeleton with hydroxylamine functionality.

 In FIG. 19 shows the synthesis of L-serinol-linked thymidine building block 166 having a hydroxylamine moiety between thymine and serinol. This building block is useful for producing nucleic acids with 4'-5 'bonds.

 In FIG. 20 shows the synthesis of glutamic acid-glycine-bound thymidine monomer 174. This monomeric building block is useful for producing nucleic acid with amide skeletons and 2′-5 ′ bonds.

 In FIG. 21 shows the synthesis of glycine-glycine-bound thymidine building block 181 and 182 having a hydroxylamine moiety between thymine and glycine. These building blocks are useful for producing nucleic acids with 2'-5 'bonds.

 In FIG. 22-24 show the synthesis of thymidine building blocks associated with a ribose amino acid 191, 199, and 207. These building blocks are useful for producing oligonucleotides having a ribose amide skeleton.

 In FIG. 25 shows solid-phase synthesis of an oligonucleotide 211 having a ribose amide skeleton.

 In FIG. 26 shows the synthesis of 1-O- (4,4'-dimethoxy-trityl) -2- [amino (thyminylacetyl)] -L-propan-3-O- (N, N-diisopropyl) -β-cyanoethylphosphoramidite.

 In FIG. 27 shows the synthesis of 1-O- (4,4'-dimethoxy-trityl) -2- [amino (thyminylacetyl)] - D-propan-3-O- (H, H-diisopropyl) -β-cyanoethylphosphoramidite.

 In FIG. Figure 28 shows the synthesis of 2 - [(β- (4,4'-dimethoxy-trityl) -O-acetyl) amino] -3-thyminyl-L-propan-1-O- (N, N-diisopropyl) -β-cyanoethylphosphoramidite .

 In FIG. 29 shows the synthesis of N- (thyminyl acetyl) -N - [[(2-isobutyryl) oxy] ethyl] -O-benzylhydroxylamine.

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

Detailed description of specific embodiments
Definitions and abbreviations
The following terms are used in the present invention and are intended to define the following.

 As used herein, the term “antisense” therapy refers to the introduction or generation of in situ DNA or RNA oligomers or their analogs that specifically bind to a complementary target nucleic acid sequence. This binding can occur by traditional complementary base pairing or by using another mechanism, for example, in the case of DNA duplex binding, using specific interactions in the large groove of the double helix. In the General case, the term "antisense" refers to a number of techniques, mainly used according to this description in the prior art, and includes any therapy based on specific binding to oligonucleotide sequences. These techniques of antisense gene regulation are well known to those skilled in the field of molecular biology; descriptions of antisense gene regulation are described, for example, in US Pat. No. 5,107,065, US Pat. No. 5,166,195, US Pat. 329-376.

 The terms "oligomer" or "oligonucleotide" are used interchangeably and include naturally occurring compounds such as RNA or DNA, as well as their synthetic analogues, including the compounds of the invention. Unless otherwise indicated, the terms “oligomer” and “oligonucleotide” refer to both DNA / RNA and their synthetic counterparts. The term "oligomer" is applied to compounds containing two or more nucleomonomers covalently attached to each other via a phosphodiester bond or other substitute bonds. Unless otherwise indicated, the term “oligomer” is not to be understood as limiting the length. Thus, the oligomer can have only two covalently linked nucleomonomers (dimer), or it can be much longer. The oligomers can be linked competently and, thus, pair with single-stranded or double-stranded nucleic acid sequences. Oligomers (e.g., dimers - hexamers) can also be used as synthons for longer oligomers, as described below. Oligomers may contain non-base sites and pseudonucleosides.

 Oligomers include oligonucleotides, oligonucleosides, polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or its modified forms), i.e. DNA, polyribonucleotides (containing D-ribose or its modified forms), i.e. RNA, and any other type of polynucleotides being N-glycosides or C-glycosides of purine or pyrimidine bases or modified purine or pyrimidine bases.

It is understood that oligomers, as the term is used here, also include compounds in which adjacent nucleomonomers are linked via hydroxamate bonds. Elements typically present in oligomers, such as a furanose ring and / or phosphodiester bond, may be replaced by any suitable functionally equivalent element. The term “oligomer” is intended to include any structure that serves as a backbone or base support, and this backbone allows binding to target nucleic acids in a sequence-dependent manner. Traditional known oligomers can be divided into four groups, which are characterized as having
(1) bonds in the form of phosphodiesters or analogues of phosphodiesters (phosphorothioates, methyl phosphonates, etc.),
(2) substitute bonds that contain non-phosphoric isosteres (riboacetal, formacetal, carbamate, etc.),
(3) morpholine residues, carbocyclic residues or other furanose sugars, such as arabinose or hexose, instead of ribose or deoxyribose,
(4) nucleomonomers linked through amide bonds, or acyclic nucleomonomers linked through any suitable replacement bond.

The term "nucleomonomer" as used herein refers to a moiety containing
(1) a base covalently linked to
(2) the second grouping.

 Nucleomonomers include nucleosides, nucleotides or bases attached to an amino alcohol. Nucleomonomers can be associated with the formation of oligomers that bind targets or complementary nucleic acid base sequences in a sequence-specific manner.

 A “second moiety,” as used herein, refers to a compound coupled to a nucleomonomer and includes an amino acid / amino alcohol moiety, typically a serine, aspartic acid, glutamic acid, glycine, and those species that contain modifications of the amino acid group, for example in which one or more hydrogens is replaced by another functionality (see formulas 24-41), or one carboxylic acid is converted to the function of alcohol, amines, thiols, hydroxylamines, etc. Nucleomonomers as defined herein also include bases associated with amino acids or amino alcohols and / or analogs of amino acids / alcohols having a free carboxyl / hydroxyl group and / or a free amino group and / or their protected forms.

 The term "nucleoside", as used here, refers to its amino acid and amino alcohol derivatives, as described below, bearing purine, pyrimidine, or their analog forms, as defined below, but in which there is no linking group, such as a phosphodiester analog or a modified internucleotide bond . By "5 '" nucleoside is meant a nucleoside enabling the linker to be bonded to 5' carbon. The "5 '" end of the linker binds to the 5' nucleoside. The "3 '" end of the linker binds to the 3' position of the next nucleoside. If a modified nucleoside is present that does not specifically include 3 'and / or 5' carbon, then it should be understood by one of ordinary skill in the art that this "3 '" and "5'" terminology is used to describe the polarity of the strand by analogy with DNA and RNA.

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

 The term "nucleotide", as used here, refers to nucleosides having a phosphate group or a phosphate analogue (groups with phosphorus in the same oxidation state as in the phosphate group, for example thiophosphate, amidate).

The term "base", as used here, refers to a wide range of nucleoside bases, including purine, pyrimidine heterocycles and analogues of heterocycles and their tautomers. Purines include adenine, guanine, and xanthine; an example of a purine analogue is 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. When “bases” are included in a suitable molecular reading frame, for example a phosphodiester skeleton, they are capable of participating in base pairing, which occurs in double-stranded DNA or other double-stranded nucleic acids with similar structures. Bases are also capable of participating in base pairing in a triple helix nucleic acid.

 The term “sugar modification,” as used herein, refers to any residue of an amino acid or amino alcohol other than 2'-deoxyribose.

 The term “amino acid / alcohol,” as used herein, refers to any naturally occurring amino acids and alcohols, both the “R '” and the “S'" isomers.

 The term "nucleoside bonds," as used herein, refers to a bond that exists within a monomer.

 The term “bond,” as used herein, refers to a moiety that is used to couple a base with an amino acid / amino alcohol and derivatives thereof.

 The term “internucleotide bonds,” as used herein, refers to a phosphodiester moiety (—O — P (O) (O) O—) or any other functionally equivalent moiety that covalently binds adjacent nucleomonomers.

 The term “substitutional bonds,” as used herein, refers to any analog of a native group or any suitable moiety that covalently binds adjacent nucleomonomers. Substitutional bonds include phosphodiester analogs, for example, such as phosphothioates and methylphosphonates, and phosphorus-free bonds, such as amides, hydroxamates, hydroxylamine. Replacement bonds include phosphorus-free bonds (2 ′, 5 ′ bonds, 3 ′, 5 ′ bonds, 4 ′, 5 ′ bonds) of the invention.

The term “cross-linking moiety,” as used herein, refers to a group or moiety in an oligomer that forms a covalent bond with a target nucleic acid. The crosslinking moiety includes covalently crosslinking species that covalently bind the oligomer to target nucleic acids either spontaneously (e.g. N ⁴ , N ⁴ ethanocytosine) or by photoactivation (e.g. psoralen), etc.

The term “blocking groups” as used herein refers to substituents other than H which are covalently linked to oligomers or nucleomonomers either as a protecting group, combining synthesis groups, OPO _3-2 , or another conventional conjugate, such as a solid support, label, antibody, monoclonal antibody or fragment thereof, and the like. It is not intended that the “blocking group” as used herein is designed only as a protecting group according to slang terminology, but it is understood that it also includes, for example, combining groups such as H-phosphonates or phosphoramidites.

 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 attached from participation in a reaction or binding. Such protective groups for N-atoms on the main group in the nucleomonomer and their introduction are well known in the art. Particular examples of suitable protecting groups are: diisobutylformamidine, benzoyl, silyl and the like. Suitable protecting groups for O atoms and S atoms are, for example, DMT, MMT, FMOC or esters. “Protecting groups”, as used herein, include any group capable of preventing the participation of the O-atom, S-atom, or N-atom to which it is attached in a reaction or binding. Such protective groups for O-, S- or N-atoms in nucleomonomers are described and methods for their introduction are traditionally known in the art. Protecting groups also include any group capable of preventing a reaction and binding to carboxylic acids, thiols, and the like.

 The term “combining group”, as used here, refers to any group capable of creating a bond or replacing a bond between nucleomonomers, such as hydrophosphonate and phosphoramidite.

 The term “conjugate” or “conjugate moiety,” as used herein, refers to any group attached to an oligomer at the terminal end or within the oligomer itself. Conjugates include solid supports such as silica gel, pore controlled glass and polystyrene; labels, such as fluorescent, chemiluminescent, radioactive atoms or molecules, enzyme groups and reporter groups; transport agents for oligomers, such as polycations, whey proteins and glycoproteins and polymers and the like. Other conjugate groups include O-cholesterol, polyethylene glycol (PEG), amino acids, intercalators, polynucleotide-derived groups, cross-linking functionalities, lipids, hydroxamates, alkylating agents, and the like.

 The term "synton", as used here, refers to a structural unit within the oligonucleotide analogue of the invention.

 The term "transfection", as used here, refers to any method suitable for enhanced delivery of oligomers to cells.

 The term “subject,” as used herein, refers to a plant or animal, including mammals, in particular humans.

The term "derivatives" and monomeric constituent oligomers thereof include all their conventionally known prior art. For example, oligonucleotides can be covalently linked to various groups, such as intercalators, substances that interact specifically with the small groove of the DNA double helix and other arbitrarily selected conjugates, such as labels (radioactive, fluorescent, enzymatic, etc.). Derivatives of these additional moieties can be (but not necessarily) obtained through a modified skeletal link as part of the link itself. For example, intercalators such as acridine can be linked via —R — CH ₂ - attached via any available —OH or SH, for example, at the terminal 5 ′ position of RNA or DNA, the 2 ′ position of RNA or OH or SH created at 5 position of pyrimidines, for example, instead of 5 methyl cytosine, a derivative of which contains -CH ₂ CH ₂ CH ₂ OH or -CH ₂ CH ₂ CH ₂ SH in 5 position. A wide variety of substituents may be attached, including those that are linked by conventional bonds. Accordingly, these OH groups in the oligomer of formula (1) can be replaced by phosphonate groups protected by standard protecting groups, or activated to obtain additional bonds with other nucleotides, or may be linked to a conjugated substituent. The 5'-terminal OH is traditionally phosphorylated; 2'-OH or OH substituents at the 3'-end can also be phosphorylated. Hydroxyl derivatives with standard protecting groups can also be prepared.

The term “phosphodiester analogue” as used herein refers to both the analogue of the conventional phosphodiester bond and 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 R is H or alkyl (1-7C) and R 'is alkyl (1-7C). Not all phosphodiester analogues in the same oligomer must be identical, the only requirement is that at least one of these bonds is a modified internucleotide bond, as described herein.

"Analogous" forms of purines and pyrimidines are forms known in the art, many of which are used as chemotherapeutic agents. Examples include (but are not limited to this list):
4-acetylcytosine, 8-hydroxy-N ⁵ -methyladenine, aziridiniltsitozin, psevdoizotsitozin, 5- (karboksigidroksimetil) uracil, 5-fluorouracil, 5-bromouracil, 5- karboksimetilaminometil-2-thiouracil, 5- karboksimetilaminometiluratsil, dihydrouracil, inosine, N ₆ - isopentenyladenine, 1-methyladenine, 1-methylpsevdouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N ₆ -methyladenine, 7-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylguanosine, 5'-methoxycarbonylme tiluracil, 5-methoxyuracil, 2-methylthio-N ₆ -isopentenyladenine, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, oxybutoxosin, pseudouracil, geosin, 2-thiouracil, 4-thiouracil, N-uracil-5-hydroxyacetic acid ester, 2-thiocytosine and 2,6-diaminopurin. A particularly preferred analogue is 5-methylcytosine (abbreviation used here: Cme).

 The term "isosteric", as used here, refers to the spatial and orientational properties of the internucleoside bond; it is a fact that these properties are so similar to those of the native phosphodiester bond that a modified oligonucleotide containing an isosteric bond will replace, replace, mimic and / or hybridize with the native oligonucleotide.

 The term "riboseamide," as used herein, refers to an internucleotide bond existing between two nucleobases. This ribose-amide internucleotide linkage contains a combination of ribose / (2'-deoxy) and amino acid functionality.

 In this description, various abbreviations for functional groups and compounds are used. These abbreviations are understood by a person skilled in the field of organic chemistry, for example, “Ph” means phenyl, “Me” means methyl, “(1-7C)” means that this chain contains from 1 to 7 carbons, etc.

Description of the invention
The present invention provides new oligonucleotide analogs containing modified amino acid / amino alcohol bonds between bases and skeletons (phosphodiester, phosphorothioates and others, as shown in Table A), also called modified nucleotide bonds. These modifications or their functional equivalents replace the sugar moiety that lies between the skeleton and the bases with amino acid derivatives, for example as shown in Formula 24. The present invention also provides novel nucleomonomers and methods for incorporating them into oligomers containing these nucleomonomers.

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

Олигомеры по изобретению являются полимерами, содержащими одно или несколько интересующих нас мономерных соединений, соединенных так, чтобы обеспечить структурный аналог ДНК или РНК. Олигомеры по изобретению содержат два или большее число нуклеомономеров и могут содержать фактически любое число нуклеомономеров, хотя олигомеры, состоящие из 200 или меньшего числа нуклеомономеров, обычно легче синтезировать. Соединения формул 1-23 могут быть присоединены друг к другу через 4'-5' связи, 3'-5' связи и 2'-5' связи, как видно из формул 24-41.

The oligomers of the invention are polymers containing one or more of the monomeric compounds of interest to us, connected to provide a structural analogue of DNA or RNA. The oligomers of the invention contain two or more nucleomonomers and can contain virtually any number of nucleomonomers, although oligomers consisting of 200 or fewer nucleomonomers are usually easier to synthesize. The compounds of formulas 1-23 can be attached to each other via 4'-5 'bonds, 3'-5' bonds and 2'-5 'bonds, as can be seen from formulas 24-41.

Нуклеотидные связи в соединениях по изобретению получены из аминокислот серина и глицина или их производных. Олигонуклеотиды по изобретению являются стабильными in vivo, устойчивыми к действию эндогенных нуклеаз и способны гибридизоваться с нуклеотидными последовательностями - мишенями. Примеры соединений по изобретению показаны в формулах 24-41, они являются конформационно более ограниченными по сравнению с фосфодиэфирными связями, имеющимися в немодифицированных ДНК и РНК. Это конформационное ограничение может частично содействовать усилению связывающих свойств интересующего нас соединения в отношении комплементарных полинуклеотидных последовательностей-мишеней; однако применение данного изобретения не зависит от этой теории усиления связывающих свойств.

The nucleotide bonds in the compounds of the invention are derived from the amino acids of serine and glycine or their derivatives. The oligonucleotides of the invention are stable in vivo, resistant to endogenous nucleases, and are capable of hybridizing with target nucleotide sequences. Examples of the compounds of the invention are shown in formulas 24-41; they are conformationally more limited than the phosphodiester bonds found in unmodified DNA and RNA. This conformational limitation may partially contribute to enhancing the binding properties of the compound of interest to us with respect to complementary target polynucleotide sequences; however, the application of the present invention is independent of this theory of enhancing binding properties.

Где каждый "R" независимо представляет собой H, ОН, SH, CN, CH₃, OCH₃, SCH₃, ONH₂, ONH(CH₃), Ph, -(CH₂)_x-F, где "x" обозначает 1-7 углерод и "F" обозначает NH₂, SH, ОН, COOH, OCH₃, SCH₃, SPh, NOH, NOH(CH₃), SNH₂, S(O)NH₂, S(O)(O)NH₂, CH₃, Ph.In another embodiment, the present invention is directed to modified oligonucleotides or their derivatives in which the furanose moiety of a natural oligonucleotide, such as DNA or RNA, is replaced by an amino acid / amino alcohol moiety, other modifications containing a substituent at amino acid positions are shown in formulas 25-41. Internucleotide bonds between adjacent nucleomonomers are bonds between the 4 'and 5' positions of neighboring nucleomonomers. In other words, the phosphodiester internucleotide bond or its functional equivalent begins at the 5'-position of one nucleomonomer and attaches to the 4'-position of the adjacent monomer, as shown in examples of compounds of formulas 24-33

Where each "R" independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and “F” means 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.

 Where each base ("base") is independently a nucleotide base.

Where each "R ₁ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₂ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₃ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₄ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "A" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "B" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "X" independently represents (CH ₂ ) _x , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" denotes 1-7 carbon.

Where each "Z" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" means 1- 7 carbon.

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (1-7C), 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 ₃ , ONH (CH ₃ ), SNH ₂ , S ( O) NH ₂ , S (O) (O) NH ₂ , CH ₃ , Ph.

 Where each "V" independently represents an analogue of phosphodiester, phosphorothioates, methylphosphonates, phosphorodithioates, borophosphonates, selenophosphonates, phosphoramidates, acetamidates, oxyformamido, oxyacetamido, diisopropylsilyl, carbamate, dimethylene sulfonyl dimethylene sulfonyl dimethylene sulfonate selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of this oligomer can vary from dimer to 200 mers or even more. Preferred modified internucleotide bonds, including structure "V", are shown in table. A.

Additionally, compounds of these formulas can be conjugated to one or more conjugation moieties. Suitable conjugation moieties include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleavable moieties (e.g. imidazole), cross-linking functionalities (e.g. psoralen), lipids, peptides, alkylating agents, hydroxams, and fluorescent labels. Such a conjugation moiety can independently replace one or more radicals from R, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ .

 In yet another embodiment, the invention provides oligomeric structures shown in formulas 34-36 and derivatives thereof.

В соединениях формул 34-36 связи между соседними нуклеомономерами являются 3'-5' связями.

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

Where each "R" independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and “F” means 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.

 Where each base ("base") is independently a nucleotide base.

Where each "R ₁ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₂ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₃ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₄ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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 ₂ , Ph.

Where each "A" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "B" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "X" independently represents (CH ₂ ) _x , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ where "x" represents 1 -7 carbon.

Where each "Y" independently represents (CH ₂ ) _x , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" denotes 1-7 carbon.

Where each "Z" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" represents 1- 7 carbon.

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (1-7C), 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.

 Where each "V" independently represents an analog of phosphodiester, phosphorothioates, methylphosphonates, phosphorodithioates, borophosphonates, selenophosphonates, phosphoramidates and / or an internucleotide bond from two to four atoms in length selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of this oligomer can vary from dimer to 200 mers or even more. Preferred modified internucleotide bonds, including structure "V", are shown in table. A.

In another embodiment according to the invention, oligomers of formulas 37-41 or variants thereof are provided, wherein these oligomers contain novel internucleotide bonds, which are 2 ′, 5 ′ bonds. These oligonucleotides are stable in vivo and have improved resistance to endogenous nucleases, and they are capable of hybridization.
with oligonucleotide target sequences.

Где каждый "R" независимо представляет собой H, OH, SH, CN, CH₃, OCH₃, SCH₃, ONH₂, ONH(CH₃), Ph, -(CH₂)_x-F, где "x" обозначает 1-7 углерод и "F" обозначает NH₂, SH, OH, COOH, OCH₃, SCH₃, SPh, NOH, NOH(CH₃), SNH₂, S(O)NH₂, S(O)(O)NH₂, CH₃, Ph.

Where each "R" independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and “F” means 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.

 Where each base ("base") is independently a nucleotide base.

Where each "R ₁ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₂ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₃ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "R ₄ " independently represents H, OH, SH, CN, CH ₃ , OCH ₃ , SCH ₃ , ONH ₂ , ONH (CH ₃ ), Ph, - (CH ₂ ) _x -F, where "x" denotes 1-7 carbon and "F" denotes 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.

Where each "A" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "B" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ , NR ₅ and Se, where "x" denotes 1-7 carbon.

Where each "X" independently represents (CH ₂ ) _x , CO, CS, O, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" denotes 1-7 carbon.

Where each "Z" independently represents (CH ₂ ) _x , CO, CS, S, S (O), S (O) (O), NH, NOH, NCH ₃ and NR ₅ , where "x" represents 1- 7 carbon.

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (1-7C), 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.

 Where each "V" independently represents an analog of phosphodiester, phosphorothioates, methylphosphonates, phosphorodithioates, borophosphonates, selenophosphonates, phosphoramidates and / or an internucleotide bond from two to four atoms in length selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of this oligomer can vary from dimer to 200 mers. Preferred modified internucleotide bonds, including structure "V", are shown in table. A.

 In another embodiment according to the invention, an oligomer of formula 42 and its monomeric components of formulas 85-90 are provided.

где X выбран из группы, состоящей из (CH₂)_n, где n=1-3, CO(CH₂)_n, где n= 0-2, и (CH₂)_nSO₂, где n=1-2,
Y выбран из группы, состоящей из CH₂, СО, COOH, CS и SO₂,
Y' выбран из группы, состоящей из CH₂, СО, COOH, CS и SO₂,
Z выбран из группы, состоящей из О, S, NH и CH₂,
R выбран из группы, состоящей из CH₂OH, CH₂NH₂, CH₂NHCHO, CONH₂ и COOH,
В является нуклеозидным основанием.

where X is selected from the group consisting of (CH ₂ ) _n , where n = 1-3, CO (CH ₂ ) _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.

где X выбран из группы, состоящей из (CH₂)_n, где n=1-3, CO(CH₂)_n, где n= 0-2, и (CH₂)_nSO₂, где n=1-2,
Y выбран из группы, состоящей из CH₂, СО, COOH, CS и SO₂,
Y' выбран из группы, состоящей из CH₂, СО, COOH, CS и SO₂,
Z выбран из группы, состоящей из О, S, NH и CH₂,
R выбран из группы, состоящей из CH₂OH, CH₂NH₂, CH₂NHCHO, CONH₂ и COOH,
B является нуклеозидным основанием.

where X is selected from the group consisting of (CH ₂ ) _n , where n = 1-3, CO (CH ₂ ) _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 other embodiments, the present invention provides methods of treating diseases mediated by the presence of a certain nucleotide sequence in which a subject in need of treatment is exposed to so many oligonucleotides modified above that are capable of specifically binding such a nucleotide sequence that is capable of inactivating this nucleotide sequence.

 In the oligonucleotides of the invention, at least one of the phosphodiester groups included in the “V” formulas 24-41 is replaced by the modified internucleotide bonds described herein. It is desirable that multiple phosphodiester bonds in unmodified oligonucleotides replaced by a modified internucleotide bond can be used several times in such a structure, or, if desired, a number of modified internucleotide bonds can be used in one individual oligonucleotide. In a preferred embodiment of the oligonucleotides of interest to us, these substitutional bonds are non-chiral and, thus, enhance the ability of these nucleotides to hybridize the desired target; however, useful compounds of the invention include those embodiments in which chiral forms are used.

 Preferred modified internucleotide bonds include the structure "V" shown in the table. A.

R ₅ is H, OH, OMe, CN, NH, NOH, ONCH ₃ , ONH ₂ , ethyl, propyl, lower alkyl (1-7C), 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, one or more conjugation moieties can be attached to this bond so that an oligomeric conjugate is obtained. Suitable conjugation moieties include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleavable moieties (e.g. imidazole), crosslinking functionalities (e.g. psoralen), lipids, peptides, alkylating agents, hydroxamates, and fluorescent labels.

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

 The oligomers of the invention are not limited to oligomers with a homogeneous type of bonds, oligomers with alternating or randomly distributed bonds, including 2 ′, 5 ′ bonds, are also included. Since, when synthesizing oligomers of the invention, the nucleomonomer residues can be added one at a time, each individual bond and / or substitutional bond and nature of each individual substituent “base” can be independently selected so that oligonucleotides of the desired sequence are obtained.

 The oligomers of the invention may contain any desired number of substitution bonds. These substitutional bonds may be identical to each other or different due to the implementation selected for "V", including other non-invention substitutional bonds. Since these oligomers are obtained sequentially, any type of bond or substitute bond, base and sugar modification may be used.

 In a preferred embodiment of the invention, the substitution bonds alternate in a regular pattern. For example, one replacement bond is followed by two phosphodiester bonds, followed by one replacement bond according to the invention, etc. Additional embodiments include, for example, alternating bonds, such as a replacement bond, followed by an analog of a phosphodiester (e.g. thioate and t etc.), followed by the replacement bond of the invention, followed by the phosphodiester analog, etc., that is, the oligomer of the invention may contain two successive types of alternating replacement bonds. The oligomers of the invention containing more than one type of bond may have any number of regular samples formed by alternating bonds of various types located between the subunits of the oligomer.

 Sugar modifications can be made for one or more nucleomonomer residues in the oligomer of the invention; however, when such modifications are to be introduced, 4'-5 ', 3'-5' and 2'-5 'nucleotide bonds between amino acid residues are preferred. In this case, the following abbreviation can be used to indicate the base sequence of an oligonucleotide analog. For example, in standard DNA (or RNA), sequences are usually indicated only by a base sequence, for example, ATG CGC TGA. As a rule, it is simply established in advance whether this is an RNA or DNA sequence. The corresponding notation is also used here to represent nucleotide analogues with a given base sequence.

Additional modifications of nucleomonomers
The oligomers of the invention may also contain a number of modifications in addition to the substitutional bonds of the invention. Additional modifications include oligomers where
(1) one or more nucleomonomer residues are modified at 2 ', 3', 4 'and 5'-positions,
(2) one or more covalent crosslinked moieties are introduced,
(3) other substitutional bonds not of the invention are included,
(4) other base analogs are included, such as 8-oxo-N ^5- methyladenine, and
(5) conjugates such as intercalating agents or polylysine are included, which respectively enhance the binding affinity for target nucleic acid sequences or enhance the association of the oligomer with cells.

 The ability of the oligomers of the invention to sequence-specific binding of polynucleotides to a single-stranded or duplex target is compatible with further modifications of the oligomers. These further modifications can also give other useful properties, such as resistance to cleavage by nucleases (for example, in the domain of the oligomer according to the invention having phosphodiester bonds) or to enhance their ability to penetrate through cell membranes, etc.

 The oligomers of the invention may contain one or more substituent bonds, such as sulfide or sulfone bonds (Benner, SA, international publication N WO 89/12060), sulfamate bonds (international publication N WO 91/15500), carbamate or other substitution bonds in morpholino -related oligomers (Stirchak, E.P. et al Nucleic Acids Res, 1989, 17, 6129-6141; Summerton, J., et al, international publication N 216 860) and related bonds.

Thus, examples of implementation of the oligomers of the invention include oligomers having
(1) at least one replacement bond and an amino acid that is linked to an adjacent monomer, and
(2) one or more non-inventive substitution bonds selected from the group consisting of phosphorothioate, methylphosphonate and thiomethylphosphonate and / or
(3) one or more phosphodiester bonds and / or
(4) purine or pyrimidine analogs that enhance the binding affinity of complementary target sequences.

Other examples of oligonucleotides will include
(1) oligomers having substitutional bonds of the invention at the 3'- and / or 5'-ends and phosphorothioate bonds in other places of the oligomer,
(2) oligomers having substituent bonds according to the invention and standard purine or pyrimidine bases (for example adenine, guanine, cytosine, thymine or uracil);
(3) oligomers having replacement bonds of the invention and one or more bases enhancing the binding affinity or penetration of the oligomer (eg 5-methylcytosine, 5 '(1-propynyl) uracil, 5- (1-propynyl) cytosine. Oligomers are also included containing nucleomonomer residues bound via hydroxamates.

Synthesis of Oligomers
The oligomers of the invention can be formed using only the nucleomonomers of the invention or in combination with conventional nucleomonomers and synthesized using standard solid phase (or solution phase) oligomer synthesis techniques that are currently commercially available. In general, the oligomers of the invention can be synthesized using a method comprising the steps of: synthesizing a nucleomonomer or oligomeric synthon having a protecting group and a base and combining a group capable of combining with the nucleomonomer or oligomer; combine a nucleomonomer or oligomeric synthon with an acceptor nucleomonomer or an acceptor oligomer; remove the protective group; repeat this cycle as necessary until the desired oligomer is synthesized.

 The oligomers of the invention can be of any length, including oligomers containing more than 40, 50, 100, 200 or 500 nucleomonomers. In general, preferred oligomers contain 2-30 nucleomonomers. Lengths equal to or greater than 8-20 nucleomonomers may be useful for therapeutic or diagnostic applications if they have a suitable base sequence. Short oligomers containing 2, 3, 4 or 5 nucleomonomers are specifically included in the present invention and can be used as synthons.

 Oligomers having a random sequence and containing about 6, 7 or 8 nucleomonomers can be used as primers that are used in cloning or amplification protocols using random primer sequences, provided that such an oligomer contains about 1 or 2 residues of 3 the 'end, which can serve as a primer for polymerases or reverse transcriptases or, otherwise, do not affect the polymerase activity.

In addition to the bonds described here first, the oligomers of the invention may contain conventional phosphodiester bonds or may contain other substitutional bonds, such as phosphoramidate bonds, in addition to the substitutional bonds of the invention. These substitutional bonds include, but are not limited to embodiments in which a group of the formula —O — P (O) (S) —O (“phosphorothioate”), —OP (O) (NR ₂ ¹¹ ) —X ² , —OP (O) (R ¹¹ ) -O-, -O-P (S) (R ¹¹ ) -O- ("thionoalkylphosphonate"), -P (O) (OR ⁹ ) -X ² , OC (O) -X ² or —OC (O) (NR ₂ ¹¹ ) —X ² -, where R ¹¹ is H (or a salt) or alkyl (1-12C, including methyl and ethyl) and R ⁹ is alkyl (1-9C), and the bond is attached to adjacent nucleomonomers via —O— or —S— bonded to the carbon of the nucleomonomer, and X ² is O or S. Phosphorothioate and phosphodiester bonds are well known. Particularly preferred substitution bonds for use in the oligomers of the invention include phosphodiester, phosphorothioate, methylphosphonate and thiomethylphosphonate substitution bonds. Phosphorothioate and methylphosphonate substituent bonds give additional stability to an oligomer that must be identical, especially preferred oligomers of the invention contain one or more phosphorothioate and methylphosphonate substituent bonds.

 The oligomers of the invention and their segments can be synthesized using methods known to those skilled in the art. Synthesis methods known in the art and described herein can be used to synthesize oligomers containing substitutional bonds of the invention, as well as other bonds or substitutional bonds known in the art, using suitably protected nucleomonomers. Methods for synthesizing oligomers having phosphorus bonds are known (Froehler, B., et al., Nucleic Acids Res., 1986, 14, 5399-5467; Nucleic Acids Res., 1988, 16, 4831-4839; Nucleosides & Nucleotides 1987, 6, 287-291; Froehler, B., Tetrahedron Letts., 1986, 27, 5575-5578; Caruthers, MH in Oligodeoxynucleotides Antisense Inhibitions of Gene Expression, 1989, JS Cohen, editor, CRC Press, Boca Raton, p. 7 -24; Reese, CB et al. Tetrahedron Letts., 1985, 26, 2245-2248. The synthesis of oligomers bound by methylphosphonate using phosphonamidite chemistry is also known (Agrawal, S. et al., Tetrahedron Letts., 1987, 28, 3539-3542; Klem, RE, et al, International Publication WO 92/07864).

 It is also convenient to synthesize oligomers containing the bonds of the invention by preparing dimeric or trimeric compounds using the chemistry of dissolved phases, followed by the conversion of synthon to a derivative, which is introduced into oligomers using the chemistry of solid or dissolved phases. Typical synthons are 5 'DMT or MMT blocked 3' phosphonate or phosphoramidate derivatives, which are obtained by standard methods (see Gait, M. J. ed., Oligonucleotide Synthesis; A Practical Approach 1984, IRL Press, Oxford).

 Synthons included in the scope of the present invention include dimers, trimers, tetramers, hexamers and longer oligomers obtained by synthesis in solid or dissolved phase. Trimers and longer synthons may contain more than one type of relationship. These synthons can include any base as described above, or 2 ′, 3 ′, 4 ′ or 5′-groups, such as OH, DMTO, MMTO, O-allyl, phosphate, phosphonate or amidite, as described above.

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

Blocking groups for the synthesis of compounds of the invention
1. Combining groups
Suitable combining groups are, for example, H-phosphonate, methylphosphonamidite or phosphoramidite. Phosphoramidites that may be used include β-cyanoethylphosphoramidites (these are preferred). Methylphosphonamidites, alkylphosphonamidites (including ethylphosphonamidites and propylphosphonamidites) may also be used. Examples of phosphoramidites are shown in FIG. 1-21.

 Suitable “combining groups” at the 2 ′, 3 ′, 4 ′ or 5 ′ position for oligomer synthesis by means of phosphoramiditriether chemistry, referred to herein as “amidite” chemistry, include N, N-diisopropylamino-β-cyanoethoxyphosphine, N, N-diisopropylamino methoxyphosphine, N, N-diethylamino-cyanoethoxyphosphine and (N-morpholino) methoxyphosphine (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; Dahl, O. Sulfur Reports, 1991, 11, 167-192). Related coupling groups, such as N, N-diisopropylamino-methylphosphine or N, N-diethylamino-methylphosphine, can also be used to produce methylphosphonates. Methylphosphonate oligomers can be conveniently synthesized using combining groups such as N, N-diisopropylamino-methylphosphoramidite. The synthesis of nucleomonomeric amidites according to the invention can be carried out by conventional methods (e.g. Gryaznov, SM, 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
Protecting groups such as diisobutylformamidine, benzoyl, isobutyryl, FMOC, dialkylformamidine, dialkylacetamidine or other groups known in the art can be used to protect exocyclic nitrogen from cytosine, adenine or guanine heterocycles. Alternatively, cytidine can be directly introduced into oligomers without a protective group on exocyclic nitrogen using the methods described (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).

 Suitable protecting groups are DMT (dimethoxytrityl), Bz (benzoyl), Bu (isobutyryl), phenoxyacetyl, MMT (monomethoxytrityl) or FMOC at the 5'-end and / or hydrophosphonate, methylphosphoramidite, methylphosphonamidite, β-cyanoethylphosphoramidylamide, ) or TBDPS (tert-butyldiphenylsilyl) 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, hydrophosphonate, methylphosphoramidite, methylphosphonamidite, β-cyanoethylphosphoramidite at the 3'end. However, it is contemplated that the positions of the protecting groups can be reversed (e.g., phosphoramidite at the 5'-position and DMT at the 3'-position). In general, derivatives of the nucleomonomers and oligomers of the invention with such “blocking groups” as shown in the corresponding formulas by methods known in the art can be obtained.

Conjugates
The invention also provides “conjugates” of the oligomers of the invention. The "conjugates" of traditional oligomers are known to those skilled in the art. For example, the oligomers of the invention can be covalently linked to various moieties, such as, for example, intercalators and compounds that specifically interact with the minor groove of the DNA double helix. Other groups for conjugation to the oligomers of interest to us include labels (for example, radioactive, fluorescence, enzyme) or groups that facilitate the association of cells using cleavable linkers, etc. Suitable radioactive labels include ³² P, ³⁵ S, ³ H, ¹³¹ I and ¹⁴ C; suitable fluorescence labels include fluorescence, resorufin, rhodamine, BODIPY (molecular probes) and Texas red; suitable enzymes include alkaline phosphatase and horseradish peroxidase. Other compounds that can be used as covalently linked moieties include biotin, antibodies or antibody fragments, azialoglycoprotein, transferrin and HIV Tat protein, which can also be covalently attached to the oligomers of the invention.

Derivatives of these additional groupings can be obtained using any convenient grouping. For example, intercalators, such as acridine or psoralen, can be coupled to the oligomer of the invention via any available —OH or —SH group, for example, at the terminal 5′-position of the oligomer, 2′-position of RNA, or OH, NH ₂ , COOH or SH introduced at the 5'-position of the pyrimidines. Convenient derivatives thereof include, for example, —CH ₂ CH ₂ CH ₂ OH or —CH ₂ CH ₂ CH ₂ SH at the 5′-position of the pyrimidines. Conjugates, including polylysine or lysine, can be synthesized as described and can further enhance the binding affinity of the oligomer to the target nucleic acid sequence (Lemaitre, M. et al., Proc NatI Acad Sci. USA, 1987, 84, 648-652; Lemaitre M. et al., Nucleosides and Nucleotides, 1987, 60, 311- 315).

 A wide variety of substituents may be attached, including those which are linked via bonds and substitute bonds. The - OH moieties in oligomers can be replaced by phosphate groups, protected by standard protecting groups, or by combination groups to obtain additional bonds with other nucleomonomers, or linked to a conjugated substituent. 5'-terminal OH can be phosphorylated; The 2'-OH or OH substituent at the 3'-end may also be phosphorylated. Hydroxyl derivatives can also be prepared using standard protecting groups.

 Covalent derivatives of the oligomers of the invention can be prepared with moieties that facilitate cell association using cleavable linkers. Suitable conjugates also include solid supports for the synthesis of oligomers and to facilitate the determination of nucleic acid sequences. Solid substrates include, but are not limited to silica gel, glass with controlled pore size, polystyrene, and ferromagnetic glass beads.

Sugar Modifications
Derivatives can be prepared by substituting sugars. Among the preferred derivatives of the oligomers of the invention, the 2'-O-allyl and 3'-allyl groups increase the penetration ability and resistance to cleavage of nucleases, but imperceptibly, they reduce the affinity of the oligomer to single-stranded or duplex targets. In particular, various functionalities at the 1 ′, 2 ′, 3 ′, 4 ′, and 5 ′ positions of the ribose moiety can be introduced into oligonucleotides with a ribose amide skeleton to improve the pharmacokinetic properties of the corresponding oligonucleotides.

Replacements
The oligomers of the invention may also contain one or more "substitutional bonds" in addition to the 2'-5, '3'-5' and 4'-5 'bonds described herein, which is generally understood from the prior art. These “substituent bonds” include phosphorothioate, methylphosphonate, thiomethylphosphonate, phosphorodithioate, alkylphosphonate, morpholinosulfamides, borophosphates (—O — P (OCH ₃ ) (BH ₃ ) —O—), siloxane (—O — Si (X ⁴ ) ( X ⁴ ) —O—; X ⁴ represents 1-6C alkyl or phenyl) and phosphoramidates (methoxyethylamine (—OP (OCH ₂ CH ₂ OCH ₃ ) (O) —O—) and the like) and can be synthesized by known methods (Sood, A., et al J. Am. Chem. Soc., 1990, 112, 9000-9001; WO 91/08213; WO 09/15065; WO 91/15500; Stirchak, EP et al Nucleic Acid Res ., 1989, 17, 6129-6141; US patent 5,034,506; US patent 5,142,047; Hewitt, JM et al., Nucleosides & Nucleotides, 1992, 11, 1661-1666; Summerton, J. et al., International publication N 216 860). Substituent bonds that can be used in the oligomers described 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'-amine (-NH-CH ₂ -, N-methylhydroxylamine (-CH ₂ -NCH ₃ -O-) and 2 ', 5' bonds (such as 2 ' 5 'carbamate (2'-N (H) -C (O) -O-5'), 5 ', 2' carbamate (2'-O-C (O) -N (H) -5 '), 5 ', 2' methyl carbamate (2'-O-C (O) -N (CH ₃ ) -5 ') and 5', 2 'thioformacetal (2'-O-CH ₂ -S-5'). Further suitable substituent bonds include amide bonds described by Buchard (Buchardt, O., et al.) (International Publication No. WO 92/20702) described by Cook (Cook, PD et al.) (International publication N WO 92/20822), described by De Mesmaeker. A., et al. (International publication N WO 92/20823) and described in PCT / US 92/04294.

Unless otherwise indicated, substitutional bonds, such as the formalcetal bond, —O — CH ₂ —O—, are attached to any of the 4 ′, 3 ′, 2 ′ carbon monomer on the left side and 5 ′ carbon nucleomonomer on the right side . The designations 4 ', 3', 2 'or 5' carbon can be modified, respectively, when a structure other than ribose, deoxyribose or arabinose is attached to the adjacent nucleomonomer. Such structures include xylose, hexose, morpholine ring, carbocyclic ring (e.g. cyclopentane) and the like.

 The use of carbamate, carbonate, sulfide, sulfoxide, sulfonic, N-methylhydroxylamine and dimethylhydrazino bonds in synthons or oligomers is known (Vaseur, JJ et al., J. Amer. Chem. Soc., 1992, 114, 4006-4007; WO 89/400/4007; 12060; Musicki, B. et al., J. Org Chem., 1990, 55, 4231-4233; Reynolds, RC et al., J. Org Chem., 1992, 51, 2983-2985; Mertes, MP, et al., J. Med. Chem., 1969, 12, 154-157; Mungall, WS, et al., J. Org Chem., 1977, 42, 703-706; Stirchak, EP, et al., J. Org Chem., 1987, 52, 4202-4206; Wang, H., et al. Tetrahedron Letts. 1991, 32, 7385-7388; international publication N PCT / US 91/03680). The substitutional bond can be used in oligomers for many purposes, such as further facilitating binding to complementary target nucleic acid sequences and / or increasing the resistance of oligomers to the action of nucleases.

Grounds
Suitable bases for use as nucleotide bases in the compounds of the invention are not only naturally occurring purine and pyrimidine bases, but also analogues of these heterocyclic bases and their tautomers. Such analogs include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Such “similar purines” or “similar pyrimidines” or analogs of purines or pyrimidines are well known in the art, some of which are used as chemotherapeutic agents. Their approximate, but not exhaustive list includes N ⁴ , N ⁴ - ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N ^6- methyladenine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-aracil, inosine, N ⁶ -isopentenyladenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine, N ⁶ -methyladenine, 5-methylguanine, 5-methylguanine methoxy aminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- (1-propynyl) -4-thiouracil, 5- (1-propynyl) -2-thiouracil, 5- (1-propynyl) -2-thiocytosine 2-thio cytosine and 2,6-diaminopurin. In addition to these base analogs, pyrimidine analogs, including 6-azacytosine, 6-azatimidine and 5-trifluoromethyluracil described by Cook (Cook, DP, et al.) In international publication WO 92/02258 can conveniently be introduced into the oligomers of the invention.

The introduction of 4-thiouridine and 2-thiothymidine into oligomers is known (Nikiforov, T.T. et al. Tetrahedron Letts., 1992, 33, 2379-2382; Clivio, P., et al. Tetrahedron Letts., 1992, 33:65 -63; Nikiforov, TT et al. Tetrahedron Letts., 1991, 32: 2505-2508; Xu, Y.-Z., et al. Tetrahedron Letts., 1991, 32: 2817-2320; Clivio, P., et al. Tetrahedron Letts., 1992, 33: 69-72; Connolly, B. A., et al., Nucleic Acids Res., 1989 17: 4957-4974). Preferred bases include adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5- (1-propynyl) uracil, 5- (1-propynyl) cytosine, 8-oxo-N ^6- methyladenine, 7-deaza- 7-methylguanine, 7-dease-7-methyladenine and 7-deazaxanthosine.

Covalently linking group
Some oligomers of the invention include a moiety capable of at least one covalent bond between the oligomer and the duplex. Multiple covalent bonds can also be formed by providing a plurality of such cross-linking moieties. A covalent bond is preferably created with the remainder of the base in the target yarn, but can also be created with another part of the target, including a saccharide or phosphodiester. The reaction nature of the group that cross-links determines the nature of the target in the duplex. Preferred cross-linking moieties include acylating and alkylating agents and, in particular, those that are positioned relative to the specificity portion of the sequence so that a reaction with the target in the strand can occur.

 Obviously, the heterocycle is not necessarily purine or pyrimidine; in fact, the pseudo base to which the reaction function is attached should not be a heterocycle at all. Any means of joining the reaction group are satisfactory as long as the position is correctly occupied.

Polarity of Oligomers
In its most general form, the symbol 3 ′ ---- 5 ′ denotes the direction of the oligomer in which bonds are sequentially formed between the 5′-hydroxyl of the amino acid residue of the nucleomonomer on the left side and 3′- (or 2′- for oligomers having 2′-5 'bonds, or 4' for oligomers having 4'-5 'bonds) with the hydroxyl of the amino acid residue of the nucleomonomer on the right side (i.e., a region of uniform polarity), thus leaving the 5'-hydroxyl of the rightmost amino acid residue of the nucleomonomer free for further conjugation. Similarly, 5 '---- 3' denotes the direction of the oligomer in the opposite orientation, when bonds are sequentially formed between the 3'-hydroxyl of the amino acid residue of the left nucleomonomer with the 5'-hydroxyl of the amino acid residue of the nucleomonomer on the right side, thus leaving the 3'-hydroxyl itself the right nucleomonomer residue free for additional conjugation. The same is true for 5 '---- 4' strands of oligomers.

Pharmaceutically Acceptable Salts
The invention also provides various salts of all of the compounds described herein, including pharmaceutically acceptable salts for use in animals or humans. Pharmaceutically acceptable salts and their constituent materials are well known in the art. The pharmaceutically acceptable salts are advantageously the metal or ammonium salts of the oligomers of the invention and include alkali and alkaline earth metal salts, for example, a sodium, potassium, magnesium or calcium salt; or predominantly easily crystallizable ammonium salts - derivatives of ammonium 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 benzyl trimethylammonium hydroxide The oligomers of the invention can form salts obtained by the addition of acids, pre appropriately therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrophilic, in particular hydrochloric or hydrobromic acid; sulfuric, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, for example formic, acetic, propionic, succinic, glycolic, lactic, malic , wine, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-hydrox ibenzoic, salicylic, 4-aminosalicylic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, sulfanilinic or cyclohexyl sulfamic acid, etc.

Applicability and Impact
Since the oligomers of the invention have significant binding activity towards single or double stranded target nucleic acids with the formation of duplexes, triplexes or other forms of stable association with naturally occurring polynucleotides or their structural analogues, the oligomers of the invention can be used in most methods, using traditional oligomers. Thus, the oligomers of the invention can be used as, 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 treatment 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 either with the development or maintenance of a pathological state using antisense oligomers. The oligomers of the invention can be used to antisense inhibition of many genetic targets. Examples of genes or RNA encoded by these genes that can be targets for antisense use of oligomers are those that encode enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules (LFA-1, GPII _a / III _a , ELAM-1, VACM-1, ICAM-1, E-selection, etc.), receptor molecules, including cytokine receptors, cytokines (IL-1, IL-2, IL-3, IL-4, IL-6, etc. .), oncogenes, growth factors and interleukins. Genes or target RNAs can be associated with any pathological condition, for example, inflammatory conditions, cardiovascular diseases, immune reactions, cancer, viral infections, bacterial infections, yeast infections, parasitic infections, and the like.

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

Indications for ex vivo applications include treating cells, such as bone marrow or peripheral blood, in conditions such as leukemia (chronic myelogenous leukemia, acute lymphocytic leukemia) or viral infection. Target genes or RNAs encoded by these genes that can serve as targets for cancer treatment include oncogenes such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl, or overexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2, and translocations such as bcr-abl. Viral gene sequences or RNA encoded by these genes, such as 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 others DNA or RNA of viruses such as HBV, HPV, VZV, influenza virus, adenoviruses, flaviviruses, rhinovirus and the like. are also suitable targets. The use of specific binding oligomers can be carried out in conjunction with other therapeutic effects. Other therapeutic uses of the oligomers of the invention include (1) modulation of inflammatory responses by modulating the expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-selection, which play a role in mediating inflammation, and (2) modulation cell proliferation in conditions such as clogged arteries (restenosis) after angioplasia by modulating the expression of (a) growth factors or mitogenic factors such as non-muscle myosin, myc, fox, PCNA, PDGF or FGF or their receptors, or (b) factors cell proliferation, such as c-myb. Other suitable proliferation factors or signal transduction factors, such as TGF _x IL-6, gINF, protein kinase C, tyrosine kinases (such as p210, p190), can be targets for the treatment of psoriasis and other conditions. Additionally, the EGF receptor, TGFa, or MHC alleles can be targets in autoimmune diseases.

 The delivery of the oligomers of the invention to cells can be enhanced by any suitable method, including calcium phosphate transfection, DMSO, glycerol or dextran transfection, electroporation or the use of cationic, anionic and / or neutral lipid compositions or liposomes by known methods (international publication N WO 90 / 14074, WO 91/16024, WO 91/17424, U.S. Patent 4,897,355). The oligomers of the invention can be introduced into cells by complexing with cationic lipids, such as DOTMA (which may or may not form liposomes), after which these complexes are brought into 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-3-dimethylaminopropyl-β-hydroxyethylammonium and its salts; and 2,2-bis (oleyloxy) -3- (trimethylammonio) propane and its salts, but are not limited to.

Enhanced delivery of oligomers according to the invention can also be achieved by using (1) viruses, such as Sendai virus (Sendai) (Bartzatt, R., Biotechnol. Appl. Biochem., 1989, 11, 133-135) or adenovirus (Wagner, E . et al., Proc Nati Acad Sci, USA, 1992, 89, 6099-6013); (2) polyamine or polycationic
conjugates using compounds such as polylysine, protamine or Na, N ₁₂ bis (ethyl) spermine (Wagner, E. et al., Proc Nati Acad Sci. USA, 1991, 88, 4255-4259; Zenke, M. et al., Proc Natl Acad Sci. USA, 1990, 87, 3655-3659; Chank, BK et al., Biochem Biophys Res Commun., 1988, 157, 264-270; U.S. Patent 5138045); (3) lipopolyamine complexes using compounds such as lipospermin (Berh, J.-P. et al., Proc Natl Acad Sci. USA, 1989, 86, 6982-6986; Loeffler, J.P. et al., J Neurochem., 1990, 54, 1812-1815); (4) anionic, neutral, or pH sensitive lipids using compounds comprising anionic phospholipids, such as phosphatidylglycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D. et al., Biochem Biophys ACTA, 1992, 1103, 185- 185- 197; Cheddar, G. et al. Arch Biochem Biophys, 1992, 294, 188-192; Yoshimura, T., et al., Biochem Int., 1990, 20, 697-706); (5) conjugates with compounds such as transferrin or biotin; or (6) conjugates with proteins (including albumin or antibodies), glycoproteins, or polymers (including polyethylene glycol), which enhances the pharmacokinetic properties of oligomers in the subject. The term transfection is used herein in relation to any method that is suitable for delivery of oligomers to cells. Any reagent, such as a lipid, or any agent, such as a virus, that can be used in a transfection protocol, is referred to herein as a “penetration enhancing agent”. Oligomers can be delivered to cells by cotransfection with other nucleic acids, such as (1) expression fragments of DNA encoding a protein (s) or a fragment of protein or (2) capable of translating RNA encoding a protein (s) or protein fragment .

 The oligomers of the invention can thus be incorporated into any suitable formulation that enhances the delivery of oligomers to cells. Suitable pharmaceutical preparations also include those which are usually used when the compounds are delivered to cells or tissues by topical application. Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonil-9, oleic acid, DMSO, polyamines or lipopoliamines can be used in topical preparations that contain oligomers.

 The oligomers of the invention can conveniently be used as reagents for research or production purposes, when inhibition of gene expression is desired. Currently, only a very small number of reagents are available that are capable of effectively and specifically inhibiting the expression of target genes in accordance with any mechanism. Oligomers whose ability to inhibit the expression of a target gene have been reported previously, in most cases, have a nonspecific effect; and / or do not reduce the expression of the target gene to very low levels (less than about 40% of uninhibited levels).

 Thus, oligomers, as described herein, constitute a reagent that can be used in methods of inhibiting the expression of a selected protein or proteins in a subject or in cells, when these proteins are encoded by DNA sequences and these proteins are translated from RNA sequences, including steps, to which: introduce the oligomer of the invention into cells; allow this oligomer to form triplex with DNA or RNA, or duplex with DNA or RNA, resulting in inhibition of protein or protein expression. The methods and compound of the invention are suitable for modulating gene expression in both prokaryotic and eukaryotic cells, such as bacteria, fungal parasites, yeast, or mammalian cells.

 RNase H "competent" or RNase H "incompetent" oligomers can be easily created using the substitution bonds of the invention. RNase H competent oligomers may contain one or more RNase H competent domains consisting of connected RNase H competent nucleomonomers. Oligomers having modifications, such as 2'-substitutions (2'-O-allyl, etc.) or some uncharged bonds (methylphosphonate, phosphoramidate, etc.), are usually incompetent as a substrate that is recognized and / or which is affected by RNase N. RNase N competence can facilitate the antisense function of the oligomer by degradation of the target RNA in the RNA oligomer duplex (Dagle, J.M. et al., Nucl Acids Res., 1990, 18, 4751-4757; Walder, JA et al., international publication N WO 89/05358). This enzyme cleaves RNA in RNA DNA duplexes.

 In order to maintain RNase H competency, the oligomer must have a RNase H competent domain of three or more competent adjacent nucleomonomers located in it (Quartin, R.S., et al., Nucl Acids Res., 1989, 17, 7253-7262). The design of oligomers resistant to the action of nucleases should contain modifications of the terminal bond, sugar and / or base to ensure resistance to nuclease. Thus, an oligomer having modified nucleomonomer residues at either one or both of the 5 ′ and / or 3 ′ ends, while having an internal RNase competent domain, can be designed. Exemplary oligomers that retain Rnase H competency should typically have uniform polarity and should contain from about 2 to about 12 nucleomonomers at the 5'-end and 3'-end, making the oligomer resistant to degradation by nucleases, and from about 3 to about 26 nucleomonomers that function as RNase H competent domain between RNase H incompetent 3'- and 5'-ends. Variations of such an oligomer should include (1) a shorter RNase H competent domain containing 1 or 2 RNase H competent bonds or substitute bonds; (2) a longer RNase H incompetent domain containing up to 15, 20 or more replacement bonds or nucleomonomers; (3) a longer RNase H competent domain containing up to 30, 40 or more bonds; (4) oligomers with only a single RNase H incompetent domain at the 3'-end or 5'-end.

 Oligomers containing only 8 nucleomonomers can be used to inhibit the expression of target protein (s) by forming duplex or triplex structures with target nucleic acid sequences. However, the linear oligomers used to inhibit the expression of the target protein by forming a duplex or triplex should preferably have from about 10 to about 20 nucleomonomer residues.

 Oligomers containing substitute bonds of the invention can conveniently be circulated in a known manner (international publication N 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 target nucleic acids. Ring oligomers can be of different sizes. Such oligomers of about 22-50 nucleomonomers in size can be conveniently prepared. Ring oligomers can have from about 3 to about 6 nucleomonomer residues in the region of the loop that separates the binding domains of the oligomer as described (Prakash, G. ibid). Oligomers can be circularly enzymatically circulated through terminal phosphates by ligase or chemically by bonding between the 5'- and 3'-terminal sugars and / or bases.

 Oligomers can be used to modulate the expression of a target gene by inhibiting the interaction of nucleic acid-binding proteins responsible for modulating transcription (Maher, L.J., et al. Science, 1989, 245, 725-730) or translation. These oligomers are thus suitable as sequence-specific agents competing with nucleic acid binding proteins (including ribosomes, RNA polymerases, DNA polymerases, translation initiation factors, transcription factors that either increase or decrease transcription, protein transcription factors hormones, etc.). Appropriately engineered oligomers can thus be used to enhance the synthesis of target proteins using a mechanism such as binding to or near the regulatory site that transcription factors use to repress expression, or by inhibiting the expression of the chosen repressor protein itself.

 The oligomers of the invention, containing additional modifications that enhance binding affinity, can be designed to contain secondary or tertiary structures such as pseudo-nodes or pseudo-half-nodes (Esker, D.J. et al. Science, 1992, 257, 958-961). Such structures may have a more stable secondary or tertiary structure than the corresponding unmodified oligomers. The enhanced stability of such structures should be based on the increased binding affinity between the regions of self-complementarity in a single oligomer or the regions of complementarity between two or more oligomers that form this structure.

 Such structures can be used to mimic structures such as the HIV TAR structure in order to inhibit the binding of HIV Tat protein (a protein that binds to TAR). A similar approach can be used for other transcription or translation factors that recognize higher-order nucleic acid structures, such as trunks, loops, hairpins, knots, etc. Alternatively, the oligomers of the invention can be used to (1) disrupt or (2) bind to structures such as (1) an obstacle or (2) enhance protein binding to nucleic acid structures.

 In addition to being used in antisense therapy or triplet helix therapy, the oligomers of the invention can also be used as therapeutic or diagnostic agents that act by directly replacing one strand in a duplex nucleic acid. Replacement of a strand in a natural duplex, such as chromosomal DNA or duplex viral DNA, RNA, or a DNA / RNA hybrid, is possible for oligomers with high binding affinities for which their complementary sequence is not so large as to effectively replace a DNA or RNA strand in a duplex. The therapeutic efficacy of oligomers that act by forming a D-loop should result from binding with high affinity to a complementary sequence, which modulates the normal biological function associated with the target nucleic acid. Types of target nucleic acids include, but are not limited to (1) gene sequences, including exons, introns, exon / intron junctions, promoter / enhancer regions, and 5 'or 3' untranslated regions, (2) nucleic acid regions that for they use secondary structures (e.g. HIV TAR stem-loop element or tRNAse), (3) nucleic acids that perform structural or other functions, such as telomeres, centromeres, or sites of origin (viruses) of replication (viruses, bacteria, etc.). ), and (4) any other duplex areas. Obviously, oligomers with discrete functional domains can be synced, in which one region of the oligomer binds the target by forming a D-loop, and the neighboring region binds the target molecule by forming a triplex helix or binding to a protein as an aptamer. Alternatively, a D-woven oligomer may bind to each strand in the duplex by switching the strand to which the oligomer binds (i.e., having one region of the oligomer that binds one strand and another region that binds the complementary strand). The control elements prescribing the type of binding (triplex, duplex or D-loop) are the oligomer sequence and the affinity characteristic of the oligomer. The basic rules for recognizing in Watson-Crick's duplex binding are different from the rules of controlled triplex binding of Hoogsteen. Because of this, the oligomer base sequence can be used to determine the type of oligomer binding rule to be used. D-loop structures are formed in nature by enzyme-mediated processes (Harris, LD et al., J. Biol Chem., 1987, 262, 9285-9292) or are associated with regions where DNA replication occurs (Jacobs, N.T. et al ., Nucl Acids Res, 1989, 17, 8949-8966). D-loops arising from the binding of oligomers can be obtained as a result of a one- or two-step process. Direct replacement of the target strand will result in a D-loop as a single binding event. However, the formation of a D-loop can also occur by the formation of a triplex helix, which facilitates the thread change event leading to the D-loop.

 Ribozymes containing substitute bonds according to the invention can be constructed in order to create species with altered characteristics. Ribozymes are known that cleave single-stranded RNA or DNA (Robertson, D.L., et al. Nature, 1990, 344, 467-468). Therapeutic uses of ribozymes are known (Sarver, N. et al. Science, 1990, 247, 1222-1225; international publication N WO 91/04319). The secondary and tertiary structure necessary for the functioning of ribozymes can be influenced by creating the corresponding oligomeric sequences. For example, ribozymes having nuclease-resistant target binding domains containing substitutional bonds of the invention may have higher affinity for target sequences while maintaining the specificity of base pairing. Due to the higher affinity and / or resistance to the action of the nuclease substitutions of the invention, shorter recognition domains can be constructed in the ribozyme (production advantage), which can lead to a more rational functional cycle of the substrate (advantage in the action of the ribozyme).

 In therapeutic applications, the oligomers of the invention can 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 various forms of use, including systemic, local or localized use. Methods and drugs are mainly described (Remington's Pharmaceutical Sciences, Merck Publishing Co., Easton, PA, latest edition). The oligomeric active ingredient is usually combined with a carrier, such as a diluent or excipient, which may include excipients, cheaper additives, binders, wetting agents, disintegrants, surfactants or lubricants, depending on the type of administration and dosage forms. Typical dosage forms include tablets, powders, liquid preparations, including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid injectable preparations, including liposome preparations.

 For systemic administration, injections are preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For injection, a preparation of the oligomers of the invention is prepared in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution. In addition, solid formulations of the oligomers of the invention can be prepared which are redissolved or suspended immediately before use. Lyophilized forms are also suitable. Doses that can be used for systemic administration are preferably in the range of 0.001 mg / kg to 50 mg / kg for administration once or twice daily. However, different dosage regimens may be used depending on (1) the ability of an individual oligomer to inhibit the activity of a target’s DNA or RNA, (2) the strength and duration of a pathological disease state associated with a given target gene, or (3) the form-kinetic properties of this oligomer.

 Systemic administration may also be via the mucous membrane or through the skin, or the compound may be administered orally. For the introduction through the mucous membrane or through the skin in the preparations, penetrating substances are used in the preparations, allowing passage through such a barrier. Such penetration promoting agents are generally known in the art and include, for example, bile salts and fusidic acid derivatives for administration through the mucosa. Additionally, detergents can be used to facilitate penetration. The introduction through the mucous membrane can be carried out using nasal aerosols, for example, or suppositories. For oral administration, oligomer preparations are prepared in a conventional oral form, such as capsules, tablets or tonics.

 For local administration, oligomer preparations of the invention are prepared in the form of ointments, gels or creams, which is well known in the art. Formulations of the oligomers of the invention for administration in eye lesions such as viral infections should be based on standard formulations known in the art.

 In addition to being used in therapy, the oligomers of the invention can be used as diagnostic reagents to determine the presence or absence of target nucleic acid sequences with which they specifically bind. The enhanced binding affinity of the oligomers of the invention provides an advantage when used as primers or probes. Diagnostic tests can be carried out by hybridization with the formation of a double or triple helix, which is then detected by traditional methods. For example, oligomers can be labeled with radioactive, fluorescent, or chromogenic labels, and the presence of the label bound to the solid support will be determined. Alternatively, the presence of a double or triple helix can be determined using antibodies that specifically recognize these forms. Means for carrying out such assays using such oligomers as probes are generally known.

 The use of oligomers with substitutional bonds according to the invention as diagnostic agents by the formation of triplexes is advantageous, since triple helices are formed under mild conditions, and analysis can be carried out without subjecting the test sample to harsh conditions. Diagnostic tests based on RNA determination to identify sequences of bacteria, fungi, or protozoa often require isolation of RNA from samples or organisms grown in the laboratory, which takes time and energy because RNA is extremely sensitive to ubiquitous nucleases.

 Oligomeric probes may also include additional modifications, such as modified sugars and / or substitution bonds, which give the oligomer particular stability to the action of nucleases, and thus, may be useful for assays performed in the presence of cell or tissue extracts, usually having nuclease activity . Oligomers containing terminal modifications often retain their ability to bind complementary sequences without loss of specificity (Uhlmann et al. Chemical Reviews, 1990, 90, 543-584). As stated above, the probes of the invention may also contain linkers that allow specific binding of alternative DNA strands by incorporating a linker allowing such binding (Froehler, BC et al. Biochemical, 1992, 31, 1603-1609; Home et al., J. Am Chem Soc., 1990, 112, 2435-2437).

 The introduction of analogues of the bases of the invention into probes, which also contain covalent cross-linking agents, makes it possible to increase the sensitivity and decrease the background in the assays for diagnosis or determination. Additionally, the use of cross-linking agents will allow new modifications of the analyzes, such as (1) the use of cross-linking to enhance the recognition of the probe, (2) the introduction of a stage of denaturation with water to reduce the background, and (3) hybridization and cross-linking at or near the melting point of the hybrid for partial destruction of the secondary structure of the target and increase the specificity of the probe. Modifications of hybridization conditions are known (Camper et al., Nucleic Acids Res., 1986, 14, 9943).

 The oligomers of the invention are suitable for use in diagnostic assays using methods in which either the oligomer or nucleic acid to be determined is covalently linked to a solid support (as described in US Pat. No. 4,775,619). These oligomers are also suitable for use in diagnostic assays that are based on the polymerase chain reaction technique for amplifying target sequences according to the prior art (European Patent Publication No. 0393744). The oligomers of the invention containing the 3'-end, which can serve as a primer, are compatible with polymerases used in the polymerase chain reaction technique, such as Taq or Vent (New England Biolabs) polymerase. The oligomers of the invention can thus be used in PCR protocols.

 The oligomers of the invention are useful as primers that have discrete sequences, or as primers with an arbitrary sequence. Random sequence primers typically have a length of about 6, 7, or 8 nucleomonomers. 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 ability of the oligomer to function as a primer. Oligomers according to the invention having 2'-modifications at sites that are not 3'-terminal residues, other modifications that give the RNase H oligomer incompetence or other resistance to the action of nucleases, can be successfully used as probes or primers for RNA or DNA sequences in cell extracts or other solutions containing nucleases. Thus, oligomers can be used in protocols for amplifying nucleic acids 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 using PCR, LCR or other suitable methods.

 Derived oligomers obtained with chelating agents such as EDTA, DTPA, or 1,2-diaminocyclohexanacetic acid analogs can be used in various in vitro diagnostic assays (US Pat. Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, derivatives of the oligomers of the invention can be prepared using cross-linking agents such as 5- (3-iodoacetamidoprop-1-yl) 2'-deoxyuridine or 5- (3- (4-bromobutyramido) prop-1-yl) - 2'-deoxyuridine, and used in various assay methods or kits (international publication WO 90/14353).

 In addition to the above applications, the ability of the oligomers of the invention to inhibit gene expression can be tested in an in vitro system by measuring expression levels in subject cells or in recombinant systems using any suitable method (Graessmann, M. et al., Nucleic Acids Res., 1991, 19, 53-59).

 The invention described above is illustrated by the following examples to better understand it. The examples are intended to illustrate the invention and should not be construed as limiting this invention.

Examples
Overview of the synthesis of nucleomonomer synthons and oligomers
The oligomers of the invention can be synthesized using reactions known in the art for the synthesis of oligonucleotide derivatives (Flandor, J. and Yam, SY, Tetrahedron Letts., 1990, 31, 597-600; Mattson, RJ 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 substitute bonds specifically listed in table. And, the substituent bonds according to the invention can be varied so as to contain one or more nitrogen, sulfur and / or oxygen atoms in their structure. The positions of these atoms in the substituent bonds can be different and can be located at the “5 '” end, “in the middle”, at the “2'” or “3 '” and “4'” end. This section provides a series of drawings representing synthesis reactions that show how to achieve different locations and combinations of nitrogen and oxygen atoms in the substituent bonds.

 The synthesis method shown in FIG. 1-25, can be modified in a manner known to those skilled in the art of oligonucleotide chemistry. For example, although the step of protecting the bases is not always indicated in the drawings, it may be desirable and carried out using reagents and techniques known in the art (see, for example, Protective Groups in Organic Synthesis, Theodora W. Greene, John Wiley and Sons, 1981). Similarly, although the use of protective groups is indicated in some cases, blocking of reagents for the synthesis of oligomers illustrating the invention is not always necessary.

Example 1
The first five stages shown in FIG. 1 relate to the preparation of isobutyryl-protected amino acid alcohol of serinol. The sixth and subsequent stages in FIG. 1 are directed to the synthesis of a serine-substituted thymine phosphoramidite building block.

In step 1 of FIG. The amino group 1 of the serine amino acid is protected by reacting 1 with di-tert-butyl dicarbonate to give compound 2. Other equivalent protecting groups may be used. In the next step, the β-hydroxyl group of compound 2 is blocked with dihydropyran to give fully protected amino acid 3. The amino acid 3 is then reacted with the diborane-dimethyl sulfide complex to give alcohol 4, which gives 5 under the influence of isobutyryl chloride. This reduction reaction can also be carried out using isobutyl chloroformate and sodium borohydride (Ramasamy, RK Olsen and T. Emery, Synthesis 1982, 42). Reaction of 5 with trifluoroacetic acid for 30 minutes followed by washing with NaHCO ₃ gives 6.

 Thymine acetic acid 7 was prepared as described in the literature (L. Kosynkina, W. Wang and T. C. Liang, Tetrahedron Letts, 1994, 35, 5173). A combination of 7 with 6 under mixed anhydride conditions gives 8. Dimethoxytrilation of compound 8 with DMTCI gives compound 9, which, after hydrolysis with a base, gives 10. Phosphitylation of compound 10 under standard conditions gives a thymine building block bound to serinol 11. This synthon can then be added to growing oligomer using traditional chemistry methods. Any DNA synthesis chemistry, such as phosphosoramidate or phosphonate chemistry, can be used to bind monomers or dimers in the same manner as above.

Example 2
In the reaction scheme of FIG. 2 thymine acetaldehyde 13 is obtained by treating thymine with bromoacetaldehyde dimethylacetal followed by hydrolysis of compound 12 with aqueous TFA. Aldehyde 13 and amine 6 are then combined and transformed into the corresponding intermediate compound into the phosphoramidite building block 17 in a manner analogous to the step used in FIG. 1.

Example 3
In the reaction scheme of FIG. 3, the starting material is a β-substituted amino acid 18. The substituted amino acid must be transformed into the phosphoramidite building block 27 with the subsequent steps used in FIG. 1 and 2.

Example 4
In FIG. 4, starting amino alcohol 21 is oxidized with a mixture of CrO ₃ / pyridine to give aldehyde 28. This aldehyde, when reacted with an alkyl halide in the presence of a base, should give compound 29. Amino alcohol 29 can then be transformed into building block 35 in a manner similar to the steps used in FIG. 1 and 2.

Example 5
In FIG. 5, the first four stages are essentially the same as the first four stages used in FIG. 1, only aspartic acid is used instead of serine. Aspartic acid methyl ester 36 gives a fully protected alcohol 40 which, after selective deprotection with acetic acid, gives 41. Oxidation of 41 with CrO ₃ / pyridine gives the corresponding aldehyde 42. The reductive amination of aldehyde 42 with o-benzylhydroxylamine in the presence of sodium triacetoxyborohydride should give 43 (T. Kolasa and M.J. 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 hydroxyl function 39. The combination of aldehyde 46 and hydroxylamine 43 in the presence of sodium triacetoxyborohydride followed by deprotection of the amino protecting groups should result in bisamine 48. Bisamine 48 can then be converted to dimer 53, followed by the steps used in FIG. 1.

Example 6
In FIG. 6, the combination of alcohol 54 with O-benzylhydroxylamine 55 under the Mitsunobu reaction (Mitsunobu, Sinthesis, 1981, 1) gives compound 56. Intermediate 56 during hydrogenation followed by acetylation should lead to 57. “TBDMSI” protective compounds are released under the action of TFA in compound 57 groups and it turns out 58. A combination of 58 with 7 followed by dimethoxytritylation should give 60. The final building block 62 is obtained from 60 by basic hydrolysis followed by phosphitylation.

Example 7
In FIG. 7, serinol 4 is converted to halide 59 and alkylated with thymine to give 63. The protecting groups of 63 are removed, combined with DMT-protected hydroxyacetic acid and phosphitylated to give 66.

Example 8
In FIG. 8, alcohol 64 is combined with N-hydroxylaminopropanoic acid 69 to give 70. Alkylation of thymine with halide 73 gives 74, which, after deprotection, combination with 76 followed by hydrolysis, should result in 78. Condensation of 78 with 70 followed by phosphitylation should give hydroxamate dimer 80.

Example 9
In FIG. 9, N-hydroxylaminopropane aldehyde 81 is used to combine with alcohol 64. Dimer 88 is obtained from 83 and 86 followed by the steps used in FIG. 8.

Example 10
In FIG. 10 alkylation (T. Kolasa and M.J. Miller, J. Org Chem., 1990, 55, 4246) of α-bromo-β-aminopropanoic acid methyl ester 89 with thymine gives 90. Intermediate 90 upon hydrolysis with sodium hydroxide gives acid 91 which is combined with 6 to give 92. Compound 92 is then converted to phosphoramidite building block 95 using the steps described in FIG. 1.

Example 11
In FIG. 11 thymine is alkylated with alkylamino halide 96 (for the production of aminoalkyl halide see RK Olsen, K. Ramasamy and T. Emery, J. Org. Chem., 1984, 49, 3527 and Islam et al., J. Med. Chem., 1994, 37, 293 -304) to give 97. Compound 97 under the influence of TFA followed by alkylation will give 100. Building block 103 is obtained from 100 by dimethoxytritylation, hydrolysis and subsequent phosphitylation.

Example 12
In FIG. 12 shows an alternative way of producing a dimer 111 with a hydroxamate skeleton from N-hydroxylamine 43 and aldehyde 107, which in turn is derived from aspartic acid.

Example 13
In FIG. 13, dimer 115 is prepared from intermediate 108 and 13, followed by the steps described in FIG. 2.

Example 14
In FIG. 14 receive N-hydroxylthymine (see Kim, CU, et al. Tetrahedron Letts., 1992, 33, 25-28) and combine with N-hydroxyphthalimide to give 117, which should give 118 under the action of hydrazine in ethanol. Treatment 118 DMT- protected glycerol epoxide 119 gives 120. Intermediate 120 is then converted to phosphoramidite 121 using standard techniques. In the second synthesis, compound 118 is combined with amino acid aldehyde 122 under reductive amination conditions to give 123. Protection of secondary amino functionality with FMOCCI followed by hydrolysis gives 125.

Example 15
In FIG. 15 1,2-hydroxypropanoic acid 126 is combined with N-hydroxylamine thymine 118 to give 127, which is then transformed into phosphoramidite synthon 129 under standard conditions. Compound 118 is also combined with adipic acid and transformed into the nucleic acid building block 133.

Example 16
In FIG. 16, the first building block 136 is synthesized from 118 and 134 similar to that described in FIG. 1 way. The combination of 139 with 118 will give 140. Processing 137 with 118 should give 138, which when condensed with 140 gives a dimer 141.

Example 17
In FIG. 17 aldehyde 142 and glycine benzyl ester are combined to give 143. Treatment 143 with 7 gives 145, which under the action of acetic acid gives 148. Mitsunda alkylation 148 with Boc-NH-O-acetylhydroxylamine gives 147, which, when hydrogenated, gives building block 150 Similarly, a combination of 143 with 13 with the same as above, subsequent reactions gives synthone 149.

Example 18
In FIG. 18 reductive amination of aldehyde 142 and Boc-NH-O-benzylhydroylamine gives 151. Hydrogenation 151 followed by alkylation of 152 with glycolic acid 153 (BC Borer and DC Balogh, Tetrahederon Letts, 1991, 32, 1039) will yield 154. Processing 154 with TFA will remove Boc is a protecting group, which when combined will give 155. Hydroxyl protecting groups at 155 can be selectively removed with acetic acid to give 156. Then the compound will be transformed into building block 157 using standard reaction conditions. Similarly, building block 158 will be obtained by combining 154 with 13 followed by the steps used to make 157.

Example 19
In FIG. 19, the alkylation of thymine-N-hydroxylamine 160 with alcohol 162 will result in 163. Compound 163 can be transformed into phosphoramidite building block 166 using the subsequent steps used in FIG. 1.

Example 20
In FIG. 20, the first intermediate 169 is synthesized from glutamic acid using standard reaction conditions. Alkylation of thymine with 169 will give 170, which, when processed with TFA, will give 171. Intermediate 171 can be combined with Boc-glycine to give 173, which upon hydrolysis gives monomeric synthon 174. Similarly, 172 is combined with 118 and Boc-aminoacetic aldehyde with subsequent hydrolysis of benzyl ether.

Example 21
In FIG. 21 intermediate 177 was prepared from Boc-NH-O-benzylhydroxylamine and 175 using standard reaction conditions. Hydrogenation of 177 followed by coupling with N-hydroxythymine 116 will give 178. Removal of the THP protecting group followed by dimethoxytritylation and phosphitylation will produce a building block synthone 181. Similarly, 182 can be obtained by following the above reactions using THP-hydroxyacetic aldehyde instead of THP-hydroxyux .

Example 22
In FIG. 22, a building block 191 can be obtained using known starting material 183 following the reaction conditions indicated below in FIG. 22.

Example 23
In FIG. 23, the synthesis of building block 199 can be carried out using starting material 183, following the reaction conditions indicated below in FIG. 23.

Example 24
In FIG. 24, the source material 200 is transformed into a building block 207, following the reaction conditions indicated below in FIG. 24.

Example 25
The compounds used and prepared in this example are shown in FIG. 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 the solution was heated in a 40 ^° C water bath, a solution of bromoacetic acid (62.5 g, 450 mmol) in 100 ml of water was added over 1 h. The reaction mixture was stirred for another 1 h at this temperature. The mixture was allowed to cool to room temperature and the pH was adjusted to 5.5 with conc. HCl. The solution was then cooled in the refrigerator for 2 hours. Any precipitate (unreacted thymine) was removed by filtration. Then the solution was adjusted to pH 2 with conc. HCl and placed in a freezer for 2 hours. A white precipitate was collected by filtration and dried in a vacuum oven at 40 ^° C for 6 hours. The yield was 44 g (88%).

N-Boc-L-Serine methyl ester (2):
L-serine methyl ester (15.6 g, 100 mmol) was suspended in a mixture of THF / DMF (100 ml each) at room temperature. Triethylamine (11.13 g, 110 mmol) was added to this stirred mixture, followed by di-tert-butyl dicarbonate (24.0 g, 110 mmol) and stirring was continued at room temperature for 30 minutes. Water (20 ml) was added and the solution was stirred at room temperature for 8 hours. The solution was evaporated to dryness. The residue was suspended in ethyl acetate (250 ml) and treated with potassium hydrosulfate (0.25 N solution, 100 ml). This purge was then 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 gave an oily residue in an amount of 26 g (90%).

N-Boc-L-Serine Methyl Ether (OTHP) (3):
Compound 2 (15 g, 68.49 mmol) was dissolved in dry CH ₂ Cl ₂ (100 ml) and treated with 3,4-dihydro-2H-pyran (8.4 g, 100 mmol) and a catalytic amount of p-toluenesulfonic acid ( 100 mg) at room temperature. The reaction mixture was allowed to stir at room temperature for 12 hours 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. Such a residue is pure enough for use in the next step, and is used as such. Yield 15 g (72%).

N-Boc-L-Serinol (OTHP) (4):
Serine methyl ester (OTHR) (10 g, 33 mmol) was dissolved in dry THF (100 ml) and cooled to 0 ^° C. in an ice bath under argon atmosphere. To this cooled mixed solution was added a boromethyl sulfide complex (2 M solution in THF, 100 ml, 200 mmol) for 1 h at 0 ^° C. After adding boron, the reaction mixture was warmed to room temperature and kept at 40 ^° C for 6 hours. The reaction mixture was cooled to 0 ^° C., neutralized with water and acetic acid to a pH of 6-7, and extracted with ether (3 x 100 ml). The ether extract was washed with water (2 x 200 ml) and brine (100 ml), dried over anhydrous Na ₂ SO ₄ and evaporated to dryness to give the crude product as an oil. From this oil, purification by flash chromatography on silica gel using hexane -> acetone as the eluent gave 8 g (88%) of pure product.

N-Boc-L-Serine (OTHP) OIb (5):
To a mixed solution of compound 4 (8 g, 29.09 mmol) in dry CH ₂ Cl ₂ (100 ml) at 0 ^° C was added TEA (3.54 g, 35 mmol), followed by isobutyryl chloride (3.71 g, 35 mmol) for 30 minutes Then the reaction mixture was 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. From this oil, purification by flash chromatography on silica gel using hexane -> acetone as the eluent gave 7.9 g (79%) of pure product.

L-Serinol (OIb) (6):
Compound 5 (10 g, 28.98 mmol) was dissolved in CH ₂ Cl ₂ (100 ml), allowed to stir at room temperature with TFA (50 ml) for 1 h 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 NaHSO ₃ solution (2 x 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 4.5 g (96%) of the product as an oil.

N- (thyminylacetyl) -L-serine (OIb) (8):
Thymine acetic acid (7) (7.3 g, 4 mmol) and N-methylmorpholine (4.4 ml, 40 mmol) were dissolved in 100 ml of DMF. Allowed the solution to cool to -20 ^o C in an argon atmosphere. To this cold stirred solution, isobutyl chloroformate (5.2 ml, 40 mmol) was added once. After 15 minutes, a solution of 6 (6.44 g, 40 mol) 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 stirring was continued for 1 hour. 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. 12 g (92%) of pure product were obtained from this crude product by purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone as eluent.

4,4'-Dimethoxytrityl-N- (thyminylacetyl) -L-serinol (OIb) (9):
Compound 8 (10 g, 30.58 mmol) was coevaporated with dry pyridine (3 x 50 ml) and dissolved in dry pyridine (100 ml). To this solution was added TEA (3.54 g, 35 mmol), followed by DMTCl (11.83 g, 35 mmol) at room temperature under argon. The reaction mixture was stirred for 12 hours, neutralized with methanol (20 ml) and stirred for 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. From this crude product, purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone as the eluent gave 17 g (88%) of pure product.

1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -L-propan-1,3-diol (10):
Compound 9 (10 g, 15.89 mmol) was dissolved in methanol (20 ml). To this solution was added 1 N NaOH solution (20 ml, 20 mmol) at 0 ^° C. The reaction mixture was stirred for 1 h, neutralized with acetic acid to pH 7. The solution was extracted with EtOAc (2 x 100 ml). The organic extract was 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 the crude product as a foam. 8.2 g (92%) of pure product were obtained from this crude product during purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone as eluent.

1'-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -L-propan-3'-O- (N, N-diisopropyl) -β-cyanoethylphosphoramidite (11):
Compound 10 (8.00 g, 14.31 mmol) was coevaporated with dry pyridine (3 x 50 ml) and dried over solid NaOH overnight under vacuum. The dried material was dissolved in dry CH ₂ Cl ₂ (100 ml) and cooled to 0 ^° C. under argon. To this cooled solution was added N, N-diisopropylethylamine (5.23 g, 25 mmol), followed by 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (4.72 g, 20.00 mmol) under argon. The reaction mixture was stirred at 0 ^° C. for 1 hour and at room temperature for 1 hour. The reaction mixture was diluted with CH ₂ Cl ₂ (100 ml). The CH ₂ Cl ₂ solution was washed with 5% NaH-CO 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. From this crude product, upon purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone containing 0.1% TEA, 10 g of pure product was obtained as eluent. This foam was dried over solid NaOH in vacuo overnight. The foam was dissolved in CH ₂ Cl ₂ (15 ml) and added dropwise to a stirred solution of dry hexane (2000 ml) in argon for 1 h. After adding the CH ₂ Cl ₂ solution, the resulting precipitate was stirred for another 1 h and filtered, washed with dry hexane (200 ml) and dried over solid NaOH overnight. Yield 9.5 g (87%).

Example 26 (see FIG. 26)
N- (tert-butyloxycarbonyl) -O-benzyl-L-serine (2):
O-benzyl-L-serine (1) (10 g, 51.28 mmol) was suspended in a mixture of THF / H ₂ O (8: 2, 100 ml) at room temperature. To this stirred mixture was added triethylamine (6.06 g, 60 mmol) and then 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 0.5 N 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- (tert-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 to -20 ^° C under argon. To this cold mixed solution were added TEA (2.32 g, 23 mmol) and isobutyl chloroformate (3.13 g, 23 mmol). Stirring was continued for 30 minutes at -20 ^° C. under argon. The reaction mixture was immediately filtered under a layer of argon, 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 a mixture of THF / water (80: 20, 200 ml) over 10 minutes. After this addition, the reaction mixture was stirred for 2 hours at 0 ^° C. and the pH was adjusted to 7 with acetic acid. The solution was evaporated to dryness, fractionated in ethyl acetate / water (300: 150 ml) and extracted in 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 on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The fractions containing the pure product were combined and dried to dryness to give 4.7 g (82%) of the pure product as an oil.

¹ H-NMR (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), 7.30-7.40 (m, 5H, Ph).

N- (tert-butyloxycarbonyl) -O-benzyl-L-serinol-O-Ib (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. To this stirred solution was added isobutyric anhydride (3.16 g, 20 mmol) and stirring was continued overnight in an argon atmosphere. The reaction mixture was evaporated to dryness, fractionated in a mixture of 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. This residue was purified by flash chromatography on silica gel using hexane -> EtOAc as eluent. The pure fractions were combined and evaporated to give 4.5 g (84%) of an oily product.

¹ H-NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH 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), 7.24-7 40 (m, 5H, Ph).

N- (thyminylacetyl) -O-benzyl-L-serinol-O-Ib (6):
N- (tert-butyloxycarbonyl) -O-benzyl-L-serinol-O-Ib (4) (4.3 g, 12.02 mmol) was allowed to stir at room temperature in trifluoroacetic acid (20 ml) and CH ₂ Cl ₂ (20 ml) for 30 minutes. The reaction mixture was evaporated to dryness, dissolved in dry CH ₃ OH (10 ml) and evaporated again to dryness. The residue was dried over solid KOH under vacuum for 12 hours. The dried residue was used as such for further reactions without analysis.

Thymine acetic acid 5 (2.76 g, 15 mmol) was dissolved in dry DMF (75 ml) and cooled to -20 ^° C in argon. To this cold mixed solution was added N-methylmorpholine (1.72 g, 17 mmol), and then isobutyl chloroformate (2.31 g, 17 mmol). After 15 minutes of stirring, a solution of the above TFA salt in dry DMF (50 ml) was neutralized with N-methylmorpholine (1.72 g, 17 mmol) and thymine acetic acid was added in a cold mixed solution. The reaction mixture was stirred at -20 ^° C for 1 h, warmed to room temperature and stirring was continued 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 in 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 a crude product. 4.8 g (94%) of pure product were obtained from this crude product by purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone as eluent. This pure product was crystallized from a mixture of CH ₂ Cl ₂ / hexane. Mp 122-124 ^o C.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH 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 ₃ Ph), 7.24-7.40 (m, 6H, C ₆ H and Ph), 8.22 (d, 1H, NH) and 11.22 (s, 1H, NH).

N- (thyminylacetyl) -L-serinol-O-Ib (7):
N- (thyminylacetyl) -O-benzyl-L-serinol-O-Ib (6) (2.08 g, 5 mmol) was dissolved in ethanol (50 ml). Pd (OH) ₂ (0.6 g) and cyclohexene (5 ml) were added to this solution at room temperature. The reaction mixture was heated at 70 ^° C. for 12 hours. 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 minimum amount of MeOH and cooled to room temperature. This product was crystallized as a fine powder. Mp 196-198 ^o C. Yield: 1.48 g (91%).

¹ H NMR (Me ₂ SO-d ₆ ): δ 1.04 (d, 6H, IbCH 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 (8 , 1H, C ₆ H), 8.12 (d, 1H, NH), 11.22 (s, 1H, NH).

4,4'-dimethoxytrityl-N- (thyminylacetyl) -L-serine-O-Ib (8):
N- (thyminylacetyl) -L-serynol-O-Ib (7) (1.48 g, 4.5 mmol) was dissolved in dry pyridine (50 ml) in argon. To this stirred solution were added TEA (0.51 g, 5 mmol) and N, N-dimethylaminopyridine (0.10 g). After 10 minutes, 4.4'-dimethoxytrityl chloride (1.69 g, 5 mmol) was added and stirring was continued at room temperature in argon overnight. The reaction mixture was neutralized with MeOH (10 ml), stirred for 10 minutes 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 chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 2.5 g (88%) of a foam.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.40 (m, 1H, IbCH), 3.38 (m, 2H), 3.72 (s, 6H, 2 • OCH ₃ ), 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), 11.28 (s, 1H, NH).

1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -L-propan-1,3-diol (9):
4,4'-dimethoxytrityl-N- (thyminylacetyl) -L-serinol-O-Ib (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 2 N NaOH (10 ml, 20 mmol) and stirring was continued for 30 minutes at 0 ^° C. The solution was adjusted to pH 7 with acetic acid and evaporated to dryness. The residue was fractionated in a mixture of water (50 ml) and CH ₂ Cl ₂ (150 ml) and extracted into CH ₂ Cl ₂ . The aqueous layer was again 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 on silica gel using CH ₂ Cl ₂ -> acetone as eluent. Yield: 3.0 g (99%).

¹ H NMR (CDCl ₃ ): δ 1.72 (s, 3H, CH ₃ ), 3.0 (m, 2H), 3.42 (m, 2H), 3.72 (s, 6H, 2 • OCH 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), 11.28 (bs, 1H, NH).

1-O- (4,4'-dimethoxytrityl) -2- [anino (thyminylacetyl)] - L-propan-3-O- (N, N-diisopropyl) -β-cyanoethylphosphoramidite (10):
1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -L-propan-1,3-diol (9) (3.1 g, 5.55 mmol) was dried over solid NaOH under vacuum overnight and dissolved in dry CH ₂ Cl ₂ (100 ml). This solution was cooled to 0 ^° C. under argon. To the cold mixed solution was added N, N-diisopropylethylamine (1.29 g, 10 mmol), followed by 2-cyanoethyl-N, N-diisopropyl-chloro-phosphoramidite (1.96 g, 8.3 mmol). The reaction mixture was stirred at 0 ^° C. for 1 hour and at room temperature for 2 hours. The reaction mixture 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 Cl ₂ extract was dried and evaporated to dryness to give an oily residue. This residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc containing 0.1% TEA as eluent. Pure fractions were combined and evaporated to give a foam. This foam was dried on solid NaOH under vacuum overnight. The dried foam was dissolved in dry CH ₂ Cl ₂ (20 ml) and added dropwise to a stirred solution of dry hexane (2000 ml) under argon for 1 h. After addition, the precipitate formed was stirred for another 1 h and filtered, washed with dry hexane (100 ml ) and the solid was dried over solid NaOH in vacuo for 4 hours. Yield: 3.5 g (83%).

Example 27 (see FIG. 27)
N- (tert-butyloxycarbonyl) -O-benzyl-D-serine (12):
O-benzyl-D-serine (11) (5 g, 25.64 mmol) was suspended in a mixture of THF / H ₂ O (8: 2, 70 ml) at room temperature. 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 at room temperature overnight. The homogeneous solution was evaporated to dryness and the residue was dissolved in ethyl acetate (150 ml). The organic extract was washed with 0.5 N 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-serynol (13):
N- (tert-butyloxycarbonyl) -O-benzyl-D-serine (12) (7.56 g, 25.63 mmol) was dissolved in dry THF and cooled to -20 ^° C under argon. To this cold mixed solution was added TEA (3.03 g, 30 mmol) and isobutyl chloroformate (4.08 g, 30 mmol). Stirring was continued for 30 minutes at -20 ^° C. under argon. The reaction mixture was immediately filtered under a layer of argon, 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 a mixture of THF / water (80: 20, 200 ml) over 10 minutes. After this addition, the reaction mixture was stirred for 2 hours at 0 ^° C. and the pH was adjusted to 7 with acetic acid. The solution was evaporated to dryness, fractionated in 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 on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The fractions containing the pure product were combined and dried to dryness to give 6.68 g (92%) of the pure product as an oil.

¹ H NMR (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), 7.30-7.40 (m, 5H, Ph).

N- (tert-butyloxycarbonyl) -O-benzyl-D-serynol-O-Ib (14):
To a dried solution of N- (tert-butyloxycarbonyl) -O-benzyl-D-serinol (13) (6.6 g, 23.5 mmol) in dry pyridine (50 ml) was added TEA (3.03 g, 30 mmol) at room temperature. Isobutyric acid 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, fractionated in a mixture of 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. This residue was purified by flash chromatography on silica gel using hexane -> EtOAc as eluent. The pure fractions were combined and evaporated to give 8.0 g (97%) of an oily product.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH 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), 7.24-7, 40 (m, 5H, Ph).

N- (thyminylacetyl) -O-benzyl-D-serynol-O-Ib (15):
N- (tert-butyloxycarbonyl) -O-benzyl-D-serynol-O-Ib (14) (5.0 g, 14.25 mmol) was allowed to stir at room temperature in trifluoroacetic acid (20 ml) and CH ₂ Cl ₂ (20 ml) for 30 minutes. 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), adjusted to pH 7 with a 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. The resulting residue was dried over solid KOH under vacuum for 12 hours. The dried residue was used as such for further reactions without analysis.

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 mixed solution was added N-methylmorpholine (1.52 g, 15 mmol), followed by isobutyl chloroformate (2.04 g, 15 mmol). After 15 minutes of stirring, a solution of the above amine in dry DMF (50 ml) was added to a cold stirred solution of thymine acetic acid. The reaction mixture was stirred at -20 ^° C for 1 h, warmed to room temperature and stirring was continued 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 in 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 a crude product. 2.8 g (54%) of pure product are obtained from this crude product by purification by flash chromatography on silica gel using CH ₂ Cl ₂ -> acetone as eluent.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH 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 obtained by using the method for the preparation of the L isomer. Reagents used: thyminacetic 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%).

N- (thyminylacetyl) -D-serynol-O-Ib (16):
N- (thyminylacetyl) -O-benzyl-D-serynol-O-Ib (15) (3.5 g, 8.39 mmol) was dissolved in ethanol (50 ml). Pd (OH) ₂ (1.00 g) and cyclohexene (10 ml) were added to this solution at room temperature. The reaction mixture was heated at 70 ^° C. for 12 hours. 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 H NMR (Me ₂ SO-d ₆ ): δ 1.04 (d, 6H, IbCH 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), 11.22 (s, 1H, NH).

4,4'-dimethoxytrityl-N- (thyminylacetyl) -D-serine-O-Ib (17):
N- (thyminylacetyl) -D-serynol-O-Ib (16) (2.7 g, 8.26 mmol) was dissolved in dry pyridine (50 ml) in argon. To this stirred solution was added TEA (1.01 g, 10 mmol) followed by 4,4'-dimethoxytrityl chloride (3.38 g, 10 mmol) and stirring was continued at room temperature in argon overnight. The reaction mixture was neutralized with MeOH (10 ml), stirred for 10 minutes 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 evaporated to dryness. The crude product was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 5.0 g (96%) of a foam.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH ₃ ), 1.72 (s, 3H, CH ₃ ), 2.40 (m, 1H, IbCH), 3.38 (m, 2H), 3.72 (s, 6H, 2 • OCH ₃ ), 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), 11.28 (s, 1H, NH).

1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -D-propan-1,3-diol (18):
4,4'-dimethoxytrityl-N- (thyminylacetyl) -D-serinol-O-Ib (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 stirred solution was added 2 N NaOH (10 ml, 20 mmol) and stirring was continued for 30 minutes at 0 ^° C. The solution was adjusted to pH 7 with acetic acid and evaporated to dryness. The residue was fractionated in a mixture of water (50 ml) and CH ₂ Cl ₂ (250 ml) and extracted into CH ₂ Cl ₂ . The aqueous layer was again 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 on silica gel using CH ₂ Cl ₂ -> acetone as eluent. Yield: 4.0 g (90%).

¹ H NMR (CDCl ₃ ): δ 1.72 (s, 3H, CH ₃ ), 3.0 (m, 2H), 3.42 (m, 2H), 3.72 (s, 6H, 2 • OCH 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), 11.28 (bs, 1H, NH).

1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -D-propan-3-0- (N, N-diisopropyl) - β-cyanoethylphosphoramidite (19):
1-O- (4,4'-dimethoxytrityl) -2- [amino (thyminylacetyl)] -D-propane-1,3-diol (18) (2.79 g, 5.0 mmol) was dried over solid NaOH under vacuum overnight and dissolved in dry CH ₂ Cl ₂ (100 ml). This solution was cooled to 0 ^° C. under argon. N, N-diisopropylethylamine (1.29 g, 10 mmol) was added to the cold stirred solution, followed by 2-cyanoethyl N, N-diisopropyl chlorophosphoramidite (1.96 g, 8.3 mmol). The reaction mixture was stirred at 0 ^° C. for 1 hour and at room temperature for 2 hours. The reaction mixture 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 Cl ₂ extract was dried and evaporated to dryness to give an oily residue. This residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc containing 0.1% TEA as eluent. Pure fractions were combined and evaporated to give a foam. This foam was dried on solid NaOH under vacuum overnight. The dried foam was dissolved in dry CH ₂ Cl ₂ (20 ml) and added dropwise to a stirred solution of dry hexane (2000 ml) under argon for 1 h. After addition, the precipitate formed was stirred for another 1 h and filtered, washed with dry hexane (100 ml ) and the solid was dried on solid NaOH under vacuum for 4 hours. Yield: 3.3 g (87%).

Example 28 (Fig. 28)
1-O-benzyl-2- [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L-propanol (21):
To a mixed solution of N ₃ -benzoylthymine (20) (5.75 g, 25 mmol) in dry THF (200 ml) under argon was added triphenylphosphine (10.48 g, 40 mmol) and Nα-tert-butyloxycarbonyl-β-benzyloxy - L-serinol 3 (5.3 g, 18.86 mmol) at room temperature. After 15 minutes, diethyl azodicarboxylate (6.96 g, 40 mmol) was added slowly over 30 minutes. The reaction mixture was covered with aluminum foil and allowed to stir at room temperature under argon for 24 hours. 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 . Dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography on silica gel using hexane -> EtOAc as eluent. Fractions containing the desired product were combined and evaporated to give a pale pink oil. Yield: 8.0 g (86%).

¹ H NMR (CDCl ₃ ): δ 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), 7.20- 7.60 (m, 10H, Ph).

2 - [(tert-butyloxycarbonyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L-propan-1-ol (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 (10%, 1 g). The reaction mixture was hydrogenated at 50 psi of hydrogen for 12 hours. The catalyst was filtered off, washed with MeOH (50 ml) and the filtrate was evaporated to dryness. The residue was crystallized from acetone / hexane to give 3.70 r (92%) of pure product. Mp 156-159 ^o C.

¹ H NMR (CDCl ₃ ): δ 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), 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 (thyminyl) -L-propan-1-ol (22) (1.60 g, 3.97 mmol) was dissolved in dry pyridine (30 ml ) and left to mix at room temperature under argon. To this mixed solution were added TEA (0.51 g, 5 mmol) and isobutyric anhydride (0.79 g, 5 mmol). The reaction mixture was stirred at room temperature for 12 hours 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 column chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 1.6 g (85%) of a foam. The pure product was crystallized from acetone / hexane. Mp 165-167 ^o C.

¹ H NMR (CDCl ₃ ): δ 1.16 (d, 6H, IbCH 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), 7.98 (d, 2H, Ph).

1-O-Isobutyryl-2- [(β-hydroxyacetyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L-propanol (24):
1-O-Isobutyryl-2- [(tert-butyloxycarbonyl) amino] -3- [N ₃ - benzoyl (thyminyl) -L-propanol (23) (1.6 g, 3.38 mmol) was allowed to mix in a mixture TFA (5 ml) and CH ₂ Cl ₂ (10 ml) at room temperature for 30 minutes and evaporated to dryness. The residue was dissolved in dry MeOH (10 ml) and evaporated again. The resulting residue was dried over solid NaOH in vacuo overnight. The dried material was used as such for the next reaction.

To a mixed 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- (3-dimethylaminopropyl) -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 hours and evaporated to dryness. The residue was fractionated in a mixture of 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 on silica gel using CH ₂ Cl ₂ -> acetone as eluent. Fractions containing the desired product were combined and evaporated to give 1.35 g (92%) of a foam.

¹ H NMR (CDCl ₃ ): δ 1.16 (d, 6H, IbCH 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); 7.94 (d, 2H, Ph).

1-O-Isobutyryl-2 - [(β- (4,4'-dimethoxytrityl) -O-acetyl) amino] - 3- [N ₃ -benzoyl (thyminyl) -L-propanol (25):
1-O-Ieobutyryl-2 - [(β-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 warm at room temperature in an argon atmosphere. To this stirred solution were 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 hours, neutralized 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 anhydrous Na ₂ SO ₄ and evaporated to dryness. The residue was purified by flash column chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 1.7 g (83%) of pure product.

¹ H NMR (CDCl ₃ ): δ 1.16 (d, 6H, IbCH ₃ ), 1.94 (s, 3H, CH ₃ ), 2.52 (m, 1H), 3.74 (s, 6H, 2 • OCH 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), 7.26-8.00 (m, 14H, Ph).

2 - [(β- (4,4'-dimethoxytrityl) -O-acetyl) amino] -3-thyminyl-L-propanol (26):
1-O-Isobutyryl-2 - [(β- (4,4'-dimethoxytrityl) -O-acetyl) amino] -3- [N ₃ -benzoyl (thyminyl) -L-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 2 N NaOH (5 ml, 10 mmol) and stirring was continued for 30 minutes at 0 ^° C. The pH of the solution was adjusted to 7 with acetic acid and evaporated to dryness. The residue was fractionated in a mixture of water (50 ml) and CH ₂ Cl ₂ (50 ml) and extracted into 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 chromatography on silica gel using CH ₂ Cl ₂ -> acetone as eluent. Yield: 1.0 g (99%).

¹ H NMR (CDCl ₃ ): δ 1.94 (s, 3H, CH ₃ ), 3.74 (s, 6H, 2 • OCH 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), 7.26-8.00 (m, 14H, Ph).

2 - [(β- (4,4'-dimethoxytrityl) -O-acetyl) amino] -3-thyminyl-L-propan-1-O- (N, N-diisopropyl) -β-cyanoethyl-phosphoramidite (27) :
2 - [(β- (4,4'-dimethoxytrityl) -O-acetyl) amino] -3-thyminyl L-propanol (26) (1.00 g, 2.09 mmol) was dried over solid NaOH under vacuum for overnight and dissolved in dry CH ₂ Cl ₂ (50 ml). The solution was cooled to 0 ^° C. under argon. To this cold mixed solution was added N, N-diisopropylethylamine (0.54 g, 4.2 mmol), followed by 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.73 g, 3.1 mmol). The reaction mixture was stirred at 0 ^° C. for 1 hour and at room temperature for 2 hours. The reaction mixture 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 Cl ₂ extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc containing 0.1 TEA as eluent. Pure fractions were combined and evaporated to give a foam. This foam was dried over solid NaOH in vacuo overnight. The dried foam was dissolved in dry CH ₂ Cl ₂ (10 ml) and was added dropwise to a mixed solution of dry hexane (800 ml) under argon for 30 minutes. After addition, the precipitate formed was stirred for another 30 minutes and filtered, washed with dry hexane (100 ml) and the solid was dried over solid NaOH under vacuum for 4 hours. Yield: 1.3 g (82%).

Example 29 (Fig. 29)
Nα-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 mixture was added TEA (15.15 g, 150 mmol) followed by di-tert-butyl dicarbonate (23.98 g, 110 mmol). The reaction mixture was stirred at room temperature for 12 hours and evaporated to dryness. The residue was fractionated in a mixture of 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 an ice bath under argon. To this stirred solution was added dihydropyran (12.6 g, 150 mmol), followed by pyridin-p-toluene-4-sulfonate (1.25 g, 5 mmol) and stirring was continued overnight. The reaction mixture was evaporated to dryness and dissolved in EtOAc (200 ml). 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 residue was purified by flash chromatography on silica gel using hexane -> CH ₂ Cl ₂ as eluent. The pure fractions were combined and evaporated to give 11 g (67%) of pure product.

N-tert-butyloxycarbonyl-N - [(tetrahydropyranyl) oxy] - ethyl-O-benzylhydroxylamine (30):
To a stirred solution of N-tert-butyloxycarbonyl-O-benzylhydroxylamine (28) (5.79 g, 25.96 mmol) in dry DMF (50 ml) was slowly added NaH (60%, 1.2 g, 30 mmol) over a 15 minutes under argon at 0 ^° C. The reaction mixture was allowed to stir at 0 ^° C. for 30 minutes and at room temperature for 1 hour. 1-Chloro-2- (tetrahydropyranyl) oxy-ethane 29 (4.95 g) was added. 30 mmol) and the reaction mixture was heated at 80 ^° C. for 12 hours. The reaction mixture 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 on silica gel using hexane -> CH ₂ Cl ₂ as eluent. The desired fractions were combined and evaporated to give 6.0 g (66%) of an oily product.

¹ H NMR (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-hydroxy) ethyl] -O-benzylhydroxylamine (31):
A mixed solution of N-tert-butyloxycarbonyl-N- [(tetrahydropyranyl) oxy] ethyl-O-benzylhydroxylamine (30) (3.51 g, 10 mmol) in THF: water: AcOH (1: 1: 1, 100 ml) was heated at 70 ^° C. for 3 hours. The reaction mixture was cooled to 0 ^° C. and the pH was adjusted to 7 with solid NaHCO ₃ . The reaction mixture was extracted with EtOAc (2 x 75 ml). The combined organic extracts were washed with water (100 ml) and brine (100 ml), dried and evaporated to dryness. The residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 2.5 g (94%) of a foam.

¹ H NMR (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] -O-benzylhydroxylamine (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 hours 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 on silica gel using CH ₂ Cl ₂ as eluent. Pure fractions were combined and evaporated to give 4.5 g (80%) of pure compound.

¹ H NMR (CDCl ₃ ): δ 1.04 (d, 6H, IbCH ₃ ), 1.48 (s, 9H, Boc), 2.44 (m, 1H, IbCH), 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- (thyminylacetyl) -N - [[(2-isobutyryl) oxy] ethyl] -O-benzyl-hydroxylamine (33):
N-tert-butyloxycarbonyl-N - [[(2-isobutyryl) oxy] ethyl] -O-benzylhydroxylamine (32) (5.0 g, 14.84 mmol) was dissolved in CH ₂ Cl ₂ (10 ml) and allowed to mix at room temperature in trifluoroacetic acid (12 ml) for 30 minutes The reaction mixture was evaporated to dryness, dissolved in dry methanol (10 ml). Again evaporated to dryness and dried over solid NaOH in vacuo overnight. The dried material was used as such for further reactions without analysis.

Thymine acetic acid 5 (3.13 g, 17 mmol) was dissolved in dry DMF (75 ml) and cooled to -20 ^° C under argon. To this cold mixed solution was added N-methylmorpholine (2.02 g, 20 mmol) and then isobutyl chloroformate (2.72 g, 20 mmol). After 15 minutes of stirring, 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 mixed solution of thymine acetic acid. The reaction mixture was stirred at -20 ^° C for 1 h, warmed to room temperature and stirring was continued 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 in 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 a crude product. This crude product was purified by flash chromatography on 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. This pure product was crystallized from a mixture of CH ₂ Cl ₂ / hexane. Mp 185-188 ^o C.

¹ H NMR (Me ₂ SO-d ₆ ): δ 1.00 (d, 6H, IbCH 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 (Fig. 30)
(2R, 4R) -2-carbomethoxy-4-hydroxypyrrolidine (35):
Dry methanol (40 ml) was placed in a 250 ml round bottom flask equipped with a magnetic stirrer and a reflux condenser and cooled in an ice bath under argon atmosphere. Acetyl chloride (4.32 g, 55 mmol) was added to this mixed solution, followed by cis-4-hydroxy-D-proline (34) (5.00 g, 38.17 mmol). The resulting solution was heated under reflux for 7-8 hours 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 under vacuum over solid NaOH. Yield: 6.9 g (100%).

¹ H NMR (CDCl ₃ ): δ 2.09 (2dd, 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-carbomethoxy-4-hydroxypyrrolidine (36):
To a mixed solution of (2R, 4R) -2-carbomethoxy-4-hydroxypyrrolidine (35) (6.9 g, 38.12 mmol) in THF / water (8: 2, 150 ml) was added TEA (10.1 g, 100 mmol) and then di-tert-butyl dicarbonate (10.9 g, 50 mmol) at room temperature. The reaction mixture was stirred at room temperature for 6 hours and evaporated to dryness. The residue was dissolved in EtOAc (200 ml) and washed with 0.5 N 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 an oily product. The oily product upon drying gave a colorless solid. Mp 75-77 ^o C.

¹ H NMR (CDCl ₃ ): δ 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 (37):
(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 in argon atmosphere. To this cold solution was added lithium borohydride (1.88 g, 85.8 mmol) in small portions over 15 minutes. After adding lithium borohydride, the reaction mixture was allowed to stir at 0 ^° C for 1 h, and then 15 min at room temperature under argon. The solution was cooled to 0 ^° C., diluted with water (50 ml) and adjusted to pH 6 with AcOH. The reaction mixture 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 on 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. This oil formed a colorless solid upon standing. Mp 95-97 ^o C.

¹ H NMR (CDCl ₃ ): δ 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-4-hydroxypyrrolidine (38):
(2R, 4R) -1- (tert-butyloxycarbonyl) -2-hydroxymethyl-4-hydroxypyrrolidine (37) (4.4 g, 20.28 mmol) was dissolved in dry pyridine (50 ml) and allowed to mix 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 hours and neutralized 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 crude product was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. The desired fractions were combined and evaporated to give 8.09 g (100%) of an orange foam.

¹ H NMR (CDCl ₃ ): δ 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40-3.62 (m, 3H) 3.74 (s, 6H, 2 • OCH ₃ ), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 6.82 (d, 4H, Ph), 7.26-8.00 (m, 9H, Ph).

(2R, 4R) -1- (tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl) -4 - [(p-toluenesulfonyl) oxy] pyrrolidine (39):
(2R, 4R) -1- (tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl-4-hydroxypyrrolidine (38) (8.09 g, 20.27 mmol) was dissolved in dry pyridine / CH ₂ Cl ₂ (2: 1, 200 ml) and cooled in an ice bath under argon atmosphere. To this cold solution was added TEA (3.03 g, 30 mmol) and then p-toluenesulfonyl chloride (5.7 g, 30 mmol). The reaction mixture was allowed to mix at 0 ^° C for 3 hours and below 30 ^° C for 8 hours. The reaction mixture was evaporated to dryness, fractionated in a mixture of 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 on 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 H NMR (CDCl ₃ ): δ 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 • OCH ₃ ), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m , 1H); 6.82 (d, 4H, Ph); 7.26-8.00 (m, 13H, Ph).

(2R, 4S) -1- (tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl) -4-azido-pyrrolidine (40):
(2R, 4R) -1- (tert-butyloxycarbonyl) -2- (4,4'-dimethoxytrityl) oxymethyl) -4- [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 hours. It was cooled and evaporated to dryness. The residue was fractionated in a mixture of CH ₂ Cl ₂ (200 ml) 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 on 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 H NMR (CDCl ₃ ): δ 1.45 (s, 9H, Boc), 1.90 (dd, 1H), 2.34 (m, 1H), 3.40-3.62 (m, 3H) 3.74 (s, 6H, 2 • OCH ₃ ), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 (m, 1H), 6.82 (d, 4H, Ph), 7.26-8.00 (m, 9H, Ph).

(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 t, 5 mmol) in methanol (75 ml) hydrogenated in the presence of 10% palladium on charcoal (0.3 g) at room temperature and a pressure of 5 atm. After 12 hours, the catalyst was filtered off, washed with methanol (20 ml) and the solvent was removed in vacuo. Yield 1.0 g (93%).

¹ H NMR (CDCl ₃ ): δ 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, 4S) -1- (tert-butyloxycarbonyl) -2-hydroxymethyl-4-phthalimidopyrrolidine (42):
(2R, 4S) -1- (tert-butyloxycarbonyl) -2-hydroxymethyl-4-amino-pyrrolidine (42) (1.00 g, 4.63 mmol) was dissolved in dry methanol (20 ml) and treated with N-ethoxycarbonylphthalimide (1.09 g, 5 mmol) at room temperature. The reaction mixture was stirred for 6 hours and evaporated to dryness. The residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ -> EtOAc as eluent. Pure fractions were combined and evaporated to give 1.5 g (94%) of pure compound as a foam.

¹ H NMR (CDCl ₃ ): δ 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); 7.3-7.6 (m, 4H, Ph).

(2R, 4S) -1- (tert-butyloxycarbonyl) -2- [N ₃ -benzoyl (thymin-1-yl)] methyl-4-phthalimido-pyrrolidine (43):
Triphenylphosphine (2.62 g, 10 mmol) and (2R, 4S) -1- (tert) were added to a mixed solution of N ₃ -benzoylthymine (20) (1.15 g, 5 mmol) in dry THF (70 ml) under argon. -butyloxycarbonyl) -2-hydroxymethyl-4-phthalimido-pyrrolidine (1.4 g, 4.05 mmol) at room temperature. After 15 minutes, diethyl azodicarboxylate (1.74 g, 10 mmol) was added slowly over 10 minutes. The reaction mixture was covered with aluminum foil and allowed to stir at room temperature under argon for 24 hours. The solvent was evaporated to dryness 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 . Dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography on silica gel using hexane -> EtOAc as eluent. The fractions containing the desired product were combined and evaporated to give a pale pink oil. Yield 2.0 g (89%).

¹ H NMR (CDCl ₃ ): δ 1.41 (s, 9H, Boc), 1.72 (s, 9H, 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 ₂ H), 7.20-7.60 (m, 9H, Ph).

(2R, 4S) -1- (hydroxyacetyl) -2- [N ₃ -benzoyl (thymin-1-yl)] methyl-4-phthalimido-pyrrolidine (44):
To a stirred solution of compound (43) (1.12 g, 2 mmol) in methylene chloride (10 ml) was added TFA (10 ml) at room temperature and stirring was continued for 1 hour. The reaction mixture was evaporated to dryness. The residue was co-evaporated three times with dry methanol (10 ml) and the resulting residue was dried over solid NaOH overnight.

A mixture of the above TFA salt, glycolic acid (0.78 g, 10 mmol) and HBTU (321 mg, 10 mmol) was allowed to mix in dry CH ₃ CN (50 ml) at room temperature under argon. To this stirred solution was added N-methylmorpholine (1.01 g, 10 mmol) and stirring was continued overnight. The reaction mixture was evaporated to dryness. The residue was partitioned between CH ₂ Cl ₂ (100 ml) and 5% NaHCO ₃ solution (50 ml) and extracted into CH ₂ Cl ₂ . The organic extract was washed with water (50 ml) and brine (50 ml), dried and evaporated to dryness. The residue was purified by flash chromatography on silica gel using CH ₂ Cl ₂ ---> EtOAc as eluent. Pure fractions were combined and evaporated to give a foamy product.

Example 31
Synthesis of oligonucleotides:
Oligonucleotides containing amino acid-modified nucleic acid skeletons were synthesized using an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry. β-Cyanoethylphosphoramidites, synthesis reagents, and a CPG polystyrene column were obtained from Applied Biosystems (Foster City, CA). For the phosphorothioate oligonucleotides, the standard oxidation bottle was replaced with tetraethylthiuram disulfide / acetonitrile, and the standard ABI phosphorothioate program was used for the stepwise introduction of sulfur into phosphorus bonds. After removal from the glass column with a controlled pore size, the protective groups were removed by treating the oligonucleotides with concentrated ammonium hydroxide at 55 ^° C for 8 hours. Oligonucleotides (with DMT) were purified using HPLC (high performance liquid chromatography) using a semi-prepared C ₃ column with reverse phase (ABI) with a linear gradient of 5% acetonitrile in 0.1 M triethylammonium acetate (buffer A) and acetonitrile (buffer B). DMT protecting groups were cleaved by treatment with 80% acetic acid and the product was precipitated with ethanol. Product purity was monitored by HPLC using an analytical C ₁₈ column (Beckman). Amino acid-modified nucleic acid monomers were included in the DNA sequence at the 3'-end, 5'-end and in the middle with 100% combination efficiency. A homopolymer containing 16 amino acid-modified thymines was also prepared without any problems.

Example 32
Hybridization Analysis:
The ability of the amino acid-modified oligonucleotides of the invention to hybridize with their complementary RNA and DNA sequences is determined by end-melting analysis. The RNA complement was synthesized by Genset Corporation (La Jolla, CA) and purified by polyacrylamide gel electrophoresis under denaturing urea (PAGE). Antisense sequences, natural or containing any functions at specific positions, were added either to RNA or to DNA complement in stoichiometric concentrations to form hybrid duplexes. The temperature dependence of the absorption (260 nm) hyperchromia during the transition from a duplex to a random coil was recorded using a Varian Cary 1E UV-Visible spectrophotometer. The measurements were carried out in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA and NaCl having an ionic strength of either 0.1 M or 1.0 M. The data was analyzed by graphically representing 1 / Tm versus ln [Ct], where Ct is the total concentration of oligonucleotides. The results of this analysis determine the thermodynamic parameters. Based on the information obtained regarding the stability of the formed duplex or heteroduplex, the position of the modified pyrimidine in the oligonucleotides is evaluated by its effect on the stability of the helix. Modifications that fundamentally change the stability of the hybrid show a decrease or increase in free energy (ΔG), and conclusions are made regarding their suitability as antisense oligonucleotides.

 Hybridization studies were conducted on oligonucleotides containing an amino acid-modified nucleic acid skeleton at both the 3'-end and 5'-end. Preliminary studies have shown that modified oligonucleotides form a duplex with complementary RNA and DNA sequences in the same way as unmodified oligonucleotides.

Example 33
Resistance to the action of nucleases. The natural, phosphorothioate and modified oligonucleotides of the invention are tested for their resistance to serum nucleases by incubating these oligonucleotides in a medium containing various concentrations of fetal calf or adult serum. Labeled oligonucleotides are incubated for various periods of time, treated with protease K and then analyzed by gel electrophoresis in 20% polyacrylamide denaturing urea gels followed by autoradiography or phosphorography. Autoradiograms are analyzed using laser densitometry. Based on the provisions of the modifications and the known length of the oligonucleotide, it is possible to determine the effect of a particular modification on nuclease cleavage. For cytoplasmic nucleases, an HL60 cell line is used. The postmitochondrial supernatant is prepared by differential centrifugation, and labeled oligonucleotides are incubated at various time intervals in this supernatant. After incubation, oligonucleotides are tested for degradation as described above for serum nucleolytic degradation. The results of autoradiography are calculated to compare unmodified, i.e. phosphorothioate, and modified oligonucleotides.

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

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

Equivalents
This description is considered sufficient to enable a person skilled in the art to make this invention. The invention may take various forms of execution, which are obvious to experts in the field of molecular biology, organic chemistry, or related fields, and they are included in the scope of the following claims.

By the methods of phosphoramidite chemistry described in Example 31 of the present application for the invention, a number of oligonucleotides were obtained in which at different positions compounds are included, which are
a) compound 27, the preparation of which is described in example 28, which is a compound of group II according to claim 1 (L and D isomer), and
b) compound 19, the preparation of which is described in example 27, which is a compound of group 1 according to claim 1.

The synthesized modified oligonucleotides are presented in table. 1 and 2 (oligonucleotides containing compound 27) and in table. 3 (oligonucleotides containing compound 19). Hybridization analysis was performed as described in example 32 of this application. For this, complementary DNA (5 'GAA AGG AAG CGG AGA GAT 3') was synthesized, and complementary RNA (5 'GAA AGG AAG CGG AGA GAU 3') was obtained from Genset, San Diego. Samples for Tm measurements contained 2 μM of modified oligonucleotides and 2.0 μM of either complementary DNA or complementary RNA. The temperature dependence of the absorption curves for duplexes was measured in the temperature range from 20 to 90 ^o C. Based on these results, the values of Tm presented in Table 1 were calculated. 1, 2 and 3. Resistance to the action of enzymes was investigated using the following methods. Oligonucleotides (0.75 OD) were incubated with snake venom phosphodiesterase (1.2 units) in 1.5 ml buffer (0.1 M Tris. HCl, pH 7.5; 0.1 M NaCl; 14 mM MgCl ₂ ) in a cuvette of a Varian UV spectrophotometer at 25 ^° C., and an increase in absorbance at 260 nm was recorded during the decomposition period every 30 seconds. From these absorption curves, half-lives of oligonucleotides were calculated. The results are presented in table. 1 and 2; Tm is the temperature at the midpoint of the melting curve; concentrations were as follows: oligomeric filaments, 2 μM each; melting points (Tm) were determined by measuring the change in absorbance at 260 nm (cuvette, path length 1 mm) as a function of temperature in sodium phosphate buffer. All values were averages of at least three experiments. The designation "t" refers to the amino alcohol-modified thymidine of the invention (compound 27). Δ Tm / mod. represents the change in Tm by one modification. Negative values indicate a decrease in the value of Tm by one inclusion of "t".

As can be seen from the results presented in table. 1, compared with unmodified DNA, replacing T with "t" at the 3'-end of the oligonucleotide has very little effect on the stability of the duplex. For one replacement, the value of Tm decreased by 0.8 ^o C (line 3). This trend continued up to three inclusions “t” at the 3'-end (line 4). A similar binding behavior was also observed when "t" was also included at the 5'-end (line 5). On the other hand, the substitution of “t” in the middle of the oligonucleotide sequence strongly affected the hybridization properties, and the melting point of the duplex decreased by 8.7 ^° C per inclusion (line 6). The duplex formed by the modified oligonucleotide and the target RNA was characterized by a similar behavior. To identify the conformational effect of aminoalcohol nucleic acid on the stability of the duplex, the authors obtained the corresponding D-isomer "t" and made it into oligonucleotide sequences, the binding properties of which were studied.

 The results are presented in table. 2.

The inclusion of the D-isomer "t" in oligonucleotide sequences favorably affected RNA binding. The presence of the D-isomer "t" in the middle of the oligos sequence (line 6) significantly reduced the melting temperature (ΔTm = -6.78 ^o C) of the duplex with DNA, while the stability of the duplex with RNA decreased less significantly (ΔTm = -3.8 ^o C). However, a single substitution at the 3 ′ or 5 ′ end of the oligonucleotide does not lead to severe destabilization of hybridization.

Oligonucleotides containing the L or D-isomer "t" at the 3'-end showed many times greater resistance to the action of exonucleases compared to unmodified DNA. The results are presented in FIG. 33 and 34. As can be seen from these drawings, the unmodified oligonucleotide completely decomposed within 20 minutes and had a half-life of 4, 68 minutes. The oligonucleotide containing the D-amino alcohol nucleoside monomer at the 3'-end (Table 2, sequence 3, FIG. 34, sequence 7) had a half-life of 72 minutes, and the oligonucleotide containing the L-amino alcohol monomer (Table 1, sequence 3, Fig. 33, sequence 1), had a half-life of about 60 minutes. Replacing more than one modified amino alcohol with a nucleoside at the 3'-end makes the oligonucleotides very resistant to the action of nucleases (t _1/2 > 180 minutes, FIG. 34, sequence 2 and FIG. 33, sequence 3).

Claims (8)

1. Derivatives of amino acids or aminoalcohols of the formula 1 in any of groups I, II

where N is nitrogen,
Group I
X is CHR ₂ OH, where R ₂ is H, (lower alkyl) amine or (lower alkyl) imidazole;
Y is Base- (CH ₂ ) _n -, where Base is a non-halogenated variable nucleoside base, where n = 1 - 7;
A is carbonyl;
Z is H or OR 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, (lower alkyl) amine or (lower alkyl) imidazole;
Group II
A is carbonyl;
X is Base- (CH ₂ ) _n- , where Base is a non-halogenated variable nucleoside base, where n = 1 to 3;
Y is —CH ₂ OH;
W is —CH ₂ OH;
Z is N.  2. The oligonucleotide, the composition of which includes compounds according to claim 1 so that the internucleotide bonds are between W and X in group I, W and Y in group II.  3. The oligonucleotide according to claim 2, where the internucleotide bond contains a phosphodiester.  4. The oligonucleotide according to claim 2, where the internucleotide bond contains phosphorothioate.  5. The oligonucleotide according to claim 2, where the internucleotide bond contains phosphoramidate.  6. The oligonucleotide according to claim 2, where the internucleotide bond contains hydroxamate.  7. The oligonucleotide according to claim 2, in which many monomers are distinguishable, where at least one of the monomers contains a 2'-deoxyribose nucleoside, and further comprises at least two different internucleotide bonds from the group consisting of phosphodiester, phosphorothioate, phosphoramidate and hydroxamate . 8. The oligonucleotide according to claim 7, which has a 2'-deoxyribononucleoside pentafuranosyl ring, in which ring oxygen is replaced by one of the groups S, CH ₂ or NR ₆ , where R ₆ is acetyl, lower alkyl, carbonyl, carbonyl (lower alkyl) amine or carbonyl (lower alkyl) imidazole.

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